CA2190528C - Primate embryonic stem cells - Google Patents
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
Abstract
A purified preparation of primate embryonic stem cells is disclosed. This preparation is characterized by the following cell surface markers: SSEA-1 (-); SSEA-3 (+); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In a paricularly advantageous embodiment, the cells of the preparation have normal karyotypes and continue to proliferate in an undifferentiated state after continuous culture for eleven months. The embryonic stem cell lines also retain the ability, throughout the culture, to form trophoblast and to differentiate into all tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). A method for isolating a primate embryonic stem cell line is also disclosed.
Description
PRIMATE EMBRYONIC STEM CELLS
Field of the Invention In general, the field of the present invention is stem cell cultures. Specifically, the field of the present invention is primate embryonic stem cell cultures.
Background of the Inventiori In general, stem cells are undifferentiated cells which can give rise to a succession of mature functional cells. For example, a hematopoietic stem cell may give rise to any of the different types of terminally differentiated blood cells. Embryonic stem (ES) cells are derived from the embryo and are pluripotent, thus possessing the capability of developing into any organ or tissue type or, at least potentially, into a complete embryo.
One of the seminal achievements of mammalian embryology of the last decade is the routine insertion of specific genes into the mouse genome through the use of mouse ES cells. This alteration has created a bridge between the in vitro manipulations of molecular biology and an understanding of gene function in the intact animal. Mouse ES cells are undifferentiated, 2 219 u 5 ?8 PCT/US96/00596 pluripotent cells derived in vitro from preimplantation embryos (Evans, et al. Nature 292:154-159, 1981; Martin, Proc. Natl. Acad. Sci. USA 78:7634-7638, 1981) or from fetal germ cells (Matsui, et al., Cell 70:841-847, 1992). Mouse ES cells maintain an undifferentiated state through serial passages when cultured in the presence of fibroblast feeder layers in the presence of Leukemia Inhibitory Factor (LIF) (Williams, et al., Nature 336:684-687, 1988). If LIF
is removed, mouse ES cells differentiate.
Mouse ES cells cultured in non-attaching conditions aggregate and differentiate into simple embryoid bodies, with an outer layer of endoderm and an inner core of primitive ectoderm. If these embryoid bodies are then allowed to attach onto a tissue culture surface, disorganized differentiation occurs of various cell types, including nerves, blood cells, muscle, and cartilage (Martin, 1981, supra;
Doetschman, et al., J. Embryol. Exp. Morph. 87:27-45, 1985). Mouse ES cells injected into syngeneic mice form teratocarcinomas that exhibit disorganized differentiation, often with representatives of all three embryonic germ layers. Mouse ES cells combined into chimeras with normal preimplantation embryos and returned to the uterus participate in normal development (Richard, et al., Cytogenet. Cell Genet.
65:169-171, 1994).
The ability of mouse ES cells to contribute to functional germ cells in chimeras provides a method for introducing site-specific mutations into mouse lines. With appropriate transfection and selection strategies, homologous recombination can be used to derive ES cell lines with planned alterations of specific genes. These genetically altered cells can be used to form chimeras with normal embryos and chimeric animals are recovered. If the ES cells contribute to the germ line in the chimeric animal, then in the next.
Field of the Invention In general, the field of the present invention is stem cell cultures. Specifically, the field of the present invention is primate embryonic stem cell cultures.
Background of the Inventiori In general, stem cells are undifferentiated cells which can give rise to a succession of mature functional cells. For example, a hematopoietic stem cell may give rise to any of the different types of terminally differentiated blood cells. Embryonic stem (ES) cells are derived from the embryo and are pluripotent, thus possessing the capability of developing into any organ or tissue type or, at least potentially, into a complete embryo.
One of the seminal achievements of mammalian embryology of the last decade is the routine insertion of specific genes into the mouse genome through the use of mouse ES cells. This alteration has created a bridge between the in vitro manipulations of molecular biology and an understanding of gene function in the intact animal. Mouse ES cells are undifferentiated, 2 219 u 5 ?8 PCT/US96/00596 pluripotent cells derived in vitro from preimplantation embryos (Evans, et al. Nature 292:154-159, 1981; Martin, Proc. Natl. Acad. Sci. USA 78:7634-7638, 1981) or from fetal germ cells (Matsui, et al., Cell 70:841-847, 1992). Mouse ES cells maintain an undifferentiated state through serial passages when cultured in the presence of fibroblast feeder layers in the presence of Leukemia Inhibitory Factor (LIF) (Williams, et al., Nature 336:684-687, 1988). If LIF
is removed, mouse ES cells differentiate.
Mouse ES cells cultured in non-attaching conditions aggregate and differentiate into simple embryoid bodies, with an outer layer of endoderm and an inner core of primitive ectoderm. If these embryoid bodies are then allowed to attach onto a tissue culture surface, disorganized differentiation occurs of various cell types, including nerves, blood cells, muscle, and cartilage (Martin, 1981, supra;
Doetschman, et al., J. Embryol. Exp. Morph. 87:27-45, 1985). Mouse ES cells injected into syngeneic mice form teratocarcinomas that exhibit disorganized differentiation, often with representatives of all three embryonic germ layers. Mouse ES cells combined into chimeras with normal preimplantation embryos and returned to the uterus participate in normal development (Richard, et al., Cytogenet. Cell Genet.
65:169-171, 1994).
The ability of mouse ES cells to contribute to functional germ cells in chimeras provides a method for introducing site-specific mutations into mouse lines. With appropriate transfection and selection strategies, homologous recombination can be used to derive ES cell lines with planned alterations of specific genes. These genetically altered cells can be used to form chimeras with normal embryos and chimeric animals are recovered. If the ES cells contribute to the germ line in the chimeric animal, then in the next.
generation a mouse line for the planned mutation is established.
Because mouse ES cells have the potential to differentiate into any cell type in the body, mouse ES
cells allow the in vitro study of the mechanisms controlling the differentiation of specific cells or tissues. Although the study of mouse ES cells provides clues to understanding the differentiation of general mammalian tissues, dramatic differences in primate and mouse development of specific lineages limits the usefulness of mouse ES cells as a model of human development. Mouse and primate embryos differ meaningfully in the timing of expression of the embryonic genome, in the formation of an egg cylinder versus an embryonic disc (Kaufman, The Atlas of Mouse Development, London: Academic Press, 1992), in the proposed derivation of some early lineages (O'Rahilly & Muller, Developmental Stages in Human Embryos, Washington: Carnegie Institution of Washington, 1987), and in the structure and function in the extraembryonic membranes and placenta (Mossman, Vertebrate Fetal Membranes, New Brunswick: Rutgers, 1987). Other tissues differ in growth factor requirements for development (e.g. the hematopoietic system(Lapidot et al., Lab An Sci 43:147-149, 1994)), and in adult structure and function (e.g. the central nervous system). Because humans are primates, and development is remarkably similar among primates, primate ES cells lines will provide a faithful model for understanding the differentiation of primate tissues in general and human tissues in particular.
The placenta provides just one example of how primate ES cells will provide an accurate model of human development that cannot be provided by ES cells from other species. The placenta and extraembryonic membranes differ dramatically between mice and humans.
Structurally, the mouse placenta is classified as U
Because mouse ES cells have the potential to differentiate into any cell type in the body, mouse ES
cells allow the in vitro study of the mechanisms controlling the differentiation of specific cells or tissues. Although the study of mouse ES cells provides clues to understanding the differentiation of general mammalian tissues, dramatic differences in primate and mouse development of specific lineages limits the usefulness of mouse ES cells as a model of human development. Mouse and primate embryos differ meaningfully in the timing of expression of the embryonic genome, in the formation of an egg cylinder versus an embryonic disc (Kaufman, The Atlas of Mouse Development, London: Academic Press, 1992), in the proposed derivation of some early lineages (O'Rahilly & Muller, Developmental Stages in Human Embryos, Washington: Carnegie Institution of Washington, 1987), and in the structure and function in the extraembryonic membranes and placenta (Mossman, Vertebrate Fetal Membranes, New Brunswick: Rutgers, 1987). Other tissues differ in growth factor requirements for development (e.g. the hematopoietic system(Lapidot et al., Lab An Sci 43:147-149, 1994)), and in adult structure and function (e.g. the central nervous system). Because humans are primates, and development is remarkably similar among primates, primate ES cells lines will provide a faithful model for understanding the differentiation of primate tissues in general and human tissues in particular.
The placenta provides just one example of how primate ES cells will provide an accurate model of human development that cannot be provided by ES cells from other species. The placenta and extraembryonic membranes differ dramatically between mice and humans.
Structurally, the mouse placenta is classified as U
labyrinthine, whereas the human and the rhesus monkey placenta are classified as villous. Chorionic gonadotropin, expressed by the trophoblast, is an essential molecule involved in maternal recognition of pregnancy in all primates, including humans (Hearn, J
Reprod Fertil 76:809-819, 1986; Hearn et al., J Reprod Fert 92:497-509, 1991). Trophoblast secretion of chorionic gonadotropin in primates maintains the corpus luteum of pregnancy and, thus, progesterone secretion. Without progesterone, pregnancy fails.
Yet mouse trophoblast produces no chorionic gonadotropin, and mice use entirely different mechanisms for pregnancy maintenance (Hearn et al., "Normal and abnormal embryo-fetal development in mammals," In: Lamming E, ed. Marshall's Physiology of Reproduction. 4th ed. Edinburgh, New York: Churchill Livingstone, 535-676, 1994). An immortal, euploid, primate ES cell line with the developmental potential to form trophoblast in vitro, will allow the study of the ontogeny and function of genes such as chorionic gonadotropin which are critically important in human pregnancy. Indeed, the differentiation of any tissue for which there are significant differences between mice and primates will be more accurately reflected in vitro by primate ES cells than by mouse ES cells.
The major in vitro models for studying trophoblast function include human choriocarcinoma cells, which are malignant cells that may not faithfully reflect normal trophectoderm; short-term primary cultures of human and non-human primate cytotrophoblast, which in present culture conditions quickly form non-dividing syncytial trophoblast; and in vitro culture of preimplantation non-human primate embryos (Hearn, et al., J. Endocrinol. 119:249-255, 1988; Coutifaris, et al., Ann. NY Acad. Sci. 191-201, 1994). An immortal, euploid, non-human primate embryonic stem (ES) cell line with the developmental ..~ WO 96/22362 2 1 / D528 PCT/US96/00596 potential to form trophectoderm offers significant advantages over present in vitro models of human trophectoderm development and function, as trophoblast-specific genes such as chorionic gonadotropin could be stably altered in the ES cells and then studied during differentiation to trophectoderm.
The cell lines currently available that resembles primate ES cells most closely are human embryonic carcinoma (EC) cells, which are pluripotent, immortal cells derived from teratocarcinomas (Andrews, et al., Lab. Invest. 50(2):147-162, 1984; Andrews, et al., in:
Robertson E., ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford: IRL press, pp.
207-246, 1987). EC cells can be induced to differentiate in culture, and the differentiation is characterized by the loss of specific cell surface markers (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) and the appearance of new markers (Andrews, et al., 1987, supra). Human EC cells will form teratocarcinomas with derivatives of multiple embryonic lineages in tumors in nude mice. However, the range of differentiation of these human EC cells is limited compared to the range of differentiation obtained with mouse ES cells, and all EC cell lines derived to date are aneuploid (Andrews, et al., 1987, supra). Similar mouse EC cell lines have been derived from teratocarcinomas, and, in general their developmental potential is much more limited than mouse ES cells (Rossant, et al., Cell Differ. 15:155-161, 1984).
Teratocarcinomas are tumors derived from germ cells, and although germ cells (like ES cells) are theoretically totipotent (i.e. capable of forming all cell types in the body), the more limited developmental potential and the abnormal karyotypes of EC cells are thought to result from selective pressures in the teratocarcinoma tumor environment 2 l , ~ '~- ~ 8 (Rossant & Papaioannou, Cell Differ 15:155-161, 1984).
ES cells, on the other hand, are thought to retain greater developmental potential because they are derived from normal embryonic cells in vitro, without the selective pressures of the teratocarcinoma environment. Nonetheless, mouse EC cells and mouse ES
cells share the same unique combination of cell surface markers (SSEA-1 (+), SSEA-3 (-), SSEA-4 (-), and alkaline phosphatase (+)).
Pluripotent cell lines have also been derived from preimplantation embryos of several domestic and laboratory animals species (Evans, et al., Theriogenoloctv 33(1):125-128, 1990; Evans, et al., Theriocrenolocrv 33 (1) :125-128, 1990; Notarianni, et al., J. Reprod. Fertil. 41(Suppl.):51-56, 1990; Giles, et al., Mol. Reprod. Dev. 36:130-138, 1993; Graves, et al., Mol. Reprod. Dev. 36:424-433, 1993; Sukoyan, et al., Mol. Reprod. Dev. 33:418-431, 1992; Sukoyan, et al., Mol. Reprod. Dev. 36:148-158, 1993; Iannaccone, et al., Dev. Biol. 163:288-292, 1994).
Whether or not these cell lines are true ES cells lines is a subject about which there may be some difference of opinion. True ES cells should: (i) be capable of indefinite proliferation in vitro in an undifferentiated state; (ii) maintain a normal karyotype through prolonged culture; and (iii) maintain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after prolonged culture.
Strong evidence of these required properties have been published only for rodents ES cells including mouse (Evans & Kaufman, Nature 292:154-156, 1981; Martin, Proc Natl Acad Sci USA 78:7634-7638, 1981) hamster (Doetschmanet al. Dev Biol 127:224-227, 1988), and rat (Iannaccone et al. Dev Biol 163:288-292, 1994), and less conclusively for rabbit ES cells (Gileset al. Mol Reprod Dev 36:130-138, 1993; Graves & Moreadith, Mol -=- WO 96/22362 PGT/US96/00596 Reprod Dev 36:424-433, 1993). However, only established ES cell lines from the rat (Iannaccone, et al., 1994, su ra) and the mouse (Bradley, et al., Nature 309:255-256, 1984) have been reported to participate in normal development in chimeras. There are no reports of the derivation of any primate ES
cell line.
Summary of the Invention The present invention is a purified preparation of primate embryonic stem cells. The primate ES cell lines are true ES cell lines in that they: (i) are capable of indefinite proliferation in vitro in an undifferentiated state; (ii) are capable of differentiation to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after prolonged culture; and (iii) maintain a normal karyotype throughout prolonged culture. The true primate ES cells lines are therefore pluripotent.
The present invention is also summarized in that primate ES cell lines are negative for the SSEA-1 marker, positive for the SSEA-3 marker, and positive for the SSEA-4 marker. Preferably, the primate ES
cell lines are also positive for the TRA-1-60, and TRA-1-81 markers, as well as positive for the alkaline phosphatase marker.
It is an advantageous feature of the present invention that the primate ES cell lines continue to proliferate in an undifferentiated state after continuous culture for at least one year. In a particularly advantageous embodiment, the cells remain euploid after proliferation in an undifferentiated state.
It is a feature of the primate ES cell lines in accordance with the present invention that the cells can differentiate to trophoblast in vitro and express chorionic gonadotropin.
r 2 u 195 2 ~s The present invention is also a purified preparation of primate embryonic stem cells that has the ability to differentiate into cells derived from mesoderm, endoderm, and ectoderm germ layers after the cells have been injected into an immunocompromised mouse, such as a SCID mouse.
The present invention is also a method of isolating a primate embryonic stem cell line. The method comprises the steps of isolating a primate blastocyst, isolating cells from the inner cellular mass (ICM) of the blastocyst, plating the ICM cells on a fibroblast layer (wherein ICM-derived cell masses are formed) removing an ICM-derived cell mass and dissociating the mass into dissociated cells, replating the dissociated cells on embryonic feeder cells and selecting colonies with compact morphology containing cells with a high nucleus/cytoplasm ratio, and prominent nucleoli. The cells of the selected colonies are then cultured.
It is an object of the present invention to provide a primate embryonic stem cell line.
It is an object of the present invention to provide a primate embryonic stem cell line characterized by the following markers: alkaline phosphatase(+); SSEA-1(-); SSEA-3(+); SSEA-4(+); TRA-1-60(+); and TRA-1-8l(+).
It is an object of the present invention to provide a primate embryonic stem cell line capable of proliferation in an undifferentiated state after continuous culture for at least one year. Preferably, these cells remain euploid.
It is another object of the present invention to provide a primate embryonic stem cell line wherein the cells differentiate into cells derived from mesoderm, endoderm, and ectoderm germ layers when the cells are injected into an immunocompromised mouse.
Other objects, features, and advantages of the 2190r~8 ..~ WO 96/22362 PCTIUS96/00596 present invention will become obvious after study of the specification, drawings, and claims.
Description of the Drawings Fig. 1 is a photomicrograph illustrating normal XY karyotype of rhesus ES cell line R278.5 after 11 months of continuous culture.
Fig. 2 is a set of phase-contrast photomicrographs demonstrating the morphology of undifferentiated rhesus ES (R278.5) cells and of cells differentiated from R278.5 in vitro (bar = 100 ).
Photograph A demonstrates the distinct cell borders, high nucleus to cytoplasm ratio, and prominent nucleoli of undifferentiated rhesus ES cells.
Photographs B-D shows differentiated cells eight days after plating R278.5 cells on gel treated tissue culture plastic (with 103 units/ml added human LIF).
Cells of these three distinct morphologies are consistently present when R278.5 cells are allowed to differentiate at low density without fibroblasts either in the presence or absence of soluble human LIF.
Fig. 3 are photomicrographs demonstrating the expression of cell surface markers on undifferentiated rhesus ES (R278.5) cells (bar = l00 ). Photograph A
shows Alkaline Phosphatase (+); Photograph B shows SSEA-1 (-); Photograph C shows SSEA-3 (+);
Photograph D shows SSEA-4 (+); Photograph E shows TRA-1-60 (+); and Photograph F shows TRA-1-81 (+).
Fig. 4 is a photograph illustrating expression of a-fetoprotein mRNA and a- and 0- chorionic gonadotrophin mRNA expression in rhesus ES cells (R278.5) allowed to differentiate in culture.
Fig. 5 includes six photomicrographs of sections of tumors formed by injection of 0.5 X 106 rhesus. ES
(R278.5) cells into the hindleg muscles of SCID mice and analyzed 15 weeks later. Photograph A shows a low power field demonstrating disorganized differentiation of multiple cell types. A gut-like structure is encircled by smooth muscle(s), and elsewhere foci of cartilage (c) are present (bar = 400 ); Photograph B
shows striated muscle (bar = 40 ); Photograph C shows stratified squamous epithelium with several hair follicles. The labeled hair follicle (f) has a visible hair shaft (bar = 200 ); Photograph D shows stratified layers of neural cells in the pattern of a developing neural tube. An upper "ventricular" layer, containing numerous mitotic figures (arrows), overlies a lower "mantle" layer. (bar = 100 ); Photograph E
shows ciliated columnar epithelium (bar = 40 );
Photograph F shows villi covered with columnar epithelium with interspersed mucus-secreting goblet cells (bar = 200 ).
Fig. 6 includes photographs of an embryoid Body.
This embryoid body was formed from a marmoset ES cell line (Cj62) that had been continuously passaged in vitro for over 6 months. Photograph A (above) shows a section of the anterior 1/3 of the embryonic disc.
Note the primitive ectoderm (E) forms a distinct cell layer from the underlying primitive endoderm (e), with no mixing of the cell layers. Note also that amnion (a) is composed of two distinct layers; the inner layer is continuous with the primitive ectoderm at the margins. Photograph B (below) shows a section in the caudal 1/3 of embryonic disc. Note central groove (arrow) and mixing of primitive ectoderm and endoderm representing early primitive streak formation, indicating the beginning of gastrulation. 400X, toluidine blue stain.
Description of the Invention (1) In General (a) Uses of Primate ES Cells The present invention is a pluripotent, immortal euploid primate ES cell line, as exemplified by the isolation of ES cell lines from two primate species, the common marmoset (Callithrix jacchus) and the rhesus monkey (Macaca mulatta). Primate embryonic stem cells are useful for:
(i) Generatincr transgenic non-human primates for models of specific human genetic diseases. Primate embryonic stem cells will allow the generation of primate tissue or animal models for any human genetic disease for which the responsible gene has been cloned. The human genome project will identify an increasing number of genes related to human disease, but will not always provide insights into gene function. Transgenic nonhuman primates will be essential for elucidating mechanisms of disease and for testing new therapies.
(ii) Tissue transplantation. By manipulating culture conditions, primate ES cells, human and non-human, can be induced to differentiate to specific cell types, such as blood cells, neuron cells, or muscle cells. Alternatively, primate ES
cells can be allowed to differentiate in tumors in SCID mice, the tumors can be disassociated, and the specific differentiated cell types of interest can be selected by the usage of lineage specific markers through the use of fluorescent activated cell sorting (FACS) or other sorting method or by direct microdissection of tissues of interest. These differentiated cells could then be transplanted back to the adult animal to treat specific diseases, such as hematopoietic disorders, endocrine deficiencies, degenerative neurological disorders or hair loss.
(b) Selection of Model Species Macaques and marmosets were used as exemplary species for isolation of a primate ES cell line.
Macaques, such as the rhesus monkey, are Old World species that are the major primates used in biomedical.
2 1 - ~ WO 96/22362 il `~ L) ~ PCT/US96/00596 research. They are relatively large (about 7-10 kg).
Males take 4-5 years to mature, and females have single young. Because of the extremely close anatomical and physiological similarities between humans and rhesus monkeys, rhesus monkey true ES cell lines provide a very accurate in vitro model for human differentiation. Rhesus monkey ES cell lines and rhesus monkeys will be particularly useful in the testing of the safety and efficacy of the transplantation of differentiated cell types into whole animals for the treatment of specific diseases or conditions. In addition, the techniques developed for the rhesus ES cell lines model the generation, characterization and manipulation of human ES cell lines.
The common marmoset (Callithrix jacchus) is a New World primate species with reproductive characteristics that make it an excellent choice for ES cell derivation. Marmosets are small (about 350-400 g), have a short gestation period (144 days), reach sexual maturity in about 18 months, and routinely have twins or triplets. Unlike in macaques, it is possible to routinely synchronize ovarian cycles in the marmoset with prostaglandin analogs, making collection of age-matched embryos from multiple females possible, and allowing efficient embryo transfer to synchronized recipients with 700-80% of embryos transferred resulting in pregnancies. Because of these reproductive characteristics that allow for the routine efficient transfer of multiple embryos, marmosets provide an excellent primate species in which to generate transgenic models for human diseases.
There are approximately 200 primate species in the world. The most fundamental division that divides higher primates is between Old World and New world species. The evolutionary distance between the rhesus monkey and the common marmoset is far greater than the evolutionary distance between humans and rhesus monkeys. Because it is here demonstrated that it is possible to isolate ES cell lines from a representative species of both the Old World and New World group using similar conditions, the techniques described below may be used successfully in deriving ES cell lines in other higher primates as well. Given the close evolutionary distance between rhesus macaques and humans, and the fact that feeder-dependent human EC cell lines can be grown in conditions similar to those that support primate ES
cell lines, the same growth conditions will allow the isolation and growth of human ES cells. In addition, human ES cell lines will be permanent cell lines that will also be distinguished from all other permanent human cell lines by their normal karyotype and the expression of the same combination of cell surface markers (alkaline phosphotase, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81) that characterize other primate ES
cell lines. A normal karyotype and the expression of this combination of cell surface markers will be defining properties of true human ES cell lines, regardless of the method used for their isolation and regardless of their tissue of origin.
No other primate (human or non-human) ES cell line is known to exist. The only published permanent, euploid, embryo-derived cell lines that have been convincingly demonstrated to differentiate into derivatives of all three germ layers have been derived from rodents (the mouse, rat, and hamster), and possibly from rabbit. The published reports of embryo-derived cell lines from domestic species have failed to convincingly demonstrate differentiation of derivatives of all three embryonic germ layers or have not been permanent cell lines. Research groups in Britain and Singapore are informally reported, later ~ ~
WO 96/22362 2 1~ ~ ~ ~ ~, ~ J . (/ _ ~ PGT/US96/00596 than the work described here, to have attempted to derive human ES cell lines from surplus in vitro fertilization-produced human embryos, although they have not yet reported success in demonstrating pluripotency of their cells and have failed to isolate permanent cell lines. In the only published report on attempts to isolate human ES cells, conditions were used (LIF in the absence of fibroblast feeder layers) that the results below will indicate will not result in primate ES cells which can remain in an undifferentiated state. It is not surprising, then that the cells grown out of human ICMs failed to continue to proliferate after 1 or 2 subcultures, Bongso et al. Hum. Reprod. 9:2100-2117 (1994).
(2) Embryonic Stem Cell Isolation A preferable medium for isolation of embryonic stem cells is "ES medium." ES medium consists of 800 Dulbecco's modified Eagle's medium (DMEM; no pyruvate, high glucose formulation, Gibco BRL), with 20% fetal bovine serum (FBS; Hyclone), 0.1 mM ,6-mercaptoethanol (Sigma), 1% non-essential amino acid stock (Gibco BRL). Preferably, fetal bovine serum batches are compared by testing clonal plating efficiency of a low passage mouse ES cell line (ES,t3), a cell line developed just for the purpose of this test. FBS
batches must be compared because it has been found that batches vary dramatically in their ability to support embryonic cell growth, but any other method of assaying the competence of FBS batches for support of embryonic cells will work as an alternative.
Primate ES cells are isolated on a confluent layer of murine embryonic fibroblast in the presence of ES cell medium. Embryonic fibroblasts are preferably obtained from 12 day old fetuses from outbred CF1 mice (SASCO), but other strains may be used as an alternative. Tissue culture dishes are ,.. WO 96/22362 ~ ~ ~ ~ ~ 28 PCT/US96/00596 preferably treated with 0.1% gelatin (type I; Sigma).
For rhesus monkey embryos, adult female rhesus monkeys (greater than four years old) demonstrating normal ovarian cycles are observed daily for evidence of menstrual bleeding (day 1 of cycle = the day of onset of inenses). Blood samples are drawn daily during the follicular phase starting from day 8 of the menstrual cycle, and serum concentrations of luteinizing hormone are determined by radioimmunoassay. The female is paired with a male rhesus monkey of proven fertility from day 9 of the menstrual cycle until 48 hours after the luteinizing hormone surge; ovulation is taken as the day following the luteinizing hormone surge. Expanded blastocysts are collected by non-surgical uterine flushing at six days after ovulation. This procedure routinely results in the recovery of an average 0.4 to 0.6 viable embryos per rhesus monkey per month, Seshagiri et al. Am J Primatol 29:81-91, 1993.
For marmoset embryos, adult female marmosets (greater than two years of age) demonstrating regular ovarian cycles are maintained in family groups, with a fertile male and up to five progeny. Ovarian cycles are controlled by intramuscular injection of 0.75 g of the prostaglandin PGF2a analog cloprostenol (Estrumate, Mobay Corp, Shawnee, KS) during the middle to late luteal phase. Blood samples are drawn on day 0 (immediately before cloprostenol injection), and on days 3, 7, 9, 11, and 13. Plasma progesterone concentrations are determined by ELISA. The day of ovulation is taken as the day preceding a plasma progesterone concentration of 10 ng/ml or more. At eight days after ovulation, expanded blastocysts are recovered by a non-surgical uterine flush procedure, Thomson et al. "Non-surgical uterine stage preimplantation embryo collection from the common marmoset," J Med Primatol, 23:333-336 (1994). This ? ~ / 9 (l~ , .., F; ;_. ?~
-L.
~
Reprod Fertil 76:809-819, 1986; Hearn et al., J Reprod Fert 92:497-509, 1991). Trophoblast secretion of chorionic gonadotropin in primates maintains the corpus luteum of pregnancy and, thus, progesterone secretion. Without progesterone, pregnancy fails.
Yet mouse trophoblast produces no chorionic gonadotropin, and mice use entirely different mechanisms for pregnancy maintenance (Hearn et al., "Normal and abnormal embryo-fetal development in mammals," In: Lamming E, ed. Marshall's Physiology of Reproduction. 4th ed. Edinburgh, New York: Churchill Livingstone, 535-676, 1994). An immortal, euploid, primate ES cell line with the developmental potential to form trophoblast in vitro, will allow the study of the ontogeny and function of genes such as chorionic gonadotropin which are critically important in human pregnancy. Indeed, the differentiation of any tissue for which there are significant differences between mice and primates will be more accurately reflected in vitro by primate ES cells than by mouse ES cells.
The major in vitro models for studying trophoblast function include human choriocarcinoma cells, which are malignant cells that may not faithfully reflect normal trophectoderm; short-term primary cultures of human and non-human primate cytotrophoblast, which in present culture conditions quickly form non-dividing syncytial trophoblast; and in vitro culture of preimplantation non-human primate embryos (Hearn, et al., J. Endocrinol. 119:249-255, 1988; Coutifaris, et al., Ann. NY Acad. Sci. 191-201, 1994). An immortal, euploid, non-human primate embryonic stem (ES) cell line with the developmental ..~ WO 96/22362 2 1 / D528 PCT/US96/00596 potential to form trophectoderm offers significant advantages over present in vitro models of human trophectoderm development and function, as trophoblast-specific genes such as chorionic gonadotropin could be stably altered in the ES cells and then studied during differentiation to trophectoderm.
The cell lines currently available that resembles primate ES cells most closely are human embryonic carcinoma (EC) cells, which are pluripotent, immortal cells derived from teratocarcinomas (Andrews, et al., Lab. Invest. 50(2):147-162, 1984; Andrews, et al., in:
Robertson E., ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford: IRL press, pp.
207-246, 1987). EC cells can be induced to differentiate in culture, and the differentiation is characterized by the loss of specific cell surface markers (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) and the appearance of new markers (Andrews, et al., 1987, supra). Human EC cells will form teratocarcinomas with derivatives of multiple embryonic lineages in tumors in nude mice. However, the range of differentiation of these human EC cells is limited compared to the range of differentiation obtained with mouse ES cells, and all EC cell lines derived to date are aneuploid (Andrews, et al., 1987, supra). Similar mouse EC cell lines have been derived from teratocarcinomas, and, in general their developmental potential is much more limited than mouse ES cells (Rossant, et al., Cell Differ. 15:155-161, 1984).
Teratocarcinomas are tumors derived from germ cells, and although germ cells (like ES cells) are theoretically totipotent (i.e. capable of forming all cell types in the body), the more limited developmental potential and the abnormal karyotypes of EC cells are thought to result from selective pressures in the teratocarcinoma tumor environment 2 l , ~ '~- ~ 8 (Rossant & Papaioannou, Cell Differ 15:155-161, 1984).
ES cells, on the other hand, are thought to retain greater developmental potential because they are derived from normal embryonic cells in vitro, without the selective pressures of the teratocarcinoma environment. Nonetheless, mouse EC cells and mouse ES
cells share the same unique combination of cell surface markers (SSEA-1 (+), SSEA-3 (-), SSEA-4 (-), and alkaline phosphatase (+)).
Pluripotent cell lines have also been derived from preimplantation embryos of several domestic and laboratory animals species (Evans, et al., Theriogenoloctv 33(1):125-128, 1990; Evans, et al., Theriocrenolocrv 33 (1) :125-128, 1990; Notarianni, et al., J. Reprod. Fertil. 41(Suppl.):51-56, 1990; Giles, et al., Mol. Reprod. Dev. 36:130-138, 1993; Graves, et al., Mol. Reprod. Dev. 36:424-433, 1993; Sukoyan, et al., Mol. Reprod. Dev. 33:418-431, 1992; Sukoyan, et al., Mol. Reprod. Dev. 36:148-158, 1993; Iannaccone, et al., Dev. Biol. 163:288-292, 1994).
Whether or not these cell lines are true ES cells lines is a subject about which there may be some difference of opinion. True ES cells should: (i) be capable of indefinite proliferation in vitro in an undifferentiated state; (ii) maintain a normal karyotype through prolonged culture; and (iii) maintain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after prolonged culture.
Strong evidence of these required properties have been published only for rodents ES cells including mouse (Evans & Kaufman, Nature 292:154-156, 1981; Martin, Proc Natl Acad Sci USA 78:7634-7638, 1981) hamster (Doetschmanet al. Dev Biol 127:224-227, 1988), and rat (Iannaccone et al. Dev Biol 163:288-292, 1994), and less conclusively for rabbit ES cells (Gileset al. Mol Reprod Dev 36:130-138, 1993; Graves & Moreadith, Mol -=- WO 96/22362 PGT/US96/00596 Reprod Dev 36:424-433, 1993). However, only established ES cell lines from the rat (Iannaccone, et al., 1994, su ra) and the mouse (Bradley, et al., Nature 309:255-256, 1984) have been reported to participate in normal development in chimeras. There are no reports of the derivation of any primate ES
cell line.
Summary of the Invention The present invention is a purified preparation of primate embryonic stem cells. The primate ES cell lines are true ES cell lines in that they: (i) are capable of indefinite proliferation in vitro in an undifferentiated state; (ii) are capable of differentiation to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after prolonged culture; and (iii) maintain a normal karyotype throughout prolonged culture. The true primate ES cells lines are therefore pluripotent.
The present invention is also summarized in that primate ES cell lines are negative for the SSEA-1 marker, positive for the SSEA-3 marker, and positive for the SSEA-4 marker. Preferably, the primate ES
cell lines are also positive for the TRA-1-60, and TRA-1-81 markers, as well as positive for the alkaline phosphatase marker.
It is an advantageous feature of the present invention that the primate ES cell lines continue to proliferate in an undifferentiated state after continuous culture for at least one year. In a particularly advantageous embodiment, the cells remain euploid after proliferation in an undifferentiated state.
It is a feature of the primate ES cell lines in accordance with the present invention that the cells can differentiate to trophoblast in vitro and express chorionic gonadotropin.
r 2 u 195 2 ~s The present invention is also a purified preparation of primate embryonic stem cells that has the ability to differentiate into cells derived from mesoderm, endoderm, and ectoderm germ layers after the cells have been injected into an immunocompromised mouse, such as a SCID mouse.
The present invention is also a method of isolating a primate embryonic stem cell line. The method comprises the steps of isolating a primate blastocyst, isolating cells from the inner cellular mass (ICM) of the blastocyst, plating the ICM cells on a fibroblast layer (wherein ICM-derived cell masses are formed) removing an ICM-derived cell mass and dissociating the mass into dissociated cells, replating the dissociated cells on embryonic feeder cells and selecting colonies with compact morphology containing cells with a high nucleus/cytoplasm ratio, and prominent nucleoli. The cells of the selected colonies are then cultured.
It is an object of the present invention to provide a primate embryonic stem cell line.
It is an object of the present invention to provide a primate embryonic stem cell line characterized by the following markers: alkaline phosphatase(+); SSEA-1(-); SSEA-3(+); SSEA-4(+); TRA-1-60(+); and TRA-1-8l(+).
It is an object of the present invention to provide a primate embryonic stem cell line capable of proliferation in an undifferentiated state after continuous culture for at least one year. Preferably, these cells remain euploid.
It is another object of the present invention to provide a primate embryonic stem cell line wherein the cells differentiate into cells derived from mesoderm, endoderm, and ectoderm germ layers when the cells are injected into an immunocompromised mouse.
Other objects, features, and advantages of the 2190r~8 ..~ WO 96/22362 PCTIUS96/00596 present invention will become obvious after study of the specification, drawings, and claims.
Description of the Drawings Fig. 1 is a photomicrograph illustrating normal XY karyotype of rhesus ES cell line R278.5 after 11 months of continuous culture.
Fig. 2 is a set of phase-contrast photomicrographs demonstrating the morphology of undifferentiated rhesus ES (R278.5) cells and of cells differentiated from R278.5 in vitro (bar = 100 ).
Photograph A demonstrates the distinct cell borders, high nucleus to cytoplasm ratio, and prominent nucleoli of undifferentiated rhesus ES cells.
Photographs B-D shows differentiated cells eight days after plating R278.5 cells on gel treated tissue culture plastic (with 103 units/ml added human LIF).
Cells of these three distinct morphologies are consistently present when R278.5 cells are allowed to differentiate at low density without fibroblasts either in the presence or absence of soluble human LIF.
Fig. 3 are photomicrographs demonstrating the expression of cell surface markers on undifferentiated rhesus ES (R278.5) cells (bar = l00 ). Photograph A
shows Alkaline Phosphatase (+); Photograph B shows SSEA-1 (-); Photograph C shows SSEA-3 (+);
Photograph D shows SSEA-4 (+); Photograph E shows TRA-1-60 (+); and Photograph F shows TRA-1-81 (+).
Fig. 4 is a photograph illustrating expression of a-fetoprotein mRNA and a- and 0- chorionic gonadotrophin mRNA expression in rhesus ES cells (R278.5) allowed to differentiate in culture.
Fig. 5 includes six photomicrographs of sections of tumors formed by injection of 0.5 X 106 rhesus. ES
(R278.5) cells into the hindleg muscles of SCID mice and analyzed 15 weeks later. Photograph A shows a low power field demonstrating disorganized differentiation of multiple cell types. A gut-like structure is encircled by smooth muscle(s), and elsewhere foci of cartilage (c) are present (bar = 400 ); Photograph B
shows striated muscle (bar = 40 ); Photograph C shows stratified squamous epithelium with several hair follicles. The labeled hair follicle (f) has a visible hair shaft (bar = 200 ); Photograph D shows stratified layers of neural cells in the pattern of a developing neural tube. An upper "ventricular" layer, containing numerous mitotic figures (arrows), overlies a lower "mantle" layer. (bar = 100 ); Photograph E
shows ciliated columnar epithelium (bar = 40 );
Photograph F shows villi covered with columnar epithelium with interspersed mucus-secreting goblet cells (bar = 200 ).
Fig. 6 includes photographs of an embryoid Body.
This embryoid body was formed from a marmoset ES cell line (Cj62) that had been continuously passaged in vitro for over 6 months. Photograph A (above) shows a section of the anterior 1/3 of the embryonic disc.
Note the primitive ectoderm (E) forms a distinct cell layer from the underlying primitive endoderm (e), with no mixing of the cell layers. Note also that amnion (a) is composed of two distinct layers; the inner layer is continuous with the primitive ectoderm at the margins. Photograph B (below) shows a section in the caudal 1/3 of embryonic disc. Note central groove (arrow) and mixing of primitive ectoderm and endoderm representing early primitive streak formation, indicating the beginning of gastrulation. 400X, toluidine blue stain.
Description of the Invention (1) In General (a) Uses of Primate ES Cells The present invention is a pluripotent, immortal euploid primate ES cell line, as exemplified by the isolation of ES cell lines from two primate species, the common marmoset (Callithrix jacchus) and the rhesus monkey (Macaca mulatta). Primate embryonic stem cells are useful for:
(i) Generatincr transgenic non-human primates for models of specific human genetic diseases. Primate embryonic stem cells will allow the generation of primate tissue or animal models for any human genetic disease for which the responsible gene has been cloned. The human genome project will identify an increasing number of genes related to human disease, but will not always provide insights into gene function. Transgenic nonhuman primates will be essential for elucidating mechanisms of disease and for testing new therapies.
(ii) Tissue transplantation. By manipulating culture conditions, primate ES cells, human and non-human, can be induced to differentiate to specific cell types, such as blood cells, neuron cells, or muscle cells. Alternatively, primate ES
cells can be allowed to differentiate in tumors in SCID mice, the tumors can be disassociated, and the specific differentiated cell types of interest can be selected by the usage of lineage specific markers through the use of fluorescent activated cell sorting (FACS) or other sorting method or by direct microdissection of tissues of interest. These differentiated cells could then be transplanted back to the adult animal to treat specific diseases, such as hematopoietic disorders, endocrine deficiencies, degenerative neurological disorders or hair loss.
(b) Selection of Model Species Macaques and marmosets were used as exemplary species for isolation of a primate ES cell line.
Macaques, such as the rhesus monkey, are Old World species that are the major primates used in biomedical.
2 1 - ~ WO 96/22362 il `~ L) ~ PCT/US96/00596 research. They are relatively large (about 7-10 kg).
Males take 4-5 years to mature, and females have single young. Because of the extremely close anatomical and physiological similarities between humans and rhesus monkeys, rhesus monkey true ES cell lines provide a very accurate in vitro model for human differentiation. Rhesus monkey ES cell lines and rhesus monkeys will be particularly useful in the testing of the safety and efficacy of the transplantation of differentiated cell types into whole animals for the treatment of specific diseases or conditions. In addition, the techniques developed for the rhesus ES cell lines model the generation, characterization and manipulation of human ES cell lines.
The common marmoset (Callithrix jacchus) is a New World primate species with reproductive characteristics that make it an excellent choice for ES cell derivation. Marmosets are small (about 350-400 g), have a short gestation period (144 days), reach sexual maturity in about 18 months, and routinely have twins or triplets. Unlike in macaques, it is possible to routinely synchronize ovarian cycles in the marmoset with prostaglandin analogs, making collection of age-matched embryos from multiple females possible, and allowing efficient embryo transfer to synchronized recipients with 700-80% of embryos transferred resulting in pregnancies. Because of these reproductive characteristics that allow for the routine efficient transfer of multiple embryos, marmosets provide an excellent primate species in which to generate transgenic models for human diseases.
There are approximately 200 primate species in the world. The most fundamental division that divides higher primates is between Old World and New world species. The evolutionary distance between the rhesus monkey and the common marmoset is far greater than the evolutionary distance between humans and rhesus monkeys. Because it is here demonstrated that it is possible to isolate ES cell lines from a representative species of both the Old World and New World group using similar conditions, the techniques described below may be used successfully in deriving ES cell lines in other higher primates as well. Given the close evolutionary distance between rhesus macaques and humans, and the fact that feeder-dependent human EC cell lines can be grown in conditions similar to those that support primate ES
cell lines, the same growth conditions will allow the isolation and growth of human ES cells. In addition, human ES cell lines will be permanent cell lines that will also be distinguished from all other permanent human cell lines by their normal karyotype and the expression of the same combination of cell surface markers (alkaline phosphotase, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81) that characterize other primate ES
cell lines. A normal karyotype and the expression of this combination of cell surface markers will be defining properties of true human ES cell lines, regardless of the method used for their isolation and regardless of their tissue of origin.
No other primate (human or non-human) ES cell line is known to exist. The only published permanent, euploid, embryo-derived cell lines that have been convincingly demonstrated to differentiate into derivatives of all three germ layers have been derived from rodents (the mouse, rat, and hamster), and possibly from rabbit. The published reports of embryo-derived cell lines from domestic species have failed to convincingly demonstrate differentiation of derivatives of all three embryonic germ layers or have not been permanent cell lines. Research groups in Britain and Singapore are informally reported, later ~ ~
WO 96/22362 2 1~ ~ ~ ~ ~, ~ J . (/ _ ~ PGT/US96/00596 than the work described here, to have attempted to derive human ES cell lines from surplus in vitro fertilization-produced human embryos, although they have not yet reported success in demonstrating pluripotency of their cells and have failed to isolate permanent cell lines. In the only published report on attempts to isolate human ES cells, conditions were used (LIF in the absence of fibroblast feeder layers) that the results below will indicate will not result in primate ES cells which can remain in an undifferentiated state. It is not surprising, then that the cells grown out of human ICMs failed to continue to proliferate after 1 or 2 subcultures, Bongso et al. Hum. Reprod. 9:2100-2117 (1994).
(2) Embryonic Stem Cell Isolation A preferable medium for isolation of embryonic stem cells is "ES medium." ES medium consists of 800 Dulbecco's modified Eagle's medium (DMEM; no pyruvate, high glucose formulation, Gibco BRL), with 20% fetal bovine serum (FBS; Hyclone), 0.1 mM ,6-mercaptoethanol (Sigma), 1% non-essential amino acid stock (Gibco BRL). Preferably, fetal bovine serum batches are compared by testing clonal plating efficiency of a low passage mouse ES cell line (ES,t3), a cell line developed just for the purpose of this test. FBS
batches must be compared because it has been found that batches vary dramatically in their ability to support embryonic cell growth, but any other method of assaying the competence of FBS batches for support of embryonic cells will work as an alternative.
Primate ES cells are isolated on a confluent layer of murine embryonic fibroblast in the presence of ES cell medium. Embryonic fibroblasts are preferably obtained from 12 day old fetuses from outbred CF1 mice (SASCO), but other strains may be used as an alternative. Tissue culture dishes are ,.. WO 96/22362 ~ ~ ~ ~ ~ 28 PCT/US96/00596 preferably treated with 0.1% gelatin (type I; Sigma).
For rhesus monkey embryos, adult female rhesus monkeys (greater than four years old) demonstrating normal ovarian cycles are observed daily for evidence of menstrual bleeding (day 1 of cycle = the day of onset of inenses). Blood samples are drawn daily during the follicular phase starting from day 8 of the menstrual cycle, and serum concentrations of luteinizing hormone are determined by radioimmunoassay. The female is paired with a male rhesus monkey of proven fertility from day 9 of the menstrual cycle until 48 hours after the luteinizing hormone surge; ovulation is taken as the day following the luteinizing hormone surge. Expanded blastocysts are collected by non-surgical uterine flushing at six days after ovulation. This procedure routinely results in the recovery of an average 0.4 to 0.6 viable embryos per rhesus monkey per month, Seshagiri et al. Am J Primatol 29:81-91, 1993.
For marmoset embryos, adult female marmosets (greater than two years of age) demonstrating regular ovarian cycles are maintained in family groups, with a fertile male and up to five progeny. Ovarian cycles are controlled by intramuscular injection of 0.75 g of the prostaglandin PGF2a analog cloprostenol (Estrumate, Mobay Corp, Shawnee, KS) during the middle to late luteal phase. Blood samples are drawn on day 0 (immediately before cloprostenol injection), and on days 3, 7, 9, 11, and 13. Plasma progesterone concentrations are determined by ELISA. The day of ovulation is taken as the day preceding a plasma progesterone concentration of 10 ng/ml or more. At eight days after ovulation, expanded blastocysts are recovered by a non-surgical uterine flush procedure, Thomson et al. "Non-surgical uterine stage preimplantation embryo collection from the common marmoset," J Med Primatol, 23:333-336 (1994). This ? ~ / 9 (l~ , .., F; ;_. ?~
-L.
~
procedure results in the average production of 1.0 viable embryos per marmoset per month.
The zona pellucida is removed from blastocysts by brief exposure to pronase (Sigma). For immunosurgery, blastocysts are exposed to a 1:50 dilution of rabbit anti-marmoset spleen cell antiserum (for marmoset blastocysts) or a 1:50 dilution of rabbit anti-rhesus monkey (for rhesus monkey blastocysts) in DMEM for 30 minutes, then washed for 5 minutes three times in DMEM, then exposed to a 1:5 dilution of Guinea pig complement (Gibco) for 3 minutes.
After two further washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM
plated on mouse inactivated (3000 rads gamma irradiation) embryonic fibroblasts.
After 7-21 days, ICM-derived masses are removed from endoderm outgrowths with a micropipette with direct observation under a stereo microscope, exposed to 0.0501 Trypsin-EDTA (Gibco) supplemented with 11i chicken serum for 3-5 minutes and gently dissociated by gentle pipetting through a flame polished micropipette.
Dissociated cells are replated on embryonic feeder layers in fresh ES medium, and observed for colony formation. Colonies demonstrating ES-like morphology are individually selected, and split again as described above. The ES-like morphology is defined as compact colonies having a high nucleus to cytoplasm ratio and prominent nucleoli. Resulting ES cells are then routinely split by brief trypsinization or exposure to Dulbecco's Phosphate Buffered Saline (without calcium or magnesium and with 2 mM EDTA) every 1-2 weeks as the cultures become dense. Early passage cells are also frozen and stored in liquid nitrogen.
Cell lines may be karyotyped with a standard G-_ banding technique (such as by the Cytogenetics Laboratory of the University of Wisconsin State Hygiene Laboratory, which provides routine karyotyping services) and compared to published karyotypes for the primate species.
Isolation of ES cell lines from other primate species would follow a similar procedure, except that the rate of development to blastocyst can vary by a few days between species, and the rate of development of the cultured ICMs will vary between species. For example, six days after ovulation, rhesus monkey embryos are at the expanded blastocyst stage, whereas marmoset embryos don't reach the same stage until 7-8 days after ovulation. The Rhesus ES cell lines were obtained by splitting the ICM-derived cells for the first time at 7-16 days after immunosurgery; whereas the marmoset ES cells were derived with the initial split at 7-10 days after immunosurgery. Because other primates also vary in their developmental rate, the timing of embryo collection, and the timing of the initial ICM split will vary between primate species, but the same techniques and culture conditions will allow ES cell isolation.
Because ethical considerations in the U.S. do not allow the recovery of human in vivo fertilized preimplantation embryos from the uterus, human ES
cells that are derived from preimplantation embryos will be derived from in vitro fertilized (IVF) embryos. Experiments on unused (spare) human IVF-produced embryos are allowed in many countries, such as Singapore and the United Kingdom, if the embryos are less than 14 days old. Only high quality embryos are suitable for ES isolation. Present defined culture conditions for culturing the one cell human embryo to the expanded blastocyst are suboptimal but practicable, Bongso et al., Hum Reprod 4:706-713, 1989. Co-culturing of human embryos with human ~ ~
19 UJ%
The zona pellucida is removed from blastocysts by brief exposure to pronase (Sigma). For immunosurgery, blastocysts are exposed to a 1:50 dilution of rabbit anti-marmoset spleen cell antiserum (for marmoset blastocysts) or a 1:50 dilution of rabbit anti-rhesus monkey (for rhesus monkey blastocysts) in DMEM for 30 minutes, then washed for 5 minutes three times in DMEM, then exposed to a 1:5 dilution of Guinea pig complement (Gibco) for 3 minutes.
After two further washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM
plated on mouse inactivated (3000 rads gamma irradiation) embryonic fibroblasts.
After 7-21 days, ICM-derived masses are removed from endoderm outgrowths with a micropipette with direct observation under a stereo microscope, exposed to 0.0501 Trypsin-EDTA (Gibco) supplemented with 11i chicken serum for 3-5 minutes and gently dissociated by gentle pipetting through a flame polished micropipette.
Dissociated cells are replated on embryonic feeder layers in fresh ES medium, and observed for colony formation. Colonies demonstrating ES-like morphology are individually selected, and split again as described above. The ES-like morphology is defined as compact colonies having a high nucleus to cytoplasm ratio and prominent nucleoli. Resulting ES cells are then routinely split by brief trypsinization or exposure to Dulbecco's Phosphate Buffered Saline (without calcium or magnesium and with 2 mM EDTA) every 1-2 weeks as the cultures become dense. Early passage cells are also frozen and stored in liquid nitrogen.
Cell lines may be karyotyped with a standard G-_ banding technique (such as by the Cytogenetics Laboratory of the University of Wisconsin State Hygiene Laboratory, which provides routine karyotyping services) and compared to published karyotypes for the primate species.
Isolation of ES cell lines from other primate species would follow a similar procedure, except that the rate of development to blastocyst can vary by a few days between species, and the rate of development of the cultured ICMs will vary between species. For example, six days after ovulation, rhesus monkey embryos are at the expanded blastocyst stage, whereas marmoset embryos don't reach the same stage until 7-8 days after ovulation. The Rhesus ES cell lines were obtained by splitting the ICM-derived cells for the first time at 7-16 days after immunosurgery; whereas the marmoset ES cells were derived with the initial split at 7-10 days after immunosurgery. Because other primates also vary in their developmental rate, the timing of embryo collection, and the timing of the initial ICM split will vary between primate species, but the same techniques and culture conditions will allow ES cell isolation.
Because ethical considerations in the U.S. do not allow the recovery of human in vivo fertilized preimplantation embryos from the uterus, human ES
cells that are derived from preimplantation embryos will be derived from in vitro fertilized (IVF) embryos. Experiments on unused (spare) human IVF-produced embryos are allowed in many countries, such as Singapore and the United Kingdom, if the embryos are less than 14 days old. Only high quality embryos are suitable for ES isolation. Present defined culture conditions for culturing the one cell human embryo to the expanded blastocyst are suboptimal but practicable, Bongso et al., Hum Reprod 4:706-713, 1989. Co-culturing of human embryos with human ~ ~
19 UJ%
oviductal cells results in the production of high blastocyst quality. IVF-derived expanded human blastocysts grown in cellular co-culture, or in improved defined medium, will allow the isolation of human ES cells with the same procedures described above for nonhuman primates.
(3) Definincr Characteristics of Primate ES Cells Primate embryonic stem cells share features with the primate ICM and with pluripotent human embryonal carcinoma cells. Putative primate ES cells may therefore be characterized by morphology and by the expression of cell surface markers characteristic of human EC cells. Additionally, putative primate ES
cells may be characterized by developmental potential, karyotype and immortality.
(a) Morphology The colony morphology of primate embryonic stem cell lines is similar to, but distinct from, mouse embryonic stem cells. Both mouse and primate ES cells have the characteristic features of undifferentiated stem cells, with high nuclear/cytoplasmic ratios, prominent nucleoli, and compact colony formation. The colonies of primate ES cells are flatter than mouse ES
cell colonies and individual primate ES cells can be easily distinguished. In Fig. 2, reference character A indicates a phase contrast photomicrograph of cell line R278.5 demonstrating the characteristic primate ES cell morphology.
(b) Cell Surface Markers A primate ES cell line of the present invention is distinct from mouse ES cell lines by the presence or absence of the cell surface markers described below.
One set of glycolipid cell surface markers is known as the Stage-specific embryonic antigens 1 through 4. These antigens can be identified using ,.. , WO 96/22362 217 0528 PCTIUS96/00596 antibodies for SSEA 1, SSEA-3 and SSEA-4 which are available from the Developmental Studies Hybridoma Bank of the National Institute of Child Health and Human Development. The cell surface markers referred to as TRA-1-60 and TRA-1-81 designate antibodies from hybridomas developed by Peter Andrews of the University of Sheffield and are described in Andrews et al., "Cell lines from human germ cell tumors," In:
Robertson E, ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford: IRL Press, 207-246, 1987. The antibodies were localized with a biotinylated secondary antibody and then an avidin/biotinylated horseradish peroxidase complex (Vectastain ABC System, Vector Laboratories).
Alternatively, it should also be understood that other antibodies for these same cell surface markers can be generated. NTERA-2 cl. Dl, a pluripotent human EC
cell line (gift of Peter Andrews), may be used as a negative control for SSEA-1, and as a positive control for SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. This cell line was chosen for positive control only because it has been extensively studied and reported in the literature, but other human EC cell lines may be used as well.
Mouse ES cells (ESj13) are used as a positive control for SSEA-1, and for a negative control for SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Other routine negative controls include omission of the primary or secondary antibody and substitution of a primary antibody with an unrelated specificity.
Alkaline phosphatase may be detected following fixation of cells with 4% para-formaldehyde using "Vector Red" (Vector Laboratories) as a substrate, as described by the manufacturer (Vector Laboratories).
The precipitate formed by this substrate is red when viewed with a rhodamine filter system, providing substantial amplification over light microscopy.
WO 96/22362 2 19~ j~~ PCT/Us96/00596 Table 1 diagrams a comparison of mouse ES cells, primate ES cells, and human EC cells. The only cells reported to express the combination of markers SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 other than primate ES
cells are human EC cells. The globo-series glycolipids SSEA-3 and SSEA-4 are consistently present on human EC cells, and are of diagnostic value in distinguishing human EC cell tumors from human yolk sac carcinomas, choriocarcinomas, and other lineages which lack these markers, Wenk et al., Int J Cancer 58:108-115, 1994. A recent survey found SSEA-3 and SSEA-4 to be present on all of over 40 human EC cell lines examined, Wenk et al. TRA-1-60 and TRA-1-81 antigens have been studied extensively on a particular pluripotent human EC cell line, NTERA-2 CL. Dl, Andrews et al, supra. Differentiation of NTERA-2 CL.
D1 cells in vitro results in the loss of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 expression and the increased expression of the lacto-series glycolipid SSEA-l, Andrews et al, supra. This contrasts with undifferentiated mouse ES cells, which express SSEA-1, and neither SSEA-3 nor SSEA-4. Although the function of these antigens are unknown, their shared expression by R278.5 cells and human EC cells suggests a close embryological similarity. Alkaline phosphatase will also be present on all primate ES cells. A successful primate ES cell culture of the present invention will correlate with the cell surface markers found in-the rhesus macaque and marmoset cell lines described in Table 1.
As disclosed below in Table 1, the rhesus macaque and marmoset cell lines are identical to human EC cell lines for the 5 described markers. Therefore, a successful primate ES cell culture will also mimic human EC cells. However, there are other ways to discriminate ES cells from EC cells. For example, the primate ES cell line has a normal karyotype and the human EC cell line is aneuploid.
In Fig. 3, the photographs labelled A through F
demonstrate the characteristic staining of these markers on a rhesus monkey ES cell line designated R278.5.
Table 1 Mouse C. jacchus M. mulatta Human EC
ES ES ES (NTERA-2 cl.Dl) SSEA-1 + - - -SSEA-3 - + + +
SSEA-4 - + + +
Tra-1-60 - + + +
Tra-1-81 - + + +
(c) Developmental Potential Primate ES cells of the present invention are pluripotent. By "pluripotent" we mean that the cell has the ability to develop into any cell derived from the three main germ cell layers or an embryo itself.
When injected into SCID mice, a successful primate ES
cell line will differentiate into cells derived from all three embryonic germ layers including: bone, cartilage, smooth muscle, striated muscle, and hematopoietic cells (mesoderm); liver, primitive gut and respiratory epithelium (endoderm); neurons, glial cells, hair follicles, and tooth buds (ectoderm).
This experiment can be accomplished by injecting approximately 0.5-1.0 X 106 primate ES cells into the rear leg muscles of 8-12 week old male SCID mice.
The resulting tumors can be fixed in 4%
paraformaldehyde and examined histologically after paraffin embedding at 8-16 weeks of development. In Fig. 4, photomicrographs designated A-F are of sections of tumors formed by injection of rhesus ES
cells into the hind leg muscles of SCID mice and analyzed 15 weeks later demonstrating cartilage, smooth muscle, and striated muscle (mesoderm);
stratified squamous epithelium with hair follicles, neural tube with ventricular, intermediate, and mantle layers (ectoderm) ; ciliated columnar epithelium and villi lined by absorptive enterocytes and mucus-secreting goblet cells (endoderm).
A successful nonhuman primate ES cell line will have the ability to participate in normal development when combined in chimeras with normal preimplantation embryos. Chimeras between preimplantation nonhuman primate embryos and nonhuman primate ES cells can be formed by routine methods in several ways. (i) injection chimeras: 10-15 nonhuman primate ES cells can be microinjected into the cavity of an expanded nonhuman primate blastocyst; (ii) aggregation chimeras: nonhuman primate morulae can be co-cultured on a lawn of nonhuman primate ES cells and allowed to aggregate; and (iii) tetraploid chimeras:
10-15 nonhuman primate ES cells can be aggregated with tetraploid nonhuman primate morulae obtained by electrofusion of 2-cell embryos, or incubation of morulae in the cytoskeletal inhibitor cholchicine.
The chimeras can be returned to the uterus of a female nonhuman primate and allowed to develop to term, and the ES cells will contribute to normal differentiated tissues derived from all three embryonic germ layers and to germ cells. Because nonhuman primate ES can be genetically manipulated prior to chimera formation by standard techniques, chimera formation followed by embryo transfer can lead to the production of transgenic nonhuman primates.
(d) Karvotylpe Successful primate ES cell lines have normal karyotypes. Both XX and XY cells lines will be derived. The normal karyotypes in primate ES cell lines will be in contrast to the abnormal karyotype found in human embryonal carcinoma (EC), which are derived from spontaneously arising human germ cell -^- WO 96/22362 214 `y 528 PCTIUS96/00596 tumors (teratocarcinomas). Human embryonal carcinoma cells have a limited ability to differentiate into multiple cell types and represent the closest existing cell lines to primate ES cells. Although tumor-derived human embryonal carcinoma cell lines have some properties in common with embryonic stem cell lines, all human embryonal carcinoma cell lines derived to date are aneuploid. Thus, primate ES cell lines and human EC cell lines can be distinguished by the normal karyotypes found in primate ES cell lines and the abnormal karyotypes found in human EC lines.
By "normal karyotype" it is meant that all chromosomes normally characteristic of the species are present and have not been noticeably altered.
Because of the abnormal karyotypes of human embryonal carcinoma cells, it is not clear how accurately their differentiation reflects normal differentiation. The range of embryonic and extra-embryonic differentiation observed with primate ES
cells will typically exceed that observed in any human embryonal carcinoma cell line, and the normal karyotypes of the primate ES cells suggests that this differentiation accurately recapitulates normal differentiation.
(e) Immortality Immortal cells are capable of continuous indefinite replication in vitro. Continued proliferation for longer than one year of culture is a sufficient evidence for immortality, as primary cell cultures without this property fail to continuously divide for this length of time (Freshney, Culture of animal cells. New York: Wiley-Liss, 1994). Primate ES cells will continue to proliferate in vitro with the culture conditions described above for longer than one year, and will maintain the developmental potential to contribute all three embryonic germ layers. This developmental 2i~0 :r 3~8 potential can be demonstrated by the injection of ES
cells that have been cultured for a prolonged period (over a year) into SCID mice and then histologically examining the resulting tumors. Although karyotypic changes can occur randomly with prolonged culture, some primate ES cells will maintain a normal karyotype for longer than a year of continuous culture.
(f) Culture Conditions Growth factor requirements to prevent differentiation are different for the primate ES cell line of the present invention than the requirements for mouse ES cell lines. In the absence of fibroblast feeder layers, Leukemia inhibitory factor (LIF) is necessary and sufficient to prevent differentiation of mouse ES cells and to allow their continuous passage. Large concentrations of cloned LIF fail to prevent differentiation of primate ES
cell lines in the absence of fibroblast feeder layers. In this regard, primate ES stem cells are again more similar to human EC cells than to mouse ES
cells, as the growth of feeder-dependent human EC
cells lines is not supported by LIF in the absence of fibroblasts.
(g) Differentiation to Extra Embryonic Tissues When grown on embryonic fibroblasts and allowed to grow for two weeks after achieving confluence (i.e., continuously covering the culture surface), primate ES cells of the present invention spontaneously differentiate and will produce chorionic gonadotropin, indicating trophoblast differentiation (a component of the placenta) and produce a-fetoprotein, indicating endoderm differentiation. Chorionic gonadotropin activity can be assayed in the medium conditioned by differentiated cells by Leydig cell bioassay, Seshagiri & Hearn, Hum Reprod 8:279-287, 1992. For mRNA analysis, RNA can be prepared by guanidine isothiocyanate-phenol/chloroform extraction (1) from approximately 0.2 X 106 differentiated cells and from 0.2 X 106 undifferentiated cells. The relative levels of the mRNA for a-fetoprotein and the a- and 0-subunit of chorionic gonadotropin relative to glyceraldehyde-3-phosphate dehydrogenase can be determined by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR).
The PCR primers for glyceraldehyde 3-phosphate dehydrogenase (G3PDH), obtained from Clontech (Palo Alto, CA), are based on the human cDNA sequence, and do not amplify mouse G3PDH mRNA under our conditions.
Primers for the a-fetoprotein mRNA are based on the human sequence and flank the 7th intron (5' primer =
(5') GCTGGATTGTCTGCAGGATGGGGAA (SEQ ID NO: 1); 3' primer = (5') TCCCCTGAAGAP,AATTGGTTAAAAT (SEQ ID NO:
2)). They amplify a cDNA of 216 nucleotides.
Primers for the 0-subunit of chorionic gonadotropin flank the second intron (5' primer =(5') ggatc CACCGTCAACACCACCATCTGTGC (SEQ ID NO: 3); 3' primer =
(5') ggatc CACAGGTCAAAGGGTGGTCCTTGGG (SEQ ID NO: 4)) (nucleotides added to the hCGb sequence to facilitate sub-cloning are shown in lower case italics). They amplify a cDNA of 262 base pairs. The primers for the CGa subunit can be based on sequences of the first and fourth exon of the rhesus gene (5' primer =
(5') gggaattc GCAGTTACTGAGAACTCACAAG (SEQ ID NO: 5);
3' primer = (5') gggaattc GAAGCATGTCAAAGTGGTATGG (SEQ
ID NO: 6)) and amplify a cDNA of 556 base pairs. The identity of the a-fetoprotein, CGa and CGO cDNAs can be verified by subcloning and sequencing.
For Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), 1 to 5 l of total R278.5 RNA can be reverse transcribed as described Golos et al.
Endocrinoloav 133(4):1744-1752, 1993, and one to 20 l of reverse transcription reaction was then 2 1~~ ~?8 subjected to the polymerase chain reaction in a mixture containing 1-12.5 pmol of each G3PDH primer, 10-25 pmol of each mRNA specific primer, 0.25 mM
dNTPs (Pharmacia, Piscataway, NJ), 1X AmpliTaq buffer (final reaction concentrations = 10 mM Tris, pH 8.3, 50 mM KC1, 1.5 mM MgC12, 0.001% (w/v) gelatin) 2.5 Ci of deoxycytidine 5'a[32P]triphosphate (DuPont, Boston, MA), 10% glycerol and 1.25 U of AmpliTaq (Perkin-Elmer, Oak Brook, IL) in a total volume of 50 l. The number of amplification rounds which produced linear increases in target cDNAs and the relation between input RNA and amount of PCR product is empirically determined as by Golos et al. Samples were fractionated in 3% Nusieve (FMC, Rockland, ME) agarose gels (1X TBE running buffer) and DNA bands of interest were cut out, melted at 65 C in 0.5 ml TE, and radioactivity determined by liquid scintillation counting. The ratio of counts per minute in a specific PCR product relative to cpm of G3PDH PCR
product is used to estimate the relative levels of a mRNAs among differentiated and undifferentiated cells.
The ability to differentiate into trophectoderm in vitro and the ability of these differentiated cells to produce chorionic gonadotropin distinguishes the primate ES cell line of the present invention from all other published ES cell lines.
Examples (1) Animals and Embryos As described above, we have developed a technique for non-surgical, uterine-stage embryo recovery from the rhesus macaque and the common marmoset.
To supply rhesus embryos to interested investigators, The Wisconsin Regional Primate Research Center (WRPRC) provides a preimplantation WO 96/22362 PCr/US96/00596 embryo recovery service for the rhesus monkey, using the non-surgical flush procedure described above.
During 1994, 151 uterine flushes were attempted from rhesus monkeys, yielding 80 viable embryos (0.53 embryos per flush attempt).
By synchronizing the reproductive cycles of several marmosets, significant numbers of in vivo produced, age-matched, preimplantation primate embryos were studied in controlled experiments for the first time. Using marmosets from the self-sustaining colony (250 animals) of the Wisconsin Regional Primate Research Center (WRPRC), we recovered 54 viable morulae or blastocysts, 7 unfertilized oocytes or degenerate embryos, and 5 empty zonae pellucidae in a total of 54 flush attempts (1.0 viable embryo-flush attempt). Marmosets have a 28 day ovarian cycle, and because this is a non-surgical procedure, females can be flushed on consecutive months, dramatically increasing the embryo yield compared to surgical techniques which require months of rest between collections.
(2) Rhesus Macaque Embryonic Stem Cells Using the techniques described above, we have derived three independent embryonic stem cell lines from two rhesus monkey blastocysts (R278.5, R366, and R367). One of these, R278.5, remains undifferentiated and continues to proliferate after continuous culture for over one year. R278.5 cells have also been frozen and successfully thawed with the recovery of viable cells.
The morphology and cell surface markers of R278.5 cells are indistinguishable from human EC
cells, and differ significantly from mouse ES cells.
R278.5 cells have a high nucleus/cytoplasm ratio and prominent nucleoli, but rather than forming compact, piled-up colonies with indistinct cell borders similar to mouse ES cells, R278.5 cells form flatter U J
(3) Definincr Characteristics of Primate ES Cells Primate embryonic stem cells share features with the primate ICM and with pluripotent human embryonal carcinoma cells. Putative primate ES cells may therefore be characterized by morphology and by the expression of cell surface markers characteristic of human EC cells. Additionally, putative primate ES
cells may be characterized by developmental potential, karyotype and immortality.
(a) Morphology The colony morphology of primate embryonic stem cell lines is similar to, but distinct from, mouse embryonic stem cells. Both mouse and primate ES cells have the characteristic features of undifferentiated stem cells, with high nuclear/cytoplasmic ratios, prominent nucleoli, and compact colony formation. The colonies of primate ES cells are flatter than mouse ES
cell colonies and individual primate ES cells can be easily distinguished. In Fig. 2, reference character A indicates a phase contrast photomicrograph of cell line R278.5 demonstrating the characteristic primate ES cell morphology.
(b) Cell Surface Markers A primate ES cell line of the present invention is distinct from mouse ES cell lines by the presence or absence of the cell surface markers described below.
One set of glycolipid cell surface markers is known as the Stage-specific embryonic antigens 1 through 4. These antigens can be identified using ,.. , WO 96/22362 217 0528 PCTIUS96/00596 antibodies for SSEA 1, SSEA-3 and SSEA-4 which are available from the Developmental Studies Hybridoma Bank of the National Institute of Child Health and Human Development. The cell surface markers referred to as TRA-1-60 and TRA-1-81 designate antibodies from hybridomas developed by Peter Andrews of the University of Sheffield and are described in Andrews et al., "Cell lines from human germ cell tumors," In:
Robertson E, ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford: IRL Press, 207-246, 1987. The antibodies were localized with a biotinylated secondary antibody and then an avidin/biotinylated horseradish peroxidase complex (Vectastain ABC System, Vector Laboratories).
Alternatively, it should also be understood that other antibodies for these same cell surface markers can be generated. NTERA-2 cl. Dl, a pluripotent human EC
cell line (gift of Peter Andrews), may be used as a negative control for SSEA-1, and as a positive control for SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. This cell line was chosen for positive control only because it has been extensively studied and reported in the literature, but other human EC cell lines may be used as well.
Mouse ES cells (ESj13) are used as a positive control for SSEA-1, and for a negative control for SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Other routine negative controls include omission of the primary or secondary antibody and substitution of a primary antibody with an unrelated specificity.
Alkaline phosphatase may be detected following fixation of cells with 4% para-formaldehyde using "Vector Red" (Vector Laboratories) as a substrate, as described by the manufacturer (Vector Laboratories).
The precipitate formed by this substrate is red when viewed with a rhodamine filter system, providing substantial amplification over light microscopy.
WO 96/22362 2 19~ j~~ PCT/Us96/00596 Table 1 diagrams a comparison of mouse ES cells, primate ES cells, and human EC cells. The only cells reported to express the combination of markers SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 other than primate ES
cells are human EC cells. The globo-series glycolipids SSEA-3 and SSEA-4 are consistently present on human EC cells, and are of diagnostic value in distinguishing human EC cell tumors from human yolk sac carcinomas, choriocarcinomas, and other lineages which lack these markers, Wenk et al., Int J Cancer 58:108-115, 1994. A recent survey found SSEA-3 and SSEA-4 to be present on all of over 40 human EC cell lines examined, Wenk et al. TRA-1-60 and TRA-1-81 antigens have been studied extensively on a particular pluripotent human EC cell line, NTERA-2 CL. Dl, Andrews et al, supra. Differentiation of NTERA-2 CL.
D1 cells in vitro results in the loss of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 expression and the increased expression of the lacto-series glycolipid SSEA-l, Andrews et al, supra. This contrasts with undifferentiated mouse ES cells, which express SSEA-1, and neither SSEA-3 nor SSEA-4. Although the function of these antigens are unknown, their shared expression by R278.5 cells and human EC cells suggests a close embryological similarity. Alkaline phosphatase will also be present on all primate ES cells. A successful primate ES cell culture of the present invention will correlate with the cell surface markers found in-the rhesus macaque and marmoset cell lines described in Table 1.
As disclosed below in Table 1, the rhesus macaque and marmoset cell lines are identical to human EC cell lines for the 5 described markers. Therefore, a successful primate ES cell culture will also mimic human EC cells. However, there are other ways to discriminate ES cells from EC cells. For example, the primate ES cell line has a normal karyotype and the human EC cell line is aneuploid.
In Fig. 3, the photographs labelled A through F
demonstrate the characteristic staining of these markers on a rhesus monkey ES cell line designated R278.5.
Table 1 Mouse C. jacchus M. mulatta Human EC
ES ES ES (NTERA-2 cl.Dl) SSEA-1 + - - -SSEA-3 - + + +
SSEA-4 - + + +
Tra-1-60 - + + +
Tra-1-81 - + + +
(c) Developmental Potential Primate ES cells of the present invention are pluripotent. By "pluripotent" we mean that the cell has the ability to develop into any cell derived from the three main germ cell layers or an embryo itself.
When injected into SCID mice, a successful primate ES
cell line will differentiate into cells derived from all three embryonic germ layers including: bone, cartilage, smooth muscle, striated muscle, and hematopoietic cells (mesoderm); liver, primitive gut and respiratory epithelium (endoderm); neurons, glial cells, hair follicles, and tooth buds (ectoderm).
This experiment can be accomplished by injecting approximately 0.5-1.0 X 106 primate ES cells into the rear leg muscles of 8-12 week old male SCID mice.
The resulting tumors can be fixed in 4%
paraformaldehyde and examined histologically after paraffin embedding at 8-16 weeks of development. In Fig. 4, photomicrographs designated A-F are of sections of tumors formed by injection of rhesus ES
cells into the hind leg muscles of SCID mice and analyzed 15 weeks later demonstrating cartilage, smooth muscle, and striated muscle (mesoderm);
stratified squamous epithelium with hair follicles, neural tube with ventricular, intermediate, and mantle layers (ectoderm) ; ciliated columnar epithelium and villi lined by absorptive enterocytes and mucus-secreting goblet cells (endoderm).
A successful nonhuman primate ES cell line will have the ability to participate in normal development when combined in chimeras with normal preimplantation embryos. Chimeras between preimplantation nonhuman primate embryos and nonhuman primate ES cells can be formed by routine methods in several ways. (i) injection chimeras: 10-15 nonhuman primate ES cells can be microinjected into the cavity of an expanded nonhuman primate blastocyst; (ii) aggregation chimeras: nonhuman primate morulae can be co-cultured on a lawn of nonhuman primate ES cells and allowed to aggregate; and (iii) tetraploid chimeras:
10-15 nonhuman primate ES cells can be aggregated with tetraploid nonhuman primate morulae obtained by electrofusion of 2-cell embryos, or incubation of morulae in the cytoskeletal inhibitor cholchicine.
The chimeras can be returned to the uterus of a female nonhuman primate and allowed to develop to term, and the ES cells will contribute to normal differentiated tissues derived from all three embryonic germ layers and to germ cells. Because nonhuman primate ES can be genetically manipulated prior to chimera formation by standard techniques, chimera formation followed by embryo transfer can lead to the production of transgenic nonhuman primates.
(d) Karvotylpe Successful primate ES cell lines have normal karyotypes. Both XX and XY cells lines will be derived. The normal karyotypes in primate ES cell lines will be in contrast to the abnormal karyotype found in human embryonal carcinoma (EC), which are derived from spontaneously arising human germ cell -^- WO 96/22362 214 `y 528 PCTIUS96/00596 tumors (teratocarcinomas). Human embryonal carcinoma cells have a limited ability to differentiate into multiple cell types and represent the closest existing cell lines to primate ES cells. Although tumor-derived human embryonal carcinoma cell lines have some properties in common with embryonic stem cell lines, all human embryonal carcinoma cell lines derived to date are aneuploid. Thus, primate ES cell lines and human EC cell lines can be distinguished by the normal karyotypes found in primate ES cell lines and the abnormal karyotypes found in human EC lines.
By "normal karyotype" it is meant that all chromosomes normally characteristic of the species are present and have not been noticeably altered.
Because of the abnormal karyotypes of human embryonal carcinoma cells, it is not clear how accurately their differentiation reflects normal differentiation. The range of embryonic and extra-embryonic differentiation observed with primate ES
cells will typically exceed that observed in any human embryonal carcinoma cell line, and the normal karyotypes of the primate ES cells suggests that this differentiation accurately recapitulates normal differentiation.
(e) Immortality Immortal cells are capable of continuous indefinite replication in vitro. Continued proliferation for longer than one year of culture is a sufficient evidence for immortality, as primary cell cultures without this property fail to continuously divide for this length of time (Freshney, Culture of animal cells. New York: Wiley-Liss, 1994). Primate ES cells will continue to proliferate in vitro with the culture conditions described above for longer than one year, and will maintain the developmental potential to contribute all three embryonic germ layers. This developmental 2i~0 :r 3~8 potential can be demonstrated by the injection of ES
cells that have been cultured for a prolonged period (over a year) into SCID mice and then histologically examining the resulting tumors. Although karyotypic changes can occur randomly with prolonged culture, some primate ES cells will maintain a normal karyotype for longer than a year of continuous culture.
(f) Culture Conditions Growth factor requirements to prevent differentiation are different for the primate ES cell line of the present invention than the requirements for mouse ES cell lines. In the absence of fibroblast feeder layers, Leukemia inhibitory factor (LIF) is necessary and sufficient to prevent differentiation of mouse ES cells and to allow their continuous passage. Large concentrations of cloned LIF fail to prevent differentiation of primate ES
cell lines in the absence of fibroblast feeder layers. In this regard, primate ES stem cells are again more similar to human EC cells than to mouse ES
cells, as the growth of feeder-dependent human EC
cells lines is not supported by LIF in the absence of fibroblasts.
(g) Differentiation to Extra Embryonic Tissues When grown on embryonic fibroblasts and allowed to grow for two weeks after achieving confluence (i.e., continuously covering the culture surface), primate ES cells of the present invention spontaneously differentiate and will produce chorionic gonadotropin, indicating trophoblast differentiation (a component of the placenta) and produce a-fetoprotein, indicating endoderm differentiation. Chorionic gonadotropin activity can be assayed in the medium conditioned by differentiated cells by Leydig cell bioassay, Seshagiri & Hearn, Hum Reprod 8:279-287, 1992. For mRNA analysis, RNA can be prepared by guanidine isothiocyanate-phenol/chloroform extraction (1) from approximately 0.2 X 106 differentiated cells and from 0.2 X 106 undifferentiated cells. The relative levels of the mRNA for a-fetoprotein and the a- and 0-subunit of chorionic gonadotropin relative to glyceraldehyde-3-phosphate dehydrogenase can be determined by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR).
The PCR primers for glyceraldehyde 3-phosphate dehydrogenase (G3PDH), obtained from Clontech (Palo Alto, CA), are based on the human cDNA sequence, and do not amplify mouse G3PDH mRNA under our conditions.
Primers for the a-fetoprotein mRNA are based on the human sequence and flank the 7th intron (5' primer =
(5') GCTGGATTGTCTGCAGGATGGGGAA (SEQ ID NO: 1); 3' primer = (5') TCCCCTGAAGAP,AATTGGTTAAAAT (SEQ ID NO:
2)). They amplify a cDNA of 216 nucleotides.
Primers for the 0-subunit of chorionic gonadotropin flank the second intron (5' primer =(5') ggatc CACCGTCAACACCACCATCTGTGC (SEQ ID NO: 3); 3' primer =
(5') ggatc CACAGGTCAAAGGGTGGTCCTTGGG (SEQ ID NO: 4)) (nucleotides added to the hCGb sequence to facilitate sub-cloning are shown in lower case italics). They amplify a cDNA of 262 base pairs. The primers for the CGa subunit can be based on sequences of the first and fourth exon of the rhesus gene (5' primer =
(5') gggaattc GCAGTTACTGAGAACTCACAAG (SEQ ID NO: 5);
3' primer = (5') gggaattc GAAGCATGTCAAAGTGGTATGG (SEQ
ID NO: 6)) and amplify a cDNA of 556 base pairs. The identity of the a-fetoprotein, CGa and CGO cDNAs can be verified by subcloning and sequencing.
For Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), 1 to 5 l of total R278.5 RNA can be reverse transcribed as described Golos et al.
Endocrinoloav 133(4):1744-1752, 1993, and one to 20 l of reverse transcription reaction was then 2 1~~ ~?8 subjected to the polymerase chain reaction in a mixture containing 1-12.5 pmol of each G3PDH primer, 10-25 pmol of each mRNA specific primer, 0.25 mM
dNTPs (Pharmacia, Piscataway, NJ), 1X AmpliTaq buffer (final reaction concentrations = 10 mM Tris, pH 8.3, 50 mM KC1, 1.5 mM MgC12, 0.001% (w/v) gelatin) 2.5 Ci of deoxycytidine 5'a[32P]triphosphate (DuPont, Boston, MA), 10% glycerol and 1.25 U of AmpliTaq (Perkin-Elmer, Oak Brook, IL) in a total volume of 50 l. The number of amplification rounds which produced linear increases in target cDNAs and the relation between input RNA and amount of PCR product is empirically determined as by Golos et al. Samples were fractionated in 3% Nusieve (FMC, Rockland, ME) agarose gels (1X TBE running buffer) and DNA bands of interest were cut out, melted at 65 C in 0.5 ml TE, and radioactivity determined by liquid scintillation counting. The ratio of counts per minute in a specific PCR product relative to cpm of G3PDH PCR
product is used to estimate the relative levels of a mRNAs among differentiated and undifferentiated cells.
The ability to differentiate into trophectoderm in vitro and the ability of these differentiated cells to produce chorionic gonadotropin distinguishes the primate ES cell line of the present invention from all other published ES cell lines.
Examples (1) Animals and Embryos As described above, we have developed a technique for non-surgical, uterine-stage embryo recovery from the rhesus macaque and the common marmoset.
To supply rhesus embryos to interested investigators, The Wisconsin Regional Primate Research Center (WRPRC) provides a preimplantation WO 96/22362 PCr/US96/00596 embryo recovery service for the rhesus monkey, using the non-surgical flush procedure described above.
During 1994, 151 uterine flushes were attempted from rhesus monkeys, yielding 80 viable embryos (0.53 embryos per flush attempt).
By synchronizing the reproductive cycles of several marmosets, significant numbers of in vivo produced, age-matched, preimplantation primate embryos were studied in controlled experiments for the first time. Using marmosets from the self-sustaining colony (250 animals) of the Wisconsin Regional Primate Research Center (WRPRC), we recovered 54 viable morulae or blastocysts, 7 unfertilized oocytes or degenerate embryos, and 5 empty zonae pellucidae in a total of 54 flush attempts (1.0 viable embryo-flush attempt). Marmosets have a 28 day ovarian cycle, and because this is a non-surgical procedure, females can be flushed on consecutive months, dramatically increasing the embryo yield compared to surgical techniques which require months of rest between collections.
(2) Rhesus Macaque Embryonic Stem Cells Using the techniques described above, we have derived three independent embryonic stem cell lines from two rhesus monkey blastocysts (R278.5, R366, and R367). One of these, R278.5, remains undifferentiated and continues to proliferate after continuous culture for over one year. R278.5 cells have also been frozen and successfully thawed with the recovery of viable cells.
The morphology and cell surface markers of R278.5 cells are indistinguishable from human EC
cells, and differ significantly from mouse ES cells.
R278.5 cells have a high nucleus/cytoplasm ratio and prominent nucleoli, but rather than forming compact, piled-up colonies with indistinct cell borders similar to mouse ES cells, R278.5 cells form flatter U J
colonies with individual, distinct cells (Fig 2 A).
R278.5 cells express the SSEA-3, SSEA-4, TRA-1-60, and TRA-81 antigens (Fig 3 and Table 1), none of which are expressed by mouse ES cells. The only cells known to express the combination of markers SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 other than primate ES cells are human EC cells. The globo-series glycolipids SSEA-3 and SSEA-4 are consistently present on human EC cells, and are of diagnostic value in distinguishing human EC cell tumors from yolk sac carcinomas, choriocarcinomas and other stem cells derived from human germ cell tumors which lack these markers, Wenk et al, Int J Cancer 58:108-115, 1994. A recent survey found SSEA-3 and SSEA-4 to be present on all of over 40 human EC cell lines examined (Wenk et al.).
TRA-1-60 and TRA-1-81 antigens have been studied extensively on a particular pluripotent human EC cell line, NTERA-2 CL. Di (Andrews et al.).
Differentiation of NTERA-2 CL. Dl cells in vitro results in the loss of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 expression and the increased expression of the lacto-series glycolipid SSEA-1. Undifferentiated mouse ES cells, on the other hand, express SSEA-1, and not SSEA-3, SSEA-4, TRA-1-60 or TRA-1-81 (Wenk et al.). Although the function of these antigens is unknown, their expression by R278.5 cells suggests a close embryological similarity between primate ES
cells and human EC cells, and fundamental differences between primate ES cells and mouse ES cells.
R278.5 cells also express alkaline phosphatase.
The expression of alkaline phosphatase is shared by both primate and mouse ES cells, and relatively few embryonic cells express this enzyme. Positive cells include the ICM and primitive ectoderm (which are the most similar embryonic cells in the intact embryo to ES cells), germ cells, (which are totipotent), and a very limited number of neural precursors, Kaufman MH.
The atlas of mouse development. London: Academic Press, 1992. Cells not expressing this enzyme will not be primate ES cells.
Although cloned human LIF was present in the medium at cell line derivation and for initial passages, R278.5 cells grown on mouse embryonic fibroblasts without exogenous LIF remain undifferentiated and continued to proliferate.
R278.5 cells plated on gelatin-treated tissue culture plates without fibroblasts differentiated to multiple cell types or failed to attach and died, regardless of the presence or absence of exogenously added human LIF (Fig 2). Up to 104units/ ml human LIF fails to prevent differentiation. In addition, added LIF
fails to increase the cloning efficiency or proliferation rate of R278.5 cells on fibroblasts.
Since the derivation of the R278.5 cell line, we have derived two additional rhesus ES cell lines (R366 and R367) on embryonic fibroblasts without any exogenously added LIF at initial derivation. R366 and R367 cells, like R278.5 cells, continue to proliferate on embryonic fibroblasts without exogenously added LIF and differentiate in the absence of fibroblasts, regardless of the presence of added LIF. RT-PCR performed on mRNA from spontaneously differentiated R278.5 cells revealed a-fetoprotein mRNA (Fig 4). a-fetoprotein is a specific marker for endoderm, and is expressed by both extra-embryonic (yolk sac) and embryonic (fetal liver and intestines) endoderm-derived tissues.
Epithelial cells resembling extraembryonic endoderm are present in cells differentiated in vitro from R278.5 cells (Fig. 2). Bioactive CG (3.89 mI
units/ml) was present in culture medium collected from differentiated cells, but not in medium collected from undifferentiated cells (less than 0.03 WO 96/22362 2 19 J~`8 PCT/US96/00596 mI units/ml), indicating the differentiation of trophoblast, a trophectoderm derivative. The relative level of the CGa mRNA increased 23.9-fold after differentiation (Fig. 4).
All SCID mice injected with R278.5 cells in either intra-muscular or intra-testicular sites formed tumors, and tumors in both sites demonstrated a similar range of differentiation. The oldest tumors examined (15 weeks) had the most advanced differentiation, and all had abundant, unambiguous derivatives of all three embryonic germ layers, including gut and respiratory epithelium (endoderm);
bone, cartilage, smooth muscle, striated muscle (mesoderm); ganglia, glia, neural precursors, and stratified squamous epithelium (ectoderm), and other unidentified cell types (Fig. 5). In addition to individual cell types, there was organized development of some structures which require complex interactions between different cell types. Such structures included gut lined by villi with both absorptive enterocytes and mucus-secreting goblet cells, and sometimes encircled by layers of smooth muscle in the same orientation as muscularis mucosae (circular) and muscularis (outer longitudinal layer and inner circular layer); neural tubes with ventricular, intermediate, and mantle layers; and hair follicles with hair shafts (Fig. 5).
The essential characteristics that define R278.5 cells as ES cells include: indefinite (greater than one year) undifferentiated proliferation in vitro, normal karyotype, and potential to differentiate to derivatives of trophectoderm and all three embryonic germ layers. In the mouse embryo, the last cells capable of contributing to derivatives of both trophectoderm and ICM are early ICM cells. The timing of commitment to ICM or trophectoderm has not been established for any primate species, but the .~- WO 96/22362 2 1 a 0528 PCT/US96/00596 potential of rhesus ES cells to contribute to derivatives of both suggests that they most closely resemble early totipotent embryonic cells. The ability of rhesus ES cells to form trophoblast in vitro distinguishes primate ES cell lines from mouse ES cells. Mouse ES cell have not been demonstrated to form trophoblast in vitro, and mouse trophoblast does not produce gonadotropin. Rhesus ES cells and mouse ES cells do demonstrate the similar wide range of differentiation in tumors that distinguishes ES
cells from EC cells. The development of structures composed of multiple cell types such as hair follicles, which require inductive interactions between the embryonic epidermis and underlying mesenchyme, demonstrates the ability of rhesus ES
cells to participate in complex developmental processes.
The rhesus ES lines R366 and R367 have also been further cultured and analyzed. Both lines have a normal XY karyotype and were proliferated in an undifferentiated state for about three months prior to freezing for later analysis. Samples of each of the cell lines R366 and R367 were injected into SCID
mice which then formed teratomas identical to those formed by R278.5 cells. An additional rhesus cell line R394 having a normal XX karyotype was also recovered. All three of these cell lines, R366, R367 and R394 are identical in morphology, growth characteristics, culture requirements and in vitro differentiation characteristics, i.e. the trait of differentiation to multiple cell types in the absence of fibroblasts, to cell line 278.5.
It has been determined that LIF is not required either to derive or proliferate these ES cultures.
Each of the cell lines R366, R367 and R394 were derived and cultured without exogenous LIF.
It has also been demonstrated that the WO 96/22362 2 19 Uj L 8 PCT/US96/00596 particular source of fibroblasts for co-culture is not critical. Several fibroblast cell lines have been tested both with rhesus line R278.5 and with the marmoset cell lines described below. The fibroblasts tested include mouse STO cells (ATCC 56-X), mouse 3T3 cells (ATCC 48-X), primary rhesus monkey embryonic fibroblasts derived from 36 day rhesus fetuses, and mouse S1/S14 cells, which are deficient in the steel factor. All these fibroblast cell lines were capable of maintaining the stem cell lines in an undifferentiated state. Most rapid proliferation of the stem cells was observed using primary mouse embryonic fibroblasts.
Unlike mouse ES cells, neither rhesus ES cells nor feeder-dependent human EC cells remain undifferentiated and proliferate in the presence of soluble human LIF without fibroblasts. The factors that fibroblasts produce that prevent the differentiation of rhesus ES cells or feeder-dependent human EC cells are unknown, but the lack of a dependence on LIF is another characteristic that distinguishes primate ES cells from mouse ES cells.
The growth of rhesus monkey ES cells in culture conditions similar to those required by feeder-dependent human EC cells, and the identical morphology and cell surface markers of rhesus ES
cells and human EC cells, suggests that similar culture conditions will support human ES cells.
Rhesus ES cells will be important for elucidating the mechanisms that control the differentiation of specific primate cell types.
Given the close evolutionary distance and the developmental and physiological similarities between humans and rhesus monkeys, the mechanisms controlling the differentiation of rhesus cells will be very similar to the mechanisms controlling the differentiation of human cells. The importance of elucidating these mechanisms is that once they are understood, it will be possible to direct primate ES
cells to differentiate to specific cell types in vitro, and these specific cell types can be used for transplantation to treat specific diseases.
Because ES cells have the developmental potential to give rise to any differentiated cell type, any disease that results in part or in whole from the failure (either genetic or acquired) of specific cell types will be potentially treatable through the transplantation of cells derived from ES
cells. Rhesus ES cells and rhesus monkeys will be invaluable for testing the efficacy and safety of the transplantation of specific cell types derived from ES cells. A few examples of human diseases potentially treatable by this approach with human ES
cells include degenerative neurological disorders such as Parkinson's disease (dopanergic neurons), juvenile onset diabetes (pancreatic (3-islet cells) or Acquired Immunodeficiency Disease (lymphocytes).
Because undifferentiated ES cells can proliferate indefinitely in vitro, they can be genetically manipulated with standard techniques either to prevent immune rejection after transplantation, or to give them new genetic properties to combat specific diseases. For specific cell types where immune rejection can be prevented, cells derived from rhesus monkey ES cells or other non-human primate ES cells could be used for transplantation to humans to treat specific diseases.
(3) Marmoset Embryonic Stem Cells Our method for creating an embryonic stem cell line is described above. Using isolated ICM's derived by immunosurgery from marmoset blastocysts, we have isolated 7 putative ES cell lines, each of which have been cultured for over 6 months.
WO 96/22362 2 1~ 0 5 2- 8 PCT/US96/00596 One of these, Cjll, was cultured continuously for over 14 months, and then frozen for later analysis. The Cjll cell line and other marmoset ES
cell lines have been successfully frozen and then thawed with the recovery of viable cells. These cells have a high nuclear/cytoplasmic ratio, prominent nucleoli, and a compact colony morphology similar to the pluripotent human embryonal carcinoma (EC) cell line NT2/D2.
Four of the cell lines we have isolated have normal XX karyotypes, and one has a normal XY
karyotype (Karyotypes were performed by Dr. Charles Harris, University of Wisconsin). These cells were positive for a series of cell surface markers (alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that in combination are definitive markers for undifferentiated human embryonal carcinoma cells (EC) cells and primate ES cells. In particular, these markers distinguish EC cells from the earliest lineages to differentiate in the human preimplantation embryo, trophectoderm (represented by BeWO choriocarcinoma cells) and extraembryonic endoderm (represented by 1411H yolk sac carcinoma cells).
When the putative marmoset ES cells were removed from fibroblast feeders, they differentiated into cells of several distinct morphologies. Among the differentiated cells, trophectoderm is indicated by the secretion of chorionic gonadotropin and the presence of the chorionic gonadotropin 0-subunit mRNA. 12.7 mIU/ml luteinizing hormone (LH) activity was measured in the WRPRC core assay lab using a mouse Leydig cell bioassay in medium conditioned 24 hours by putative ES cells allowed to differentiate for one week. Note that chorionic gonadotrophin has both LH and FSH activity, and is routinely measured by LH assays. Control medium from undifferentiated 219~528 ES cells had less than 1 mIU/ml LH activity.
Chorionic gonadotropin 0-subunit mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). DNA sequencing confirmed the identity of the chorionic gonadotrophin 0-subunit.
Endoderm differentiation (probably extraembryonic endoderm) was indicated by the presence of a-fetoprotein mRNA, detected by RT-PCR.
When the marmoset ES cells were grown in high densities, over a period of weeks epithelial cells differentiated and covered the culture dish. The remaining groups of undifferentiated cells rounded up into compact balls and then formed embryoid bodies (as shown in Fig. 6) that recapitulated early development with remarkable fidelity. Over 3-4 weeks, some of the embryoid bodies formed a bilaterally symmetric pyriform embryonic disc, an amnion, a yolk sac, and a mesoblast outgrowth attaching the caudal pole of the amnion to the culture dish.
Histological and ultrastructural examination of one of these embryoid bodies (formed from a cell line that had been passaged continuously for 6 months) revealed a remarkable resemblance to a stage 6-7 post-implantation embryo. The embryonic disc was composed of a polarized, columnar epithelial epiblast (primitive ectoderm) layer separated from a visceral endoderm (primitive endoderm) layer. Electron microscopy of the epiblast revealed apical junctional complexes, apical microvilli, subapical intermediate filaments, and a basement membrane separating the epiblast from underlying visceral endoderm. All of these elements are features of the normal embryonic disc. In the caudal third of the embryonic disc, there was a midline groove, disruption of the basement membrane, and mixing of epiblast cells with underlying endodermal cells (early primitive streak).
WO 96/22362 PCI'/US96/00596 The amnion was composed of an inner squamous (ectoderm) layer continuous with the epiblast and an outer mesoderm layer. The bilayered yolk sac had occasional endothelial-lined spaces containing possible hematopoietic precursors.
The morphology, immortality, karyotype, and cell surface markers of these marmoset cells identify these marmoset cells as primate ES cells similar to the rhesus ES cells. Since the last cells in the mammalian embryo capable of contributing to both trophectoderm derivatives and endoderm derivatives are the totipotent cells of the early ICM, the ability of marmoset ES cells to contribute to both trophoblast and endoderm demonstrates their similarities to early totipotent embryonic cells of the intact embryo. The formation of embryoid bodies by marmoset ES cells, with remarkable structural similarities to the early post-implantation primate embryo, demonstrates the potential of marmoset ES
cells to participate in complex developmental processes requiring the interaction of multiple cell types.
Given the reproductive characteristics of the common marmoset described above (efficient embryo transfer, multiple young, short generation time), marmoset ES cells will be particularly useful for the generation of transgenic primates. Although mice have provided invaluable insights into gene function and regulation, the anatomical and physiological differences between humans and mice limit the usefulness of transgenic mouse models of human diseases. Transgenic primates, in addition to providing insights into the pathogenesis of specific diseases, will provide accurate animal models to test the efficacy and safety of specific treatments.
R278.5 cells express the SSEA-3, SSEA-4, TRA-1-60, and TRA-81 antigens (Fig 3 and Table 1), none of which are expressed by mouse ES cells. The only cells known to express the combination of markers SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 other than primate ES cells are human EC cells. The globo-series glycolipids SSEA-3 and SSEA-4 are consistently present on human EC cells, and are of diagnostic value in distinguishing human EC cell tumors from yolk sac carcinomas, choriocarcinomas and other stem cells derived from human germ cell tumors which lack these markers, Wenk et al, Int J Cancer 58:108-115, 1994. A recent survey found SSEA-3 and SSEA-4 to be present on all of over 40 human EC cell lines examined (Wenk et al.).
TRA-1-60 and TRA-1-81 antigens have been studied extensively on a particular pluripotent human EC cell line, NTERA-2 CL. Di (Andrews et al.).
Differentiation of NTERA-2 CL. Dl cells in vitro results in the loss of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 expression and the increased expression of the lacto-series glycolipid SSEA-1. Undifferentiated mouse ES cells, on the other hand, express SSEA-1, and not SSEA-3, SSEA-4, TRA-1-60 or TRA-1-81 (Wenk et al.). Although the function of these antigens is unknown, their expression by R278.5 cells suggests a close embryological similarity between primate ES
cells and human EC cells, and fundamental differences between primate ES cells and mouse ES cells.
R278.5 cells also express alkaline phosphatase.
The expression of alkaline phosphatase is shared by both primate and mouse ES cells, and relatively few embryonic cells express this enzyme. Positive cells include the ICM and primitive ectoderm (which are the most similar embryonic cells in the intact embryo to ES cells), germ cells, (which are totipotent), and a very limited number of neural precursors, Kaufman MH.
The atlas of mouse development. London: Academic Press, 1992. Cells not expressing this enzyme will not be primate ES cells.
Although cloned human LIF was present in the medium at cell line derivation and for initial passages, R278.5 cells grown on mouse embryonic fibroblasts without exogenous LIF remain undifferentiated and continued to proliferate.
R278.5 cells plated on gelatin-treated tissue culture plates without fibroblasts differentiated to multiple cell types or failed to attach and died, regardless of the presence or absence of exogenously added human LIF (Fig 2). Up to 104units/ ml human LIF fails to prevent differentiation. In addition, added LIF
fails to increase the cloning efficiency or proliferation rate of R278.5 cells on fibroblasts.
Since the derivation of the R278.5 cell line, we have derived two additional rhesus ES cell lines (R366 and R367) on embryonic fibroblasts without any exogenously added LIF at initial derivation. R366 and R367 cells, like R278.5 cells, continue to proliferate on embryonic fibroblasts without exogenously added LIF and differentiate in the absence of fibroblasts, regardless of the presence of added LIF. RT-PCR performed on mRNA from spontaneously differentiated R278.5 cells revealed a-fetoprotein mRNA (Fig 4). a-fetoprotein is a specific marker for endoderm, and is expressed by both extra-embryonic (yolk sac) and embryonic (fetal liver and intestines) endoderm-derived tissues.
Epithelial cells resembling extraembryonic endoderm are present in cells differentiated in vitro from R278.5 cells (Fig. 2). Bioactive CG (3.89 mI
units/ml) was present in culture medium collected from differentiated cells, but not in medium collected from undifferentiated cells (less than 0.03 WO 96/22362 2 19 J~`8 PCT/US96/00596 mI units/ml), indicating the differentiation of trophoblast, a trophectoderm derivative. The relative level of the CGa mRNA increased 23.9-fold after differentiation (Fig. 4).
All SCID mice injected with R278.5 cells in either intra-muscular or intra-testicular sites formed tumors, and tumors in both sites demonstrated a similar range of differentiation. The oldest tumors examined (15 weeks) had the most advanced differentiation, and all had abundant, unambiguous derivatives of all three embryonic germ layers, including gut and respiratory epithelium (endoderm);
bone, cartilage, smooth muscle, striated muscle (mesoderm); ganglia, glia, neural precursors, and stratified squamous epithelium (ectoderm), and other unidentified cell types (Fig. 5). In addition to individual cell types, there was organized development of some structures which require complex interactions between different cell types. Such structures included gut lined by villi with both absorptive enterocytes and mucus-secreting goblet cells, and sometimes encircled by layers of smooth muscle in the same orientation as muscularis mucosae (circular) and muscularis (outer longitudinal layer and inner circular layer); neural tubes with ventricular, intermediate, and mantle layers; and hair follicles with hair shafts (Fig. 5).
The essential characteristics that define R278.5 cells as ES cells include: indefinite (greater than one year) undifferentiated proliferation in vitro, normal karyotype, and potential to differentiate to derivatives of trophectoderm and all three embryonic germ layers. In the mouse embryo, the last cells capable of contributing to derivatives of both trophectoderm and ICM are early ICM cells. The timing of commitment to ICM or trophectoderm has not been established for any primate species, but the .~- WO 96/22362 2 1 a 0528 PCT/US96/00596 potential of rhesus ES cells to contribute to derivatives of both suggests that they most closely resemble early totipotent embryonic cells. The ability of rhesus ES cells to form trophoblast in vitro distinguishes primate ES cell lines from mouse ES cells. Mouse ES cell have not been demonstrated to form trophoblast in vitro, and mouse trophoblast does not produce gonadotropin. Rhesus ES cells and mouse ES cells do demonstrate the similar wide range of differentiation in tumors that distinguishes ES
cells from EC cells. The development of structures composed of multiple cell types such as hair follicles, which require inductive interactions between the embryonic epidermis and underlying mesenchyme, demonstrates the ability of rhesus ES
cells to participate in complex developmental processes.
The rhesus ES lines R366 and R367 have also been further cultured and analyzed. Both lines have a normal XY karyotype and were proliferated in an undifferentiated state for about three months prior to freezing for later analysis. Samples of each of the cell lines R366 and R367 were injected into SCID
mice which then formed teratomas identical to those formed by R278.5 cells. An additional rhesus cell line R394 having a normal XX karyotype was also recovered. All three of these cell lines, R366, R367 and R394 are identical in morphology, growth characteristics, culture requirements and in vitro differentiation characteristics, i.e. the trait of differentiation to multiple cell types in the absence of fibroblasts, to cell line 278.5.
It has been determined that LIF is not required either to derive or proliferate these ES cultures.
Each of the cell lines R366, R367 and R394 were derived and cultured without exogenous LIF.
It has also been demonstrated that the WO 96/22362 2 19 Uj L 8 PCT/US96/00596 particular source of fibroblasts for co-culture is not critical. Several fibroblast cell lines have been tested both with rhesus line R278.5 and with the marmoset cell lines described below. The fibroblasts tested include mouse STO cells (ATCC 56-X), mouse 3T3 cells (ATCC 48-X), primary rhesus monkey embryonic fibroblasts derived from 36 day rhesus fetuses, and mouse S1/S14 cells, which are deficient in the steel factor. All these fibroblast cell lines were capable of maintaining the stem cell lines in an undifferentiated state. Most rapid proliferation of the stem cells was observed using primary mouse embryonic fibroblasts.
Unlike mouse ES cells, neither rhesus ES cells nor feeder-dependent human EC cells remain undifferentiated and proliferate in the presence of soluble human LIF without fibroblasts. The factors that fibroblasts produce that prevent the differentiation of rhesus ES cells or feeder-dependent human EC cells are unknown, but the lack of a dependence on LIF is another characteristic that distinguishes primate ES cells from mouse ES cells.
The growth of rhesus monkey ES cells in culture conditions similar to those required by feeder-dependent human EC cells, and the identical morphology and cell surface markers of rhesus ES
cells and human EC cells, suggests that similar culture conditions will support human ES cells.
Rhesus ES cells will be important for elucidating the mechanisms that control the differentiation of specific primate cell types.
Given the close evolutionary distance and the developmental and physiological similarities between humans and rhesus monkeys, the mechanisms controlling the differentiation of rhesus cells will be very similar to the mechanisms controlling the differentiation of human cells. The importance of elucidating these mechanisms is that once they are understood, it will be possible to direct primate ES
cells to differentiate to specific cell types in vitro, and these specific cell types can be used for transplantation to treat specific diseases.
Because ES cells have the developmental potential to give rise to any differentiated cell type, any disease that results in part or in whole from the failure (either genetic or acquired) of specific cell types will be potentially treatable through the transplantation of cells derived from ES
cells. Rhesus ES cells and rhesus monkeys will be invaluable for testing the efficacy and safety of the transplantation of specific cell types derived from ES cells. A few examples of human diseases potentially treatable by this approach with human ES
cells include degenerative neurological disorders such as Parkinson's disease (dopanergic neurons), juvenile onset diabetes (pancreatic (3-islet cells) or Acquired Immunodeficiency Disease (lymphocytes).
Because undifferentiated ES cells can proliferate indefinitely in vitro, they can be genetically manipulated with standard techniques either to prevent immune rejection after transplantation, or to give them new genetic properties to combat specific diseases. For specific cell types where immune rejection can be prevented, cells derived from rhesus monkey ES cells or other non-human primate ES cells could be used for transplantation to humans to treat specific diseases.
(3) Marmoset Embryonic Stem Cells Our method for creating an embryonic stem cell line is described above. Using isolated ICM's derived by immunosurgery from marmoset blastocysts, we have isolated 7 putative ES cell lines, each of which have been cultured for over 6 months.
WO 96/22362 2 1~ 0 5 2- 8 PCT/US96/00596 One of these, Cjll, was cultured continuously for over 14 months, and then frozen for later analysis. The Cjll cell line and other marmoset ES
cell lines have been successfully frozen and then thawed with the recovery of viable cells. These cells have a high nuclear/cytoplasmic ratio, prominent nucleoli, and a compact colony morphology similar to the pluripotent human embryonal carcinoma (EC) cell line NT2/D2.
Four of the cell lines we have isolated have normal XX karyotypes, and one has a normal XY
karyotype (Karyotypes were performed by Dr. Charles Harris, University of Wisconsin). These cells were positive for a series of cell surface markers (alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that in combination are definitive markers for undifferentiated human embryonal carcinoma cells (EC) cells and primate ES cells. In particular, these markers distinguish EC cells from the earliest lineages to differentiate in the human preimplantation embryo, trophectoderm (represented by BeWO choriocarcinoma cells) and extraembryonic endoderm (represented by 1411H yolk sac carcinoma cells).
When the putative marmoset ES cells were removed from fibroblast feeders, they differentiated into cells of several distinct morphologies. Among the differentiated cells, trophectoderm is indicated by the secretion of chorionic gonadotropin and the presence of the chorionic gonadotropin 0-subunit mRNA. 12.7 mIU/ml luteinizing hormone (LH) activity was measured in the WRPRC core assay lab using a mouse Leydig cell bioassay in medium conditioned 24 hours by putative ES cells allowed to differentiate for one week. Note that chorionic gonadotrophin has both LH and FSH activity, and is routinely measured by LH assays. Control medium from undifferentiated 219~528 ES cells had less than 1 mIU/ml LH activity.
Chorionic gonadotropin 0-subunit mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). DNA sequencing confirmed the identity of the chorionic gonadotrophin 0-subunit.
Endoderm differentiation (probably extraembryonic endoderm) was indicated by the presence of a-fetoprotein mRNA, detected by RT-PCR.
When the marmoset ES cells were grown in high densities, over a period of weeks epithelial cells differentiated and covered the culture dish. The remaining groups of undifferentiated cells rounded up into compact balls and then formed embryoid bodies (as shown in Fig. 6) that recapitulated early development with remarkable fidelity. Over 3-4 weeks, some of the embryoid bodies formed a bilaterally symmetric pyriform embryonic disc, an amnion, a yolk sac, and a mesoblast outgrowth attaching the caudal pole of the amnion to the culture dish.
Histological and ultrastructural examination of one of these embryoid bodies (formed from a cell line that had been passaged continuously for 6 months) revealed a remarkable resemblance to a stage 6-7 post-implantation embryo. The embryonic disc was composed of a polarized, columnar epithelial epiblast (primitive ectoderm) layer separated from a visceral endoderm (primitive endoderm) layer. Electron microscopy of the epiblast revealed apical junctional complexes, apical microvilli, subapical intermediate filaments, and a basement membrane separating the epiblast from underlying visceral endoderm. All of these elements are features of the normal embryonic disc. In the caudal third of the embryonic disc, there was a midline groove, disruption of the basement membrane, and mixing of epiblast cells with underlying endodermal cells (early primitive streak).
WO 96/22362 PCI'/US96/00596 The amnion was composed of an inner squamous (ectoderm) layer continuous with the epiblast and an outer mesoderm layer. The bilayered yolk sac had occasional endothelial-lined spaces containing possible hematopoietic precursors.
The morphology, immortality, karyotype, and cell surface markers of these marmoset cells identify these marmoset cells as primate ES cells similar to the rhesus ES cells. Since the last cells in the mammalian embryo capable of contributing to both trophectoderm derivatives and endoderm derivatives are the totipotent cells of the early ICM, the ability of marmoset ES cells to contribute to both trophoblast and endoderm demonstrates their similarities to early totipotent embryonic cells of the intact embryo. The formation of embryoid bodies by marmoset ES cells, with remarkable structural similarities to the early post-implantation primate embryo, demonstrates the potential of marmoset ES
cells to participate in complex developmental processes requiring the interaction of multiple cell types.
Given the reproductive characteristics of the common marmoset described above (efficient embryo transfer, multiple young, short generation time), marmoset ES cells will be particularly useful for the generation of transgenic primates. Although mice have provided invaluable insights into gene function and regulation, the anatomical and physiological differences between humans and mice limit the usefulness of transgenic mouse models of human diseases. Transgenic primates, in addition to providing insights into the pathogenesis of specific diseases, will provide accurate animal models to test the efficacy and safety of specific treatments.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Wisconsin Alumni Research Foundation (B) STREET: 614 North Walnut Street, P.O. Box 7365 (C) CITY: Madison (D) STATE: Wisconsin (E) COUNTRY: U.S.A.
(F) POSTAL CODE (ZIP): 53707-7365 (G) TELEPHONE: (416)368-2400 (H) TELEFAX: (416)363-8246 (ii) TITLE OF INVENTION: PRIMATE EMBRYONIC STEM CELLS
(iii) NUMBER OF SEQUENCES: 6 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: CA 2,190,528 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/376,327 (B) FILING DATE: 20-JAN-1995 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) A
= .
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Wisconsin Alumni Research Foundation (B) STREET: 614 North Walnut Street, P.O. Box 7365 (C) CITY: Madison (D) STATE: Wisconsin (E) COUNTRY: U.S.A.
(F) POSTAL CODE (ZIP): 53707-7365 (G) TELEPHONE: (416)368-2400 (H) TELEFAX: (416)363-8246 (ii) TITLE OF INVENTION: PRIMATE EMBRYONIC STEM CELLS
(iii) NUMBER OF SEQUENCES: 6 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: CA 2,190,528 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/376,327 (B) FILING DATE: 20-JAN-1995 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) A
= .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
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Claims (11)
1. Primate embryonic stem cells in culture which (i) will proliferate in an undifferentiated state in an in vitro culture for over one year, (ii) maintain a normal karyotype through prolonged culture, (iii) maintain the potential to differentiate to derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture, and (iv) will not differentiate when cultured on a fibroblast feeder layer.
2. The cells of claim 1 wherein the stem cells will spontaneously differentiate to trophoblast and produce chorionic gonadotropin when cultured beyond confluence.
3. Primate embryonic stem cells in culture wherein the cells are negative for SSEA-1 marker, positive for SSEA-3 marker, positive for SSEA-4 marker, express alkaline phosphatase activity, are pluripotent, retain normal karyotypes and will not differentiate when cultured on a fibroblast feeder layer.
4. The cells of claim 3 wherein the cells are positive for TRA-1-60, and TRA-1-81 markers.
5. The cells of claim 3 wherein the cells continue to proliferate in an undifferentiated state after continuous culture for at least one year.
6. The cells of claim 3 wherein the cells will differentiate to trophoblast when cultured beyond confluence and will produce chorionic gonadotropin.
7. The cells of claim 3 wherein the cells remain euploid for more than one year of continuous culture.
8. The cells of claim 3 wherein the cells differentiate into cells derived from mesoderm, endoderm and ectoderm germ layers when the cells are injected into a SCID mouse.
9. A method of isolating a primate embryonic stem cell line, comprising the steps of:
(a) isolating cells from the inner cell mass of a primate blastocyst;
(b) plating the inner cell mass cells on embryonic fibroblasts, wherein inner cell mass-derived cell masses are formed;
(c) dissociating the mass into dissociated cells;
(d) replating the dissociated cells on embryonic feeder cells;
(e) selecting colonies with compact morphologies and cells with high nucleus to cytoplasm ratios and prominant nucleoli;
and (f) culturing the cells of the selected colonies on a feeder layer to produce an isolated primate embryonic stem cell.
(a) isolating cells from the inner cell mass of a primate blastocyst;
(b) plating the inner cell mass cells on embryonic fibroblasts, wherein inner cell mass-derived cell masses are formed;
(c) dissociating the mass into dissociated cells;
(d) replating the dissociated cells on embryonic feeder cells;
(e) selecting colonies with compact morphologies and cells with high nucleus to cytoplasm ratios and prominant nucleoli;
and (f) culturing the cells of the selected colonies on a feeder layer to produce an isolated primate embryonic stem cell.
10. A method as claimed in claim 9 further comprising maintaining the isolated cells on a fibroblast feeder layer to prevent differentiation.
11. An isolated primate embryonic stem cell line developed by the method of claim 9, wherein the cell line is negative for SSEA-1 marker, positive for SSEA-3 marker, positive for SSEA-4 marker, express alkaline phosphatase activity, are pluripotent, retain normal karyotypes and will not differentiate when cultured on a fibroblast feeder layer.
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US37632795A | 1995-01-20 | 1995-01-20 | |
US08/376,327 | 1995-01-20 | ||
PCT/US1996/000596 WO1996022362A1 (en) | 1995-01-20 | 1996-01-19 | Primate embryonic stem cells |
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Publication Number | Publication Date |
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CA2190528A1 CA2190528A1 (en) | 1996-07-25 |
CA2190528C true CA2190528C (en) | 2010-04-27 |
Family
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CA2190528A Expired - Lifetime CA2190528C (en) | 1995-01-20 | 1996-01-19 | Primate embryonic stem cells |
Country Status (5)
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US (11) | US5843780A (en) |
EP (2) | EP1640448A3 (en) |
AU (1) | AU4758496A (en) |
CA (1) | CA2190528C (en) |
WO (1) | WO1996022362A1 (en) |
Families Citing this family (806)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030032178A1 (en) * | 1988-08-04 | 2003-02-13 | Williams Robert Lindsay | In vitro propagation of embryonic stem cells |
US7153684B1 (en) * | 1992-10-08 | 2006-12-26 | Vanderbilt University | Pluripotential embryonic stem cells and methods of making same |
EP0688358A4 (en) * | 1993-03-12 | 1997-10-01 | Univ Creighton | Improved vectors for gene therapy |
US20040071637A1 (en) * | 1993-04-27 | 2004-04-15 | Elia James P. | Method for repairing a damaged portion of a human organ |
US5874301A (en) * | 1994-11-21 | 1999-02-23 | National Jewish Center For Immunology And Respiratory Medicine | Embryonic cell populations and methods to isolate such populations |
US5843780A (en) * | 1995-01-20 | 1998-12-01 | Wisconsin Alumni Research Foundation | Primate embryonic stem cells |
US7410773B2 (en) * | 1995-02-02 | 2008-08-12 | Ghazi Jaswinder Dhoot | Method of preparing an undifferentiated cell |
US9068164B1 (en) | 1995-02-02 | 2015-06-30 | Tristem Trading (Cyprus) Limited | Method of preparing an undifferentiated cell |
GB9502022D0 (en) * | 1995-02-02 | 1995-03-22 | Abuljadayel Ilham M S | A method for preparing lymphohaematopoietic progenitor cells |
US7112440B2 (en) | 1995-02-02 | 2006-09-26 | Ghazi Jaswinder Dhoot | Method of increasing the relative number of CD45 low cells in a cell population |
GB9518606D0 (en) * | 1995-09-12 | 1995-11-15 | Inst Of Psychiatry | Neural transplantation |
US20030190753A1 (en) * | 1995-11-09 | 2003-10-09 | Nature Technology Corporation | Vectors for gene transfer |
US7544511B2 (en) * | 1996-09-25 | 2009-06-09 | Neuralstem Biopharmaceuticals Ltd. | Stable neural stem cell line methods |
WO1997047734A1 (en) * | 1996-06-14 | 1997-12-18 | The Regents Of The University Of California | In vitro derivation and culture of primate pluripotent stem cells and therapeutic uses thereof |
US20020194637A1 (en) * | 2001-06-06 | 2002-12-19 | University Of Massachussetts | Embryonic or stem-like cell lines produced by cross species nuclear transplantation |
US7696404B2 (en) * | 1996-08-19 | 2010-04-13 | Advanced Cell Technology, Inc. | Embryonic or stem-like cell lines produced by cross species nuclear transplantation and methods for enhancing embryonic development by genetic alteration of donor cells or by tissue culture conditions |
US6331406B1 (en) | 1997-03-31 | 2001-12-18 | The John Hopkins University School Of Medicine | Human enbryonic germ cell and methods of use |
US6090622A (en) * | 1997-03-31 | 2000-07-18 | The Johns Hopkins School Of Medicine | Human embryonic pluripotent germ cells |
CA2289277C (en) | 1997-04-24 | 2013-02-12 | University Of Washington | Targeted gene modification by parvoviral vectors |
WO1999001763A2 (en) * | 1997-07-02 | 1999-01-14 | Board Of Regents, The University Of Texas System | p53 AS A REGULATOR OF CELL DIFFERENTIATION |
JP3880795B2 (en) * | 1997-10-23 | 2007-02-14 | ジェロン・コーポレーション | Method for growing primate-derived primordial stem cells in a culture that does not contain feeder cells |
WO1999027076A1 (en) * | 1997-11-25 | 1999-06-03 | Arc Genomic Research | Pluripotent embryonic stem cells and methods of obtaining them |
US20050096274A1 (en) * | 1998-04-07 | 2005-05-05 | Lough John W. | Bone morphogenetic protein and fibroblast growth factor compositions and methods for the induction of cardiogenesis |
ES2246085T3 (en) | 1998-05-07 | 2006-02-01 | University Of South Florida | OSEA MEDULA CELLS AS A SOURCE OF NEURONS USEFUL TO REPAIR THE SPINAL MEDULA AND THE BRAIN. |
US20080206206A1 (en) | 1998-05-07 | 2008-08-28 | University Of South Florida | Bone marrow-derived neuronal cells |
US6767737B1 (en) * | 1998-08-31 | 2004-07-27 | New York University | Stem cells bearing an FGF receptor on the cell surface |
JP2002523084A (en) * | 1998-09-01 | 2002-07-30 | ウイスコンシン アラムニ リサーチ ファンデーション | Primate embryonic stem cells with compatible histocompatibility genes |
US6630349B1 (en) | 1998-09-23 | 2003-10-07 | Mount Sinai Hospital | Trophoblast cell preparations |
US20020168766A1 (en) * | 2000-01-11 | 2002-11-14 | Gold Joseph D. | Genetically altered human pluripotent stem cells |
US7410798B2 (en) | 2001-01-10 | 2008-08-12 | Geron Corporation | Culture system for rapid expansion of human embryonic stem cells |
US7413904B2 (en) * | 1998-10-23 | 2008-08-19 | Geron Corporation | Human embryonic stem cells having genetic modifications |
US6667176B1 (en) * | 2000-01-11 | 2003-12-23 | Geron Corporation | cDNA libraries reflecting gene expression during growth and differentiation of human pluripotent stem cells |
JP2002529070A (en) * | 1998-11-09 | 2002-09-10 | モナシュ・ユニヴァーシティ | Embryonic stem cells |
GB9907243D0 (en) | 1999-03-29 | 1999-05-26 | Reneuron Ltd | Therapy |
US7759113B2 (en) * | 1999-04-30 | 2010-07-20 | The General Hospital Corporation | Fabrication of tissue lamina using microfabricated two-dimensional molds |
DE60017900T2 (en) * | 1999-04-30 | 2006-04-06 | Massachusetts General Hospital, Boston | PREPARATION OF THREE-DIMENSIONAL VASCULARIZED TISSUE BY USING TWO-DIMENSIONAL MICRO-MADE SHAPES |
IL129966A (en) * | 1999-05-14 | 2009-12-24 | Technion Res & Dev Foundation | ISOLATED HUMAN EMBRYOID BODIES (hEB) DERIVED FROM HUMAN EMBRYONIC STEM CELLS |
US10638734B2 (en) | 2004-01-05 | 2020-05-05 | Abt Holding Company | Multipotent adult stem cells, sources thereof, methods of obtaining and maintaining same, methods of differentiation thereof, methods of use thereof and cells derived thereof |
US8252280B1 (en) | 1999-08-05 | 2012-08-28 | Regents Of The University Of Minnesota | MAPC generation of muscle |
US7015037B1 (en) | 1999-08-05 | 2006-03-21 | Regents Of The University Of Minnesota | Multiponent adult stem cells and methods for isolation |
EP1214404A4 (en) * | 1999-09-14 | 2003-09-03 | Univ Massachusetts | Embryonic or stem-like cell lines produced by cross species nuclear transplantation and methods for enhancing embryonic development by genetic alteration of donor cells or by tissue culture conditions |
AUPQ307399A0 (en) * | 1999-09-24 | 1999-10-21 | Luminis Pty Limited | Cell cycle control |
EP1218489B1 (en) * | 1999-09-24 | 2009-03-18 | Cybios LLC | Pluripotent embryonic-like stem cells, compositions, methods and uses thereof |
EP1226239A4 (en) * | 1999-10-15 | 2003-02-12 | Advanced Cell Tech Inc | Methods of producing differentiated progenitor cells and lineage-defective embryonic stem cells |
US20030129745A1 (en) * | 1999-10-28 | 2003-07-10 | Robl James M. | Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues |
BR0015264A (en) * | 1999-11-02 | 2002-10-15 | Univ Massachusetts Public Inst | Use of haploid genomes for diagnosis, genetic modification and multiplication |
US6280718B1 (en) | 1999-11-08 | 2001-08-28 | Wisconsin Alumni Reasearch Foundation | Hematopoietic differentiation of human pluripotent embryonic stem cells |
WO2001043540A2 (en) * | 1999-12-17 | 2001-06-21 | Oregon Health And Science University | Methods for producing transgenic animals |
EP1248517A2 (en) * | 2000-01-07 | 2002-10-16 | Oregon Health and Science University | Clonal propagation of primate offspring by embryo splitting |
US20050042749A1 (en) * | 2001-05-16 | 2005-02-24 | Carpenter Melissa K. | Dopaminergic neurons and proliferation-competent precursor cells for treating Parkinson's disease |
US7455983B2 (en) * | 2000-01-11 | 2008-11-25 | Geron Corporation | Medium for growing human embryonic stem cells |
US20030134413A1 (en) * | 2000-01-14 | 2003-07-17 | Rathjen Peter David | Cell production |
US6602711B1 (en) | 2000-02-21 | 2003-08-05 | Wisconsin Alumni Research Foundation | Method of making embryoid bodies from primate embryonic stem cells |
US7541184B2 (en) * | 2000-02-24 | 2009-06-02 | Invitrogen Corporation | Activation and expansion of cells |
US7439064B2 (en) | 2000-03-09 | 2008-10-21 | Wicell Research Institute, Inc. | Cultivation of human embryonic stem cells in the absence of feeder cells or without conditioned medium |
US7005252B1 (en) * | 2000-03-09 | 2006-02-28 | Wisconsin Alumni Research Foundation | Serum free cultivation of primate embryonic stem cells |
US7226057B2 (en) * | 2000-03-10 | 2007-06-05 | Days Corporation | Apparatus and method for automatically leveling an object |
US6619693B1 (en) * | 2000-03-10 | 2003-09-16 | Days Corporation | Apparatus and method for automatically leveling an object |
US7504257B2 (en) * | 2000-03-14 | 2009-03-17 | Es Cell International Pte Ltd. | Embryonic stem cells and neural progenitor cells derived therefrom |
US6458589B1 (en) | 2000-04-27 | 2002-10-01 | Geron Corporation | Hepatocyte lineage cells derived from pluripotent stem cells |
US7776021B2 (en) | 2000-04-28 | 2010-08-17 | The Charles Stark Draper Laboratory | Micromachined bilayer unit for filtration of small molecules |
NL1017973C2 (en) | 2000-05-10 | 2002-11-08 | Tristem Trading Cyprus Ltd | Design. |
US6828145B2 (en) | 2000-05-10 | 2004-12-07 | Cedars-Sinai Medical Center | Method for the isolation of stem cells by immuno-labeling with HLA/MHC gene product marker |
US8273570B2 (en) * | 2000-05-16 | 2012-09-25 | Riken | Process of inducing differentiation of embryonic cell to cell expressing neural surface marker using OP9 or PA6 cells |
US7250294B2 (en) * | 2000-05-17 | 2007-07-31 | Geron Corporation | Screening small molecule drugs using neural cells differentiated from human embryonic stem cells |
CA2409698C (en) * | 2000-05-17 | 2010-10-26 | Geron Corporation | Neural progenitor cell populations |
US7270998B2 (en) * | 2000-06-01 | 2007-09-18 | Japan Science And Technology Corporation | Method of concentrating and separating dopaminergic neurons |
CA2411849A1 (en) * | 2000-06-05 | 2001-12-13 | The Burnham Institute | Methods of differentiating and protecting cells by modulating the p38/mef2 pathway |
CA2412361C (en) | 2000-06-14 | 2011-08-23 | Vistagen, Inc. | Toxicity typing using liver stem cells |
WO2001096532A2 (en) * | 2000-06-15 | 2001-12-20 | Tanja Dominko | Method of generating pluripotent mammalian cells by fusion of a cytoplast fragment with a karyoplast |
AU2001274540A1 (en) * | 2000-06-15 | 2001-12-24 | Tanabe Seiyaku Co., Ltd. | Monkey-origin embryonic stem cells |
JP5014535B2 (en) * | 2000-06-15 | 2012-08-29 | 田辺三菱製薬株式会社 | Cynomolgus monkey-derived embryonic stem cells |
WO2001098463A1 (en) * | 2000-06-20 | 2001-12-27 | Es Cell International Pte Ltd | Method of controlling differentiation of embryonic stem (es) cells by culturing es cells in the presence of bmp-2 pathway antagonists |
IL154159A0 (en) | 2000-08-01 | 2003-07-31 | Yissum Res Dev Co | Directed differentiation of ebryonic cells |
WO2002014469A2 (en) * | 2000-08-15 | 2002-02-21 | Geron Corporation | Reprogramming cells for enhanced differentiation capacity using pluripotent stem cells |
AU2001284160A1 (en) * | 2000-08-19 | 2002-03-04 | Axordia Limited | Modulation of stem cell differentiation |
AU785428B2 (en) | 2000-08-30 | 2007-05-17 | Maria Biotech Co., Ltd | Human embryonic stem cells derived from frozen-thawed embryo |
US6534052B1 (en) | 2000-09-05 | 2003-03-18 | Yong-Fu Xiao | Cardiac function comprising implantation of embryonic stem cell in which differentiation has been initiated |
US6607720B1 (en) | 2000-09-05 | 2003-08-19 | Yong-Fu Xiao | Genetically altered mammalian embryonic stem cells, their living progeny, and their therapeutic application for improving cardiac function after myocardial infarction |
AU2001286222A1 (en) * | 2000-09-14 | 2002-03-26 | Atsushi Yuki | Process for producing normal parenchymal cells, tissue or organ by bioincubator |
AUPR095200A0 (en) * | 2000-10-24 | 2000-11-16 | Bresagen Limited | Cell production |
AU1368402A (en) * | 2000-11-09 | 2002-05-21 | Bresagen Ltd | Cell reprogramming |
JP2004521877A (en) * | 2000-11-22 | 2004-07-22 | ジェロン コーポレイション | Pluripotent stem cell allograft tolerization |
US6576464B2 (en) * | 2000-11-27 | 2003-06-10 | Geron Corporation | Methods for providing differentiated stem cells |
US6921665B2 (en) * | 2000-11-27 | 2005-07-26 | Roslin Institute (Edinburgh) | Selective antibody targeting of undifferentiated stem cells |
US20030027331A1 (en) * | 2000-11-30 | 2003-02-06 | Yan Wen Liang | Isolated homozygous stem cells, differentiated cells derived therefrom, and materials and methods for making and using same |
EP1395652A2 (en) * | 2000-11-30 | 2004-03-10 | Stemron, Inc. | Isolated homozygous stem cells differentiated cells derived therefrom and materials and methods for making and using same |
US20020068046A1 (en) * | 2000-12-04 | 2002-06-06 | Jianwu Dai | Use of stem cells derived from dermal skin |
WO2002063938A2 (en) * | 2000-12-05 | 2002-08-22 | Layton Bioscience Inc. | Production and use of dopaminergic cells to treat dopaminergic deficiencies |
WO2002053193A2 (en) | 2001-01-02 | 2002-07-11 | The Charles Stark Draper Laboratory, Inc. | Tissue engineering of three-dimensional vascularized using microfabricated polymer assembly technology |
NZ568250A (en) | 2001-01-02 | 2009-07-31 | Stemron Inc | A method for producing a population of homozygous stem cells having a pre-selected immunotype and/or genotype, cells suitable for transplant derived therefrom, and materials and methods using same |
CA2435826A1 (en) * | 2001-01-24 | 2002-08-01 | The Government Of The United States Of America | Differentiation of stem cells to pancreatic endocrine cells |
JP2004529621A (en) * | 2001-02-14 | 2004-09-30 | ティー ファークト,レオ | Pluripotent adult stem cells, their origin, methods of obtaining and maintaining them, methods of differentiating them, methods of their use, and cells derived therefrom |
AUPR349501A0 (en) * | 2001-03-02 | 2001-03-29 | Bresagen Limited | Cellular production control |
US7126039B2 (en) * | 2001-03-21 | 2006-10-24 | Geron Corporation | Animal tissue with carbohydrate antigens compatible for human transplantation |
WO2002079457A1 (en) * | 2001-03-29 | 2002-10-10 | Ixion Biotechnology, Inc. | Method for transdifferentiation of non-pancreatic stem cells to the pancreatic differentiation pathway |
US7838292B1 (en) * | 2001-03-29 | 2010-11-23 | University Of Louisville Research Foundation, Inc. | Methods for obtaining adult human olfactory progenitor cells |
WO2002087627A1 (en) * | 2001-04-27 | 2002-11-07 | Xcyte Therapies, Inc. | Maturation of antigen-presenting cells using activated t cells |
US20030211605A1 (en) * | 2001-05-01 | 2003-11-13 | Lee Sang-Hun | Derivation of midbrain dopaminergic neurons from embryonic stem cells |
US20050176665A1 (en) * | 2001-05-18 | 2005-08-11 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of hairless (HR) gene expression using short interfering nucleic acid (siNA) |
WO2003004605A2 (en) * | 2001-07-06 | 2003-01-16 | Geron Corporation | Mesenchymal cells and osteoblasts from human embryonic stem cell |
US7732199B2 (en) | 2001-07-12 | 2010-06-08 | Geron Corporation | Process for making transplantable cardiomyocytes from human embryonic stem cells |
KR101073411B1 (en) * | 2001-07-12 | 2011-10-17 | 제론 코포레이션 | Cells of the cardiomyocyte lineage produced from human pluripotent stem cells |
US20030017587A1 (en) * | 2001-07-18 | 2003-01-23 | Rader William C. | Embryonic stem cells, clinical applications and methods for expanding in vitro |
DE10136719A1 (en) * | 2001-07-27 | 2003-02-20 | Georg S Wengler | Recovering omnipotent human embryonic stem cells, comprises separating them from a blastocyte which remains viable, useful for gene therapy especially autotransplants |
WO2003014313A2 (en) | 2001-08-06 | 2003-02-20 | Bresagen, Ltd. | Alternative compositions and methods for the culture of stem cells |
US20040092013A1 (en) * | 2001-08-14 | 2004-05-13 | Snyder Evan Y. | Method of treating alzheimer's disease with cell therapy |
US20030211603A1 (en) * | 2001-08-14 | 2003-11-13 | Earp David J. | Reprogramming cells for enhanced differentiation capacity using pluripotent stem cells |
CA2458362A1 (en) * | 2001-08-23 | 2003-03-06 | Reliance Life Sciences Pvt., Ltd. | Isolation of inner cell mass for the establishment of human embryonic stem cell (hesc) lines |
DE10144326B4 (en) * | 2001-09-10 | 2005-09-22 | Siemens Ag | Method and system for monitoring a tire air pressure |
US7588937B2 (en) | 2001-10-03 | 2009-09-15 | Wisconsin Alumni Research Foundation | Method of in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells |
US6887706B2 (en) * | 2001-10-03 | 2005-05-03 | Wisconsin Alumni Research Foundation | Method of in vitro differentiation of transplantable neural precursor cells from primate embryonic stem cells |
US8153424B2 (en) * | 2001-10-03 | 2012-04-10 | Wisconsin Alumni Research Foundation | Method of in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells |
EP1446476A4 (en) * | 2001-11-02 | 2005-01-26 | Wisconsin Alumni Res Found | Endothelial cells derived from primate embryonic stem cells |
US6759244B2 (en) * | 2001-11-08 | 2004-07-06 | Art Institute Of New York And New Jersey, Inc. | Composite blastocysts (CBs) from aggregates of dissociated cells of non-viable pre-embryos |
WO2003054146A2 (en) * | 2001-11-14 | 2003-07-03 | Northwestern University | Self-assembly and mineralization of peptide-amphiphile nanofibers |
DE60233248D1 (en) * | 2001-11-15 | 2009-09-17 | Childrens Medical Center | PROCESS FOR THE ISOLATION, EXPANSION AND DIFFERENTIATION OF FEDERAL STRAIN CELLS FROM CHORION ZOTTE, FRUIT WATER AND PLAZENTA AND THERAPEUTIC USES THEREOF |
CA2469483C (en) | 2001-12-07 | 2017-07-25 | Geron Corporation | Hematopoietic cells from human embryonic stem cells |
AU2002366602B2 (en) * | 2001-12-07 | 2008-07-03 | Asterias Biotherapeutics, Inc. | Chondrocyte precursors derived from human embryonic stem cells |
US7799324B2 (en) * | 2001-12-07 | 2010-09-21 | Geron Corporation | Using undifferentiated embryonic stem cells to control the immune system |
EP1463798A4 (en) * | 2001-12-07 | 2005-01-19 | Geron Corp | Islet cells from human embryonic stem cells |
US20040224403A1 (en) * | 2001-12-07 | 2004-11-11 | Robarts Research Institute | Reconstituting hematopoietic cell function using human embryonic stem cells |
US20030113910A1 (en) * | 2001-12-18 | 2003-06-19 | Mike Levanduski | Pluripotent stem cells derived without the use of embryos or fetal tissue |
AU2002357410A1 (en) | 2001-12-21 | 2003-07-09 | Thromb-X Nv | Compositions for the in vitro derivation and culture of embryonic stem (es) cell lines with germline transmission capability and for the culture of adult stem cells |
GB0220145D0 (en) * | 2002-08-30 | 2002-10-09 | Thromb X Nv | Novel compositions for the in vitro derivation and culture of embryonic stem (ES) cell lines with germline transmission capability |
CN1671835A (en) * | 2001-12-28 | 2005-09-21 | 塞拉提斯股份公司 | A method for the establishment of a pluripotent human blastocyst-derived stem cell line |
US7190781B2 (en) * | 2002-01-04 | 2007-03-13 | Telefonaktiebolaget Lm Ericsson (Publ) | Message transfer part point code mapping method and node |
US20040043482A1 (en) * | 2002-01-04 | 2004-03-04 | Kye-Hyung Paik | Method of producing stem cell lines |
US20050170506A1 (en) * | 2002-01-16 | 2005-08-04 | Primegen Biotech Llc | Therapeutic reprogramming, hybrid stem cells and maturation |
US20050090004A1 (en) * | 2003-01-16 | 2005-04-28 | Sayre Chauncey B. | Stem cell maturation for all tissue lines |
US20030134422A1 (en) * | 2002-01-16 | 2003-07-17 | Sayre Chauncey Bigelow | Stem cell maturation for all tissue lines |
US20030162290A1 (en) * | 2002-01-25 | 2003-08-28 | Kazutomo Inoue | Method for inducing differentiation of embryonic stem cells into functioning cells |
EP1471140A4 (en) * | 2002-01-31 | 2005-02-16 | Asahi Techno Glass Cosporation | Liquid for frozen storage of primate embryo stem cells and frozen storage method |
WO2003066839A1 (en) * | 2002-02-05 | 2003-08-14 | Rappaport Family Institute For Research In The Medical Sciences | Lineage committed stem cells selected for telomerase promoter activity |
US20050106724A1 (en) * | 2002-02-06 | 2005-05-19 | Joerg Schierholz | Pluripotent embryonic-like stem cells derived from teeth and uses thereof |
US7371719B2 (en) * | 2002-02-15 | 2008-05-13 | Northwestern University | Self-assembly of peptide-amphiphile nanofibers under physiological conditions |
US7736892B2 (en) * | 2002-02-25 | 2010-06-15 | Kansas State University Research Foundation | Cultures, products and methods using umbilical cord matrix cells |
US20030161818A1 (en) * | 2002-02-25 | 2003-08-28 | Kansas State University Research Foundation | Cultures, products and methods using stem cells |
WO2003078967A2 (en) * | 2002-03-12 | 2003-09-25 | Oregon Health & Science University | Stem cell selection and differentiation |
AU2002246132A1 (en) * | 2002-03-13 | 2003-09-22 | Fundacion Ivi Para El Estudio De La Reproduccion Humana (Fivier) | Method of producing cell lines |
IL163949A0 (en) * | 2002-03-15 | 2005-12-18 | Wisconsin Alumni Res Found | Method of identifying genes controlling differentiation |
JP4264360B2 (en) | 2002-03-15 | 2009-05-13 | ウィセル リサーチ インスティテュート インコーポレイテッド | Production method of primate trophoblast |
GB0207440D0 (en) * | 2002-03-28 | 2002-05-08 | Ppl Therapeutics Scotland Ltd | Tolerogenic antigen-presenting cells |
US20040111285A1 (en) * | 2002-04-09 | 2004-06-10 | Mark Germain | Method for human pluripotent stem cells |
CA2484223A1 (en) * | 2002-04-25 | 2003-11-06 | Wisconsin Alumni Research Foundation | Use of human neural stem cells secreting gdnf for treatment of parkinson's and other neurodegenerative diseases |
US20030207448A1 (en) * | 2002-05-06 | 2003-11-06 | Revera Gregory Henry | Methodologies for the creation of pluripotent or multipotent human stem cells without creating or destroying a human embryo |
US6816665B2 (en) * | 2002-05-09 | 2004-11-09 | Lynx Photonic Networks Inc. | Constant power operation thermo-optic switch |
US20060003446A1 (en) | 2002-05-17 | 2006-01-05 | Gordon Keller | Mesoderm and definitive endoderm cell populations |
US7763466B2 (en) * | 2002-05-17 | 2010-07-27 | Mount Sinai School Of Medicine Of New York University | Mesoderm and definitive endoderm cell populations |
WO2003100018A2 (en) | 2002-05-24 | 2003-12-04 | Advanced Cell Technology, Inc. | A bank of stem cells for transplantation |
AU2003247514A1 (en) * | 2002-06-11 | 2003-12-22 | Roy Ogle | Meningeal-derived stem cells |
EP1558086A4 (en) * | 2002-06-14 | 2008-03-05 | Univ Case Western Reserve | Cell targeting methods and compositions |
US7285415B2 (en) * | 2002-07-11 | 2007-10-23 | The Regents Of The University Of California | Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury |
US20050101014A1 (en) * | 2002-07-11 | 2005-05-12 | Keirstead Hans S. | Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury |
WO2007116408A2 (en) * | 2006-04-11 | 2007-10-18 | Nanodiagnostics Israel Ltd. | Pluripotent stem cells characterized by expression of germline specific genes |
US20110158966A1 (en) * | 2002-07-23 | 2011-06-30 | Judith Seligman | Stem cells characterized by expression of germline specific genes |
AU2003247135A1 (en) * | 2002-07-23 | 2004-02-09 | Nanodiagnostics, Inc. | Embryonic stem cell markers and uses thereof |
US7422736B2 (en) * | 2002-07-26 | 2008-09-09 | Food Industry Research And Development Institute | Somatic pluripotent cells |
US8876532B2 (en) | 2002-07-31 | 2014-11-04 | Dentsply International Inc. | Bone repair putty |
AU2003254298A1 (en) | 2002-08-02 | 2004-02-23 | Stratatech Corporation | Species specific dna detection |
US20060121607A1 (en) * | 2002-08-08 | 2006-06-08 | Thomas Schulz | Compositions and methods for neural differentiation of embryonic stem cells |
IL166636A0 (en) * | 2002-08-09 | 2006-01-15 | Innovationsagentur | Method for producing cell lines and organs by means of differentiable cells |
AU2003262760A1 (en) | 2002-08-21 | 2004-03-11 | Northwestern University | Charged peptide-amphiphile solutions and self-assembled peptide nanofiber networks formed therefrom |
US20040058310A1 (en) * | 2002-09-20 | 2004-03-25 | Grailhe Regls Christian | Method for measuring a marker indicative of the exposure of a patient to nicotine; a kit for measuring such a marker |
WO2004029203A2 (en) * | 2002-09-25 | 2004-04-08 | Bresagen, Inc. | Compositions and methods for enrichment of neural stem cells using ceramide analogs |
US7267981B2 (en) | 2002-10-07 | 2007-09-11 | Technion Research & Development Foundation Ltd. | Human foreskin fibroblasts for culturing ES cells |
CN1717478A (en) * | 2002-10-25 | 2006-01-04 | 湖南惠霖生命科技有限公司 | Be used for the feeder layer of hESC's vitro culture and the method for cultivating embryonic stem cell |
US7554021B2 (en) * | 2002-11-12 | 2009-06-30 | Northwestern University | Composition and method for self-assembly and mineralization of peptide amphiphiles |
US7683025B2 (en) | 2002-11-14 | 2010-03-23 | Northwestern University | Synthesis and self-assembly of ABC triblock bola peptide amphiphiles |
WO2004050826A2 (en) * | 2002-11-29 | 2004-06-17 | Technion Research & Development Foundation Ltd. | Method of dynamically culturing embryonic stem cells |
US20040110286A1 (en) * | 2002-12-06 | 2004-06-10 | The John P. Robarts Research Institute | Method for making hematopoietic cells |
HUE028026T2 (en) | 2002-12-16 | 2016-11-28 | Technion Res & Dev Foundation | Feeder-free, xeno-free culture system for human embryonic stem cells |
AU2003303741A1 (en) * | 2002-12-18 | 2004-09-17 | Bresagen, Inc. | Compositions and methods for neural cell production and stabilization |
WO2004065616A2 (en) | 2003-01-16 | 2004-08-05 | The General Hospital Corporation | Use of three-dimensional microfabricated tissue engineered systems for pharmacologic applications |
WO2004072104A2 (en) * | 2003-02-11 | 2004-08-26 | Northwestern University | Methods and materials for nanocrystalline surface coatings and attachment of peptide amphiphile nanofibers thereon |
BRPI0408733A (en) * | 2003-03-12 | 2006-03-07 | Reliance Life Sciences Pvt Ltd | derivation of terminally differentiated dopaminergic neurons from human embryonic stem cells |
US20030224411A1 (en) * | 2003-03-13 | 2003-12-04 | Stanton Lawrence W. | Genes that are up- or down-regulated during differentiation of human embryonic stem cells |
US7153650B2 (en) * | 2003-03-13 | 2006-12-26 | Geron Corporation | Marker system for preparing and characterizing high-quality human embryonic stem cells |
US20070020242A1 (en) * | 2003-03-27 | 2007-01-25 | Ixion Biotechnology, Inc. | Method for transdifferentiation of non-pancreatic stem cells to the pancreatic pathway |
US20040191839A1 (en) * | 2003-03-28 | 2004-09-30 | National Institute Of Agrobiological Sciences Japan | Methods for sorting undifferentiated cells and uses thereof |
JP4502317B2 (en) * | 2003-03-28 | 2010-07-14 | 独立行政法人農業生物資源研究所 | Method for selecting undifferentiated cells and use thereof |
JP2004298108A (en) * | 2003-03-31 | 2004-10-28 | Japan Science & Technology Agency | Method for producing lens cell, and lens cell obtained by the method |
CA2520023C (en) | 2003-04-08 | 2013-02-05 | Yeda Research And Development Co. Ltd | Stem cells having increased sensitivity to sdf-1 and methods of generating and using same |
FR2853551B1 (en) | 2003-04-09 | 2006-08-04 | Lab Francais Du Fractionnement | STABILIZING FORMULATION FOR IMMUNOGLOBULIN G COMPOSITIONS IN LIQUID FORM AND LYOPHILIZED FORM |
EP1615492A4 (en) * | 2003-04-09 | 2010-07-14 | Magee Womens Health Corp | Methods for correcting mitotic spindle defects associated with somatic cell nuclear transfer in animals |
US20060037086A1 (en) * | 2003-04-09 | 2006-02-16 | Schatten Gerald P | Methods for correcting mitotic spindle defects and optimizing preimplantation embryonic developmental rates associated with somatic cell nuclear transfer in animals |
IL155783A (en) | 2003-05-05 | 2010-11-30 | Technion Res & Dev Foundation | Multicellular systems of pluripotent human embryonic stem cells and cancer cells and uses thereof |
WO2004099394A2 (en) * | 2003-05-08 | 2004-11-18 | Cellartis Ab | A method for efficient transfer of human blastocyst-derived stem cells (hbs cells) from a feeder-supported to a feeder-free culture system |
WO2004099395A2 (en) * | 2003-05-08 | 2004-11-18 | Cellartis Ab | A method for the generation of neural progenitor cells |
US20090203141A1 (en) * | 2003-05-15 | 2009-08-13 | Shi-Lung Lin | Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant RNA agents |
US9567591B2 (en) | 2003-05-15 | 2017-02-14 | Mello Biotechnology, Inc. | Generation of human embryonic stem-like cells using intronic RNA |
WO2005034624A2 (en) | 2003-05-21 | 2005-04-21 | The General Hospital Corporation | Microfabricated compositions and processes for engineering tissues containing multiple cell types |
EP1702062A2 (en) * | 2003-06-11 | 2006-09-20 | Jan Remmereit | Differentiation of stem cells for therapeutic use |
WO2004108882A2 (en) * | 2003-06-11 | 2004-12-16 | Yeda Research And Development Co. Ltd. | Neural stem cells and methods of generating and utilizing same |
WO2005001080A2 (en) * | 2003-06-27 | 2005-01-06 | Ethicon, Incorporated | Postpartum-derived cells for use in treatment of disease of the heart and circulatory system |
US8518390B2 (en) | 2003-06-27 | 2013-08-27 | Advanced Technologies And Regenerative Medicine, Llc | Treatment of stroke and other acute neural degenerative disorders via intranasal administration of umbilical cord-derived cells |
US7875272B2 (en) | 2003-06-27 | 2011-01-25 | Ethicon, Incorporated | Treatment of stroke and other acute neuraldegenerative disorders using postpartum derived cells |
US9572840B2 (en) | 2003-06-27 | 2017-02-21 | DePuy Synthes Products, Inc. | Regeneration and repair of neural tissue using postpartum-derived cells |
US9592258B2 (en) | 2003-06-27 | 2017-03-14 | DePuy Synthes Products, Inc. | Treatment of neurological injury by administration of human umbilical cord tissue-derived cells |
US8491883B2 (en) | 2003-06-27 | 2013-07-23 | Advanced Technologies And Regenerative Medicine, Llc | Treatment of amyotrophic lateral sclerosis using umbilical derived cells |
US8790637B2 (en) | 2003-06-27 | 2014-07-29 | DePuy Synthes Products, LLC | Repair and regeneration of ocular tissue using postpartum-derived cells |
US20050019907A1 (en) * | 2003-07-22 | 2005-01-27 | Santiago Munne | Obtaining normal disomic stem cells from chromosomally abnormal embryos |
EP3473102A1 (en) | 2003-08-01 | 2019-04-24 | Stratatech Corporation | Methods for providing human skin equivalents |
US7569385B2 (en) * | 2003-08-14 | 2009-08-04 | The Regents Of The University Of California | Multipotent amniotic fetal stem cells |
US7820439B2 (en) * | 2003-09-03 | 2010-10-26 | Reliance Life Sciences Pvt Ltd. | In vitro generation of GABAergic neurons from pluripotent stem cells |
US20050054100A1 (en) * | 2003-09-08 | 2005-03-10 | Rennard Stephen I. | Methods for fibroblast differentiation |
WO2005033297A1 (en) * | 2003-09-19 | 2005-04-14 | The Rockefeller University | Compositions, methods and kits relating to reprogramming adult differentiated cells and production of embryonic stem cell-like cells |
WO2005032251A1 (en) * | 2003-10-09 | 2005-04-14 | I.M.T. Interface Multigrad Technology Ltd. | Method for freezing, thawing and transplantation of viable cartilage |
US20050100924A1 (en) * | 2003-11-06 | 2005-05-12 | National University Of Singapore | C/EBPalpha gene targeting constructs and uses thereof |
IL158868A0 (en) | 2003-11-13 | 2004-05-12 | Yeda Res & Dev | Methods of generating and using stem cells enriched with immature primitive progenitor |
US20050123525A1 (en) * | 2003-11-13 | 2005-06-09 | Ulrich Martin | Composition and method for inducing immune tolerance towards cell, tissue and/or organ transplants |
US20080220520A1 (en) * | 2003-11-19 | 2008-09-11 | Palecek Sean P | Cryopreservation of human embryonic stem cells in microwells |
US20050106554A1 (en) * | 2003-11-19 | 2005-05-19 | Palecek Sean P. | Cryopreservation of pluripotent stem cells |
US20050106725A1 (en) * | 2003-11-19 | 2005-05-19 | Palecek Sean P. | Method of reducing cell differentiation |
US7790039B2 (en) * | 2003-11-24 | 2010-09-07 | Northwest Biotherapeutics, Inc. | Tangential flow filtration devices and methods for stem cell enrichment |
US7682828B2 (en) * | 2003-11-26 | 2010-03-23 | Whitehead Institute For Biomedical Research | Methods for reprogramming somatic cells |
US7632681B2 (en) * | 2003-12-02 | 2009-12-15 | Celavie Biosciences, Llc | Compositions and methods for propagation of neural progenitor cells |
US20070269412A1 (en) * | 2003-12-02 | 2007-11-22 | Celavie Biosciences, Llc | Pluripotent cells |
CA2549391A1 (en) * | 2003-12-05 | 2005-06-23 | Northwestern University | Branched peptide amphiphiles, related epitope compounds and self assembled structures thereof |
CA2549164A1 (en) * | 2003-12-05 | 2005-06-23 | Northwestern University | Self-assembling peptide amphiphiles and related methods for growth factor delivery |
TWI280280B (en) * | 2003-12-09 | 2007-05-01 | Ind Tech Res Inst | Culture system and method for expansion and undifferentiated growth of human embryonic stem cells |
US20060030042A1 (en) * | 2003-12-19 | 2006-02-09 | Ali Brivanlou | Maintenance of embryonic stem cells by the GSK-3 inhibitor 6-bromoindirubin-3'-oxime |
US8647873B2 (en) | 2004-04-27 | 2014-02-11 | Viacyte, Inc. | PDX1 expressing endoderm |
WO2007059007A2 (en) | 2005-11-14 | 2007-05-24 | Cythera, Inc. | Markers of definitive endoderm |
US20050214257A1 (en) | 2003-12-23 | 2005-09-29 | Northwestern University | Compositions and methods for controlling stem cell and tumor cell differentiation, growth, and formation |
US7541185B2 (en) * | 2003-12-23 | 2009-06-02 | Cythera, Inc. | Methods for identifying factors for differentiating definitive endoderm |
US7625753B2 (en) * | 2003-12-23 | 2009-12-01 | Cythera, Inc. | Expansion of definitive endoderm cells |
US20050266554A1 (en) * | 2004-04-27 | 2005-12-01 | D Amour Kevin A | PDX1 expressing endoderm |
CN103898045B (en) | 2003-12-23 | 2019-02-01 | 维亚希特公司 | Definitive entoderm |
US7985585B2 (en) | 2004-07-09 | 2011-07-26 | Viacyte, Inc. | Preprimitive streak and mesendoderm cells |
WO2005065354A2 (en) * | 2003-12-31 | 2005-07-21 | The Burnham Institute | Defined media for pluripotent stem cell culture |
US20060112438A1 (en) | 2004-01-02 | 2006-05-25 | West Michael D | Novel culture systems for ex vivo development |
AU2005209169B2 (en) | 2004-01-16 | 2010-12-16 | Carnegie Mellon University | Cellular labeling for nuclear magnetic resonance techniques |
US20080241107A1 (en) * | 2004-01-23 | 2008-10-02 | Copland Iii John A | Methods and Compositions For Preparing Pancreatic Insulin Secreting Cells |
US20050186672A1 (en) * | 2004-01-27 | 2005-08-25 | Reliance Life Sciences Pvt. Ltd. | Tissue system with undifferentiated stem cells derived from corneal limbus |
US8196416B2 (en) * | 2004-02-02 | 2012-06-12 | Core Dynamics Limited | Device for directional cooling of biological matter |
EP1711053A2 (en) * | 2004-02-02 | 2006-10-18 | I.M.T. Interface Multigrad Technology Ltd. | Biological material and methods and solutions for preservation thereof |
WO2005080551A2 (en) * | 2004-02-12 | 2005-09-01 | University Of Newcastle Upon Tyne | Stem cells |
US20060216821A1 (en) * | 2004-02-26 | 2006-09-28 | Reliance Life Sciences Pvt. Ltd. | Pluripotent embryonic-like stem cells derived from corneal limbus, methods of isolation and uses thereof |
KR20080036636A (en) * | 2004-02-26 | 2008-04-28 | 리라이언스 라이프 사이언시스 프라이빗. 리미티드 | Pluripotent embryonic-like stem cells derived from corneal limbus, methods of isolation and uses thereof |
US8187875B2 (en) | 2004-02-26 | 2012-05-29 | Reliance Life Sciences Pvt. Ltd. | Dopaminergic neurons derived from corneal limbus, methods of isolation and uses thereof |
US20050214938A1 (en) * | 2004-03-26 | 2005-09-29 | Gold Joseph D | Cardiac bodies: clusters of spontaneously contracting cells for regenerating cardiac function |
US7452718B2 (en) * | 2004-03-26 | 2008-11-18 | Geron Corporation | Direct differentiation method for making cardiomyocytes from human embryonic stem cells |
US7670596B2 (en) | 2004-04-23 | 2010-03-02 | Bioe, Inc. | Multi-lineage progenitor cells |
US7622108B2 (en) | 2004-04-23 | 2009-11-24 | Bioe, Inc. | Multi-lineage progenitor cells |
EP2377922B1 (en) | 2004-04-27 | 2020-04-08 | Viacyte, Inc. | PDX1 expressing endoderm |
GB2428044B (en) * | 2004-05-07 | 2008-08-06 | Wisconsin Alumni Res Found | Method of forming mesenchymal stem cells from embryonic stem cells |
ATE460947T1 (en) * | 2004-06-07 | 2010-04-15 | Core Dynamics Ltd | METHOD FOR STERILIZING BIOLOGICAL PREPARATIONS |
EP3000877B1 (en) * | 2004-06-09 | 2019-08-14 | The University Court of the University of Edinburgh | Neural stem cells |
US20060008451A1 (en) * | 2004-07-06 | 2006-01-12 | Michigan State University | In vivo methods for effecting tissue specific differentiation of embryonic stem cells |
NZ582597A (en) | 2004-07-09 | 2011-08-26 | Viacyte Inc | Preprimitive streak and mesendoderm cells |
JP5687816B2 (en) | 2004-07-09 | 2015-03-25 | ヴィアサイト,インコーポレイテッド | Methods for identifying factors for differentiating definitive endoderm |
AU2005271723B2 (en) | 2004-07-13 | 2010-12-16 | Asterias Biotherapeutics, Inc. | Medium for growing human embryonic stem cells |
EP1781776A2 (en) * | 2004-07-29 | 2007-05-09 | Stem Cell Innovations, Inc. | Differentiation of stem cells |
US8037696B2 (en) * | 2004-08-12 | 2011-10-18 | Core Dynamics Limited | Method and apparatus for freezing or thawing of a biological material |
MX2007001772A (en) * | 2004-08-13 | 2007-07-11 | Univ Georgia Res Found | Compositions and methods for self-renewal and differentiation in human embryonic stem cells. |
WO2006025802A1 (en) * | 2004-09-03 | 2006-03-09 | Agency For Science, Technology And Research | Method for maintaining pluripotency of stem/progenitor cells |
US20060177926A1 (en) * | 2004-09-07 | 2006-08-10 | Erika Sasaki | Common marmoset embryonic stem cell lines |
CA2580754A1 (en) * | 2004-09-21 | 2006-03-30 | Nvr Labs, Ltd. | Compositions and methods for stem cell expansion and differentiation |
ATE373081T1 (en) * | 2004-09-30 | 2007-09-15 | Reneuron Ltd | CELL LINE |
US20070077654A1 (en) * | 2004-11-01 | 2007-04-05 | Thomson James A | Platelets from stem cells |
CA3015835A1 (en) | 2004-11-04 | 2006-05-18 | Astellas Institute For Regenerative Medicine | Derivation of embryonic stem cells |
US7893315B2 (en) * | 2004-11-04 | 2011-02-22 | Advanced Cell Technology, Inc. | Derivation of embryonic stem cells and embryo-derived cells |
WO2006053378A1 (en) * | 2004-11-16 | 2006-05-26 | Sydney Ifv Limited | Derivation and culture of human embryo-derived cells |
CN104042629A (en) | 2004-11-17 | 2014-09-17 | 神经干公司 | Transplantation of human neural cells for treatment of neurodegenerative conditions |
US8017395B2 (en) | 2004-12-17 | 2011-09-13 | Lifescan, Inc. | Seeding cells on porous supports |
US20060171930A1 (en) * | 2004-12-21 | 2006-08-03 | Agnieszka Seyda | Postpartum cells derived from umbilical cord tissue, and methods of making, culturing, and using the same |
US20060153815A1 (en) * | 2004-12-21 | 2006-07-13 | Agnieszka Seyda | Tissue engineering devices for the repair and regeneration of tissue |
US20060166361A1 (en) * | 2004-12-21 | 2006-07-27 | Agnieszka Seyda | Postpartum cells derived from placental tissue, and methods of making, culturing, and using the same |
EP1833496B1 (en) | 2004-12-23 | 2013-07-31 | Ethicon, Incorporated | Treatment of stroke and other acute neural degenerative disorders using postpartum derived cells |
DE102004062184B4 (en) * | 2004-12-23 | 2013-08-01 | Wolfgang Würfel | Embryo-preserving production of pluripotent embryonic stem cells, stem cells thus obtained and use thereof |
ES2621847T3 (en) | 2004-12-23 | 2017-07-05 | DePuy Synthes Products, Inc. | Postpartum cells derived from umbilical cord tissue, and methods of making and using them |
EP1838843B1 (en) | 2004-12-23 | 2019-07-10 | Viacyte, Inc. | Expansion of definitive endoderm cells |
US8597947B2 (en) | 2004-12-29 | 2013-12-03 | Hadasit Medical Research Services & Development Limited | Undifferentiated stem cell culture systems |
EP1844136B1 (en) * | 2004-12-29 | 2014-08-27 | Hadasit Medical Research Services And Development Ltd. | Stem cells culture systems |
EP1841857A1 (en) * | 2004-12-30 | 2007-10-10 | Stemlifeline, Inc. | Methods and compositions relating to embryonic stem cell lines |
WO2006073966A1 (en) * | 2004-12-30 | 2006-07-13 | Stemlifeline, Inc. | Methods and systems relating to embryonic stem cell lines |
AU2006206194A1 (en) * | 2005-01-21 | 2006-07-27 | Northwestern University | Methods and compositions for encapsulation of cells |
US7022518B1 (en) * | 2005-01-31 | 2006-04-04 | Glen Feye | Apparatus and method for co-culturing of cells |
WO2006084314A1 (en) * | 2005-02-09 | 2006-08-17 | Australian Stem Cell Centre Limited | Stem cell populations and classification system |
EP1850661A2 (en) * | 2005-02-22 | 2007-11-07 | Interface Multigrad Technology (IMT) Ltd. | Preserved viable cartilage, method for its preservation, and system and devices used therefor |
JP2008531733A (en) * | 2005-03-04 | 2008-08-14 | ノースウエスタン ユニバーシティ | Angiogenic heparin-binding epitopes, peptide amphiphiles, self-assembling compositions, and related uses |
US20070048865A1 (en) * | 2005-03-15 | 2007-03-01 | Kenzaburo Tani | Method for acceleration of stem cell differentiation |
US9295543B2 (en) | 2005-03-17 | 2016-03-29 | Stratatech Corporation | Skin substitutes with improved purity |
US8012751B2 (en) * | 2005-03-31 | 2011-09-06 | Wisconsin Alumni Research Foundation | Differentiation of pluripotent embryonic stem cells |
US7883847B2 (en) | 2005-04-01 | 2011-02-08 | Wake Forest University Health Sciences | Transcriptional profiling of stem cells and their multilineage differentiation |
CN101180389A (en) * | 2005-05-17 | 2008-05-14 | 利莱恩斯生命科学有限公司 | Establishment of a human embryonic stem cell line using mammalian cells |
AU2006202209B2 (en) * | 2005-05-27 | 2011-04-14 | Lifescan, Inc. | Amniotic fluid derived cells |
CA2613889A1 (en) * | 2005-06-08 | 2006-12-14 | Centocor, Inc. | A cellular therapy for ocular degeneration |
US20060292695A1 (en) * | 2005-06-22 | 2006-12-28 | Roslin Institute | Methods and kits for drug screening and toxicity testing using promoter-reporter cells derived from embryonic stem cells |
US20060292694A1 (en) * | 2005-06-22 | 2006-12-28 | Roslin Institute | Reporter hepatocytes and other cells for drug screening and toxicity testing |
US20080152632A1 (en) * | 2005-06-22 | 2008-06-26 | Roslin Institute | Promoter-reporter cells for determining drug metabolism, drug interactions, and the effects of allotype variation |
US9062289B2 (en) | 2005-06-22 | 2015-06-23 | Asterias Biotherapeutics, Inc. | Differentiation of primate pluripotent stem cells to cardiomyocyte-lineage cells |
KR20130100221A (en) | 2005-06-22 | 2013-09-09 | 제론 코포레이션 | Suspension culture of human embryonic stem cells |
US20070026520A1 (en) * | 2005-07-29 | 2007-02-01 | Kelly James H | Novel cells, compositions, and methods |
US8198085B2 (en) * | 2005-08-03 | 2012-06-12 | Core Dynamics Limited | Somatic cells for use in cell therapy |
WO2007025166A2 (en) | 2005-08-25 | 2007-03-01 | Repair Technologies, Inc. | Devices, compositions and methods for the protection and repair of cells and tissues |
AU2006286149B2 (en) | 2005-08-29 | 2012-09-13 | Technion Research And Development Foundation Ltd. | Media for culturing stem cells |
EP1940423B1 (en) | 2005-09-09 | 2014-04-16 | Duke University | Tissue engineering methods and compositions |
CA2869687C (en) | 2005-10-27 | 2024-01-23 | Viacyte, Inc. | Pdx1-expressing dorsal and ventral foregut endoderm |
US7413900B2 (en) * | 2005-10-31 | 2008-08-19 | President And Fellows Of Harvard College | Immortalized fibroblasts |
CN101374537B (en) | 2005-11-10 | 2016-04-20 | 健能万生物制药公司 | The MNTF differentiation of stem cell and growth |
US7521221B2 (en) * | 2005-11-21 | 2009-04-21 | Board Of Trustees Of The University Of Arknasas | Staphylococcus aureus strain CYL1892 |
PL2535403T3 (en) | 2005-12-08 | 2019-10-31 | Univ Louisville Res Found Inc | Very small embryonic-like (VSEL) stem cells and methods of isolating and using the same |
US9155762B2 (en) * | 2005-12-08 | 2015-10-13 | University Of Louisville Research Foundation, Inc. | Uses and isolation of stem cells from bone marrow |
US8278104B2 (en) * | 2005-12-13 | 2012-10-02 | Kyoto University | Induced pluripotent stem cells produced with Oct3/4, Klf4 and Sox2 |
US20090227032A1 (en) * | 2005-12-13 | 2009-09-10 | Kyoto University | Nuclear reprogramming factor and induced pluripotent stem cells |
CN101864392B (en) * | 2005-12-13 | 2016-03-23 | 国立大学法人京都大学 | Nuclear reprogramming factor |
US8129187B2 (en) * | 2005-12-13 | 2012-03-06 | Kyoto University | Somatic cell reprogramming by retroviral vectors encoding Oct3/4. Klf4, c-Myc and Sox2 |
EP1971681B1 (en) | 2005-12-16 | 2017-08-23 | DePuy Synthes Products, Inc. | Compositions and methods for inhibiting adverse immune response in histocompatibility-mismatched transplantation |
ES2391034T3 (en) | 2005-12-19 | 2012-11-20 | Ethicon, Inc. | In vitro expansion of postpartum derived cells in rotary bottles |
JP5599568B2 (en) * | 2005-12-28 | 2014-10-01 | エシコン・インコーポレイテッド | Treatment of peripheral vascular disease using postpartum-derived cells |
US9125906B2 (en) | 2005-12-28 | 2015-09-08 | DePuy Synthes Products, Inc. | Treatment of peripheral vascular disease using umbilical cord tissue-derived cells |
DK2420565T3 (en) | 2006-02-23 | 2017-12-04 | Viacyte Inc | APPLICABLE COMPOSITIONS AND METHODS FOR CULTIVATING DIFFERENTIBLE CELLS |
US7695965B2 (en) | 2006-03-02 | 2010-04-13 | Cythera, Inc. | Methods of producing pancreatic hormones |
US11254916B2 (en) | 2006-03-02 | 2022-02-22 | Viacyte, Inc. | Methods of making and using PDX1-positive pancreatic endoderm cells |
EP4112718A1 (en) * | 2006-03-02 | 2023-01-04 | ViaCyte, Inc. | Endocrine precursor cells, pancreatic hormone-expressing cells and methods of production |
JP5468782B2 (en) | 2006-03-02 | 2014-04-09 | エージェンシー フォー サイエンス,テクノロジー アンド リサーチ | Methods for cancer treatment and stem cell regulation |
MX347303B (en) | 2006-03-07 | 2017-04-21 | Shroff Geeta | Compositions comprising human embryonic stem cells and their derivatives, methods of use, and methods of preparation. |
WO2007106351A2 (en) * | 2006-03-10 | 2007-09-20 | The University Of Rochester | Porous silicon materials and devices |
SE0850071L (en) * | 2006-04-10 | 2008-12-19 | Wisconsin Alumni Res Found | Reagents and methods for using human embryonic stem cells to evaluate the toxicity of pharmaceutical compounds and other chemicals. |
EP2010646A2 (en) * | 2006-04-14 | 2009-01-07 | Carnegie Mellon University | Cellular labeling and quantification for nuclear magnetic resonance techniques |
AU2007314614A1 (en) * | 2006-04-14 | 2008-05-08 | Celsense, Inc. | Methods for assessing cell labeling |
CA2646491A1 (en) | 2006-04-17 | 2007-10-25 | Bioe, Inc. | Differentiation of multi-lineage progenitor cells to respiratory epithelial cells |
WO2007127927A2 (en) | 2006-04-28 | 2007-11-08 | Lifescan, Inc. | Differentiation of human embryonic stem cells |
US8741643B2 (en) | 2006-04-28 | 2014-06-03 | Lifescan, Inc. | Differentiation of pluripotent stem cells to definitive endoderm lineage |
BRPI0710949A2 (en) * | 2006-04-28 | 2012-03-06 | Asubio Pharma Co., Ltd. | METHOD FOR INDUCING PLURIPOTENT STEM CELL DIFFERENTIATION IN CARDIOMYOCYTES |
US7989204B2 (en) * | 2006-04-28 | 2011-08-02 | Viacyte, Inc. | Hepatocyte lineage cells |
US8877253B2 (en) * | 2006-05-11 | 2014-11-04 | Regenics As | Cellular extracts |
RU2421208C2 (en) | 2006-05-11 | 2011-06-20 | Ридженикс Ас | Cell and cell extract introduction for rejuvenation |
WO2007139929A2 (en) | 2006-05-25 | 2007-12-06 | The Burnham Institute For Medical Research | Methods for culture and production of single cell populations of human embryonic stem cells |
US20090298169A1 (en) * | 2006-06-02 | 2009-12-03 | The University Of Georgia Research Foundation | Pancreatic and Liver Endoderm Cells and Tissue by Differentiation of Definitive Endoderm Cells Obtained from Human Embryonic Stems |
US8415153B2 (en) | 2006-06-19 | 2013-04-09 | Geron Corporation | Differentiation and enrichment of islet-like cells from human pluripotent stem cells |
WO2007149926A1 (en) * | 2006-06-20 | 2007-12-27 | Wisconsin Alumni Research Foundation | Method for culturing stem cells |
US20080003676A1 (en) * | 2006-06-26 | 2008-01-03 | Millipore Corporation | Growth of embryonic stem cells |
KR101335884B1 (en) | 2006-07-24 | 2013-12-12 | 인터내셔날 스템 셀 코포레이션 | Synthetic cornea from retinal stem cells |
EP2617428A1 (en) | 2006-08-15 | 2013-07-24 | Agency for Science, Technology and Research | Mesenchymal stem cell conditioned medium |
CA2661232A1 (en) * | 2006-08-31 | 2008-03-06 | The University Of Louisville Research Foundation, Inc. | Transcription factors for differentiation of adult human olfactory progenitor cells |
US20100323442A1 (en) * | 2006-10-17 | 2010-12-23 | Emmanuel Edward Baetge | Modulation of the phosphatidylinositol-3-kinase pathway in the differentiation of human embryonic stem cells |
US8685720B2 (en) * | 2006-11-03 | 2014-04-01 | The Trustees Of Princeton University | Engineered cellular pathways for programmed autoregulation of differentiation |
US20080108044A1 (en) * | 2006-11-08 | 2008-05-08 | Deepika Rajesh | In vitro differentiation of hematopoietic cells from primate embryonic stem cells |
EP2087101A2 (en) * | 2006-11-24 | 2009-08-12 | Regents of the University of Minnesota | Endodermal progenitor cells |
US7883698B2 (en) * | 2007-01-17 | 2011-02-08 | Maria Michejda | Isolation and preservation of fetal hematopoietic and mesencymal system cells from non-controversial materials and/or tissues resulting from miscarriages and methods of therapeutic use |
JP2010516240A (en) | 2007-01-18 | 2010-05-20 | スオメン プナイネン リスティ,ヴェリパルベル | Novel carbohydrates from human cells and methods for their analysis and modification |
EP2108043A4 (en) | 2007-01-18 | 2010-04-21 | Suomen Punainen Risti Veripalv | Novel methods and reagents directed to production of cells |
US8084023B2 (en) | 2007-01-22 | 2011-12-27 | The Board Of Trustees Of The University Of Arkansas | Maintenance and propagation of mesenchymal stem cells |
EP2126045A4 (en) * | 2007-01-30 | 2010-05-26 | Univ Georgia | Early mesoderm cells, a stable population of mesendoderm cells that has utility for generation of endoderm and mesoderm lineages and multipotent migratory cells (mmc) |
WO2008121437A2 (en) * | 2007-02-08 | 2008-10-09 | The Burnham Institute For Medical Research | Trophinin-binding peptides and uses thereof |
JP2010518857A (en) | 2007-02-23 | 2010-06-03 | アドバンスド セル テクノロジー, インコーポレイテッド | A highly efficient method for the reprogramming of differentiated cells and the generation of animal and embryonic stem cells from the reprogrammed cells |
CA2684242C (en) | 2007-03-23 | 2019-11-12 | Wisconsin Alumni Research Foundation | Somatic cell reprogramming |
EP2129771B1 (en) | 2007-03-26 | 2018-09-12 | The Government of the U.S.A. as represented by the Secretary of the Department of Health & Human Services | Methods for modulating embryonic stem cell differentiation |
US20080267874A1 (en) * | 2007-03-28 | 2008-10-30 | The Buck Institute For Age Research | Targeted Neuronal And Glial Human Embryonic Stem Cell Line |
CA2683056C (en) * | 2007-04-07 | 2020-03-24 | Whitehead Institute For Biomedical Research | Reprogramming of somatic cells |
US8076295B2 (en) * | 2007-04-17 | 2011-12-13 | Nanotope, Inc. | Peptide amphiphiles having improved solubility and methods of using same |
EP2554661B2 (en) | 2007-04-18 | 2018-02-21 | Hadasit Medical Research Services & Development Limited | Stem cell-derived retinal pigment epithelial cells |
CN101720355A (en) * | 2007-06-07 | 2010-06-02 | 福田惠一 | Method of inducing differentiation into myocardial cells using g-csf |
JP2008307007A (en) * | 2007-06-15 | 2008-12-25 | Bayer Schering Pharma Ag | Human pluripotent stem cell induced from human tissue-originated undifferentiated stem cell after birth |
US9213999B2 (en) * | 2007-06-15 | 2015-12-15 | Kyoto University | Providing iPSCs to a customer |
KR20160005142A (en) | 2007-06-29 | 2016-01-13 | 셀룰러 다이내믹스 인터내셔널, 인코포레이티드 | Automated method and apparatus for embryonic stem cell culture |
CN101978044B (en) * | 2007-07-01 | 2017-10-24 | 生命扫描有限公司 | Single pluripotent stem cell culture |
US9080145B2 (en) * | 2007-07-01 | 2015-07-14 | Lifescan Corporation | Single pluripotent stem cell culture |
US8227610B2 (en) * | 2007-07-10 | 2012-07-24 | Carnegie Mellon University | Compositions and methods for producing cellular labels for nuclear magnetic resonance techniques |
EP2562248B1 (en) | 2007-07-18 | 2021-06-09 | Janssen Biotech, Inc. | Differentiation of human embryonic stem cells |
WO2009015343A2 (en) * | 2007-07-25 | 2009-01-29 | Bioe, Inc. | Differentiation of multi-lineage progenitor cells to chondrocytes |
WO2009048675A1 (en) | 2007-07-31 | 2009-04-16 | Lifescan, Inc. | Pluripotent stem cell differentiation by using human feeder cells |
EP2610336A1 (en) | 2007-07-31 | 2013-07-03 | Lifescan, Inc. | Differentiation of human embryonic stem cells |
US20110105393A1 (en) | 2007-08-16 | 2011-05-05 | Andreas Androutsellis-Theotokis | Methods for promoting stem cell proliferation and survival |
CN101855338B (en) | 2007-08-31 | 2013-07-17 | 怀特黑德生物医学研究所 | WNT pathway stimulation in reprogramming somatic cells |
US10925903B2 (en) | 2007-09-13 | 2021-02-23 | Reprobiogen Inc. | Use of cells derived from first trimester umbilical cord tissue |
US7695963B2 (en) | 2007-09-24 | 2010-04-13 | Cythera, Inc. | Methods for increasing definitive endoderm production |
US20110236971A2 (en) * | 2007-09-25 | 2011-09-29 | Maksym Vodyanyk | Generation of Clonal Mesenchymal Progenitors and Mesenchymal Stem Cell Lines Under Serum-Free Conditions |
UA99152C2 (en) | 2007-10-05 | 2012-07-25 | Этикон, Инкорпорейтед | Repair and regeneration of renal tissue using human umbilical cord tissue-derived cells |
US8623650B2 (en) * | 2007-10-19 | 2014-01-07 | Viacyte, Inc. | Methods and compositions for feeder-free pluripotent stem cell media containing human serum |
EP2214497A4 (en) * | 2007-10-30 | 2010-11-24 | Univ Louisville Res Found | Uses and isolation of very small embryonic-like (vsel) stem cells |
JP5710264B2 (en) | 2007-11-27 | 2015-04-30 | ライフスキャン・インコーポレイテッドLifescan,Inc. | Differentiation of human embryonic stem cells |
EP2235161A1 (en) * | 2007-12-11 | 2010-10-06 | Research Development Foundation | Small molecules for neuronal differentiation of embryonic stem cells |
US8236538B2 (en) | 2007-12-20 | 2012-08-07 | Advanced Technologies And Regenerative Medicine, Llc | Methods for sterilizing materials containing biologically active agents |
CA2712496C (en) | 2008-01-18 | 2021-01-12 | Wei-Shou Hu | Stem cell aggregates and methods for making and using |
EP2240536B1 (en) * | 2008-01-30 | 2019-09-04 | Asterias Biotherapeutics, Inc. | Synthetic surfaces for culturing stem cell derived oligodendrocyte progenitor cells |
KR20110005682A (en) | 2008-01-30 | 2011-01-18 | 제론 코포레이션 | Synthetic surfaces for culturing stem cell derived cardiomyocytes |
US10071185B2 (en) * | 2008-02-07 | 2018-09-11 | Nayacure Therapeutics Ltd. | Compartmental extract compositions for tissue engineering |
US20090202978A1 (en) * | 2008-02-13 | 2009-08-13 | Ginadi Shaham | Method and apparatus for freezing of a biological material |
CA2959401C (en) | 2008-02-21 | 2021-12-07 | Centocor Ortho Biotech Inc. | Methods, surface modified plates and compositions for cell attachment, cultivation and detachment |
US20100087002A1 (en) * | 2008-02-21 | 2010-04-08 | Benjamin Fryer | Methods, Surface Modified Plates and Compositions for Cell Attachment, Cultivation and Detachment |
EP2100954A1 (en) | 2008-03-10 | 2009-09-16 | Assistance Publique - Hopitaux de Paris | Method for generating primate cardiac progenitor cells for clinical use from primate embryonic stem cells, and their applications |
DE102008013854A1 (en) * | 2008-03-12 | 2009-09-24 | Siemens Aktiengesellschaft | Catheter and associated medical examination and treatment facility |
US8716018B2 (en) | 2008-03-17 | 2014-05-06 | Agency For Science, Technology And Research | Microcarriers for stem cell culture |
AU2009225665B9 (en) * | 2008-03-17 | 2015-01-15 | The Scripps Research Institute | Combined chemical and genetic approaches for generation of induced pluripotent stem cells |
US8093049B2 (en) | 2008-03-27 | 2012-01-10 | Geron Corporation | Differentiation of primate pluripotent stem cells to hematopoietic lineage cells |
US8338170B2 (en) | 2008-04-21 | 2012-12-25 | Viacyte, Inc. | Methods for purifying endoderm and pancreatic endoderm cells derived from human embryonic stem cells |
DK2283117T3 (en) | 2008-04-21 | 2014-01-20 | Viacyte Inc | PROCEDURE FOR CLEANING PANCREATIC ENDODERM CELLS DERIVED FROM HUMAN EMBRYONIC STEM CELLS |
US7939322B2 (en) * | 2008-04-24 | 2011-05-10 | Centocor Ortho Biotech Inc. | Cells expressing pluripotency markers and expressing markers characteristic of the definitive endoderm |
US8623648B2 (en) | 2008-04-24 | 2014-01-07 | Janssen Biotech, Inc. | Treatment of pluripotent cells |
CN102027118B (en) | 2008-04-30 | 2014-07-30 | 桑比欧公司 | Neural regenerating cells with alterations in DNA methylation |
US20110110863A1 (en) * | 2008-05-02 | 2011-05-12 | Celsense, Inc. | Compositions and methods for producing emulsions for nuclear magnetic resonance techniques and other applications |
KR101661940B1 (en) | 2008-05-02 | 2016-10-04 | 고쿠리츠 다이가쿠 호진 교토 다이가쿠 | Method of nuclear reprogramming |
WO2009136867A1 (en) * | 2008-05-06 | 2009-11-12 | Agency For Science, Technology And Research | Method of effecting de-differentiation of a cell |
US9394538B2 (en) | 2008-05-07 | 2016-07-19 | Shi-Lung Lin | Development of universal cancer drugs and vaccines |
EP2297298A4 (en) * | 2008-05-09 | 2011-10-05 | Vistagen Therapeutics Inc | Pancreatic endocrine progenitor cells derived from pluripotent stem cells |
EP2300023A2 (en) * | 2008-05-16 | 2011-03-30 | Genelux Corporation | Microorganisms for preventing and treating neoplasms accompanying cellular therapy |
EP2294187A2 (en) * | 2008-05-21 | 2011-03-16 | BioE LLC | Differentiation of multi-lineage progenitor cells to pancreatic cells |
EP2297319B1 (en) | 2008-06-03 | 2015-10-07 | Viacyte, Inc. | Growth factors for production of definitive endoderm |
US20090298178A1 (en) * | 2008-06-03 | 2009-12-03 | D Amour Kevin Allen | Growth factors for production of definitive endoderm |
KR101648019B1 (en) | 2008-06-04 | 2016-08-16 | 셀룰러 다이내믹스 인터내셔널, 인코포레이티드 | Methods for the production of iPS cells using non-viral approach |
US9497943B2 (en) | 2008-06-13 | 2016-11-22 | Whitehead Institute For Biomedical Research | Nucleic acid constructs encoding reprogramming factors linked by self-cleaving peptides |
US8871900B2 (en) | 2008-06-16 | 2014-10-28 | University Of Rochester | Fibroblast growth factor (FGF) analogs and uses thereof |
WO2010008486A2 (en) | 2008-06-24 | 2010-01-21 | Parkinsons Institute | Pluripotent cell lines and methods of use thereof |
EP2942392B1 (en) | 2008-06-30 | 2018-10-03 | Janssen Biotech, Inc. | Differentiation of pluripotent stem cells |
US20110305672A1 (en) | 2008-07-25 | 2011-12-15 | University Of Georgia Research Foundation, Inc. | COMPOSITIONS FOR MESODERM DERIVED ISL1+ MULTIPOTENT CELLS (IMPs), EPICARDIAL PROGENITOR CELLS (EPCs) AND MULTIPOTENT CD56C CELLS (C56Cs) AND METHODS OF PRODUCING AND USING SAME |
US20100028307A1 (en) * | 2008-07-31 | 2010-02-04 | O'neil John J | Pluripotent stem cell differentiation |
AU2009308967C1 (en) | 2008-10-31 | 2017-04-20 | Janssen Biotech, Inc. | Differentiation of human embryonic stem cells to the pancreatic endocrine lineage |
KR101798474B1 (en) * | 2008-10-31 | 2017-11-16 | 얀센 바이오테크 인코포레이티드 | Differentiation of human embryonic stem cells to the pancreatic endocrine lineage |
WO2010053472A1 (en) | 2008-11-04 | 2010-05-14 | Novocell, Inc. | Stem cell aggregate suspension compositions and methods for differentiation thereof |
US8956867B2 (en) * | 2008-11-07 | 2015-02-17 | Wisconsin Alumni Research Foundation | Method for culturing stem cells |
CA3229301A1 (en) * | 2008-11-14 | 2010-05-20 | Viacyte, Inc. | Encapsulation of pancreatic cells derived from human pluripotent stem cells |
BRPI0921996A2 (en) * | 2008-11-20 | 2015-08-18 | Centocor Ortho Biotech Inc | Methods and compositions for culturing and binding cells on flat substrates. |
ES2642070T3 (en) | 2008-11-20 | 2017-11-15 | Janssen Biotech, Inc. | Cultivation of pluripotent stem cells in microcarriers |
BRPI0922572A2 (en) | 2008-12-17 | 2019-09-24 | Scripps Research Inst | method for culturing pluripotent cells, pluripotent mammalian cell culture, cell culture medium, isolated pluripotent animal cell, and method for increasing pluripotence of a mammalian cell. |
CA2747794C (en) | 2008-12-19 | 2018-10-30 | Advanced Technologies And Regenerative Medicine, Llc | Treatment of lung and pulmonary diseases and disorders |
US10179900B2 (en) | 2008-12-19 | 2019-01-15 | DePuy Synthes Products, Inc. | Conditioned media and methods of making a conditioned media |
BRPI0923070A2 (en) * | 2008-12-19 | 2016-06-14 | Atrm Llc | "Uses of compositions for regeneration and repair of neural tissue after injury, said compositions, and kit" |
WO2010071862A1 (en) * | 2008-12-19 | 2010-06-24 | Ethicon, Incorporated | Umbilical cord tissue derived cells for treating neuropathic pain and spasticity |
WO2010075500A1 (en) | 2008-12-23 | 2010-07-01 | Stemcells California, Inc | Target populations of oligodendrocyte precursor cells and methods of making and using same |
WO2010084300A2 (en) | 2009-01-22 | 2010-07-29 | Iti Scotland Limited | Stem cell culture methods |
US20100209399A1 (en) * | 2009-02-13 | 2010-08-19 | Celavie Biosciences, Llc | Brain-derived stem cells for repair of musculoskeletal system in vertebrate subjects |
EP2233566A1 (en) | 2009-03-17 | 2010-09-29 | Vrije Universiteit Brussel | Generation of pancreatic progenitor cells |
ES2629003T3 (en) | 2009-03-26 | 2017-08-07 | DePuy Synthes Products, Inc. | Human umbilical cord tissue cells as a treatment for Alzheimer's disease |
GB0906424D0 (en) | 2009-04-09 | 2009-05-20 | Antoxis Ltd | Use of compounds for differentiation of cells |
WO2010120830A1 (en) * | 2009-04-13 | 2010-10-21 | Northwestern University | Novel peptide-based scaffolds for cartilage regeneration and methods for their use |
US9109245B2 (en) | 2009-04-22 | 2015-08-18 | Viacyte, Inc. | Cell compositions derived from dedifferentiated reprogrammed cells |
US20100272695A1 (en) * | 2009-04-22 | 2010-10-28 | Alan Agulnick | Cell compositions derived from dedifferentiated reprogrammed cells |
US20110002897A1 (en) | 2009-06-11 | 2011-01-06 | Burnham Institute For Medical Research | Directed differentiation of stem cells |
US8193235B2 (en) * | 2009-06-12 | 2012-06-05 | University Of Kansas | Compositions and methods for establishing and maintaining stem cells in an undifferentiated state |
KR20120065966A (en) | 2009-06-12 | 2012-06-21 | 난양 폴리테크닉 | Novel uses |
GB0911060D0 (en) | 2009-06-26 | 2009-08-12 | Ge Healthcare Uk Ltd | Methods for predicting the toxicity of a chemical |
WO2011005326A1 (en) | 2009-07-09 | 2011-01-13 | Massachusetts Institute Of Technology | Methods and compositions for increased safety of stem cell-derived populations |
CA2768643C (en) | 2009-07-20 | 2018-09-18 | Janssen Biotech, Inc. | Differentiation of human embryonic stem cells |
EP2456859A4 (en) * | 2009-07-20 | 2015-03-18 | Janssen Biotech Inc | Differentiation of human embryonic stem cells |
WO2011011302A2 (en) | 2009-07-20 | 2011-01-27 | Centocor Ortho Biotech Inc. | Differentiation of human embryonic stem cells |
CN104962512B (en) | 2009-07-21 | 2022-09-09 | Abt控股公司 | Use of stem cells for reducing leukocyte extravasation |
DK2456860T3 (en) | 2009-07-21 | 2019-01-07 | Abt Holding Co | USE OF STEM CELLS FOR REDUCING LEUKOCYTE EXTRAVASATION |
DK2462230T3 (en) * | 2009-08-03 | 2015-10-19 | Recombinetics Inc | METHODS AND COMPOSITIONS FOR TARGETED RE-MODIFICATION |
SG178365A1 (en) | 2009-08-12 | 2012-04-27 | Univ Kyoto | Method for inducing differentiation of pluripotent stem cells into neural precursor cells |
US20120148546A1 (en) | 2009-08-17 | 2012-06-14 | Technion Research & Development Foundation Ltd. | Pericyte progenitor cells and methods of generating and using same |
US8721521B2 (en) | 2009-08-22 | 2014-05-13 | The Board Of Trustees Of The Leland Stanford Junior University | Imaging and evaluating embryos, oocytes, and stem cells |
AU2010289423B2 (en) | 2009-09-04 | 2014-03-27 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods for enhancing genome stability and telomere elongation in embryonic stem cells |
AU2010306627B2 (en) | 2009-10-16 | 2014-07-17 | The Scripps Research Institute | Induction of pluripotent cells |
WO2011056416A2 (en) | 2009-10-19 | 2011-05-12 | Cellular Dynamics International, Inc. | Cardiomyocyte production |
CN107058389A (en) * | 2009-10-29 | 2017-08-18 | 詹森生物科技公司 | Multipotential stem cell |
EP2498796B1 (en) | 2009-11-09 | 2017-12-27 | AAL Scientifics, Inc. | Treatment of heart disease |
ES2932664T3 (en) | 2009-11-12 | 2023-01-23 | Technion Res & Dev Foundation | Culture media, cell cultures and methods of culturing pluripotent stem cells in the undifferentiated state |
CA2782577A1 (en) * | 2009-12-02 | 2011-06-09 | Research Development Foundation | Selection of stem cell clones with defined differentiation capabilities |
FI20096288A0 (en) | 2009-12-04 | 2009-12-04 | Kristiina Rajala | Formulations and Methods for Culturing Stem Cells |
WO2011072461A1 (en) | 2009-12-18 | 2011-06-23 | Shanghai He Chen Biotechnology Co., Ltd. | Materials and methods for generating pluripotent stem cells |
RU2610176C2 (en) | 2009-12-23 | 2017-02-08 | Янссен Байотек, Инк. | Differentiation of human embryonic stem cells |
CA2784425A1 (en) | 2009-12-23 | 2011-06-30 | Centocor Ortho Biotech Inc. | Differentiation of human embryonic stem cells |
EP2529008A1 (en) | 2010-01-26 | 2012-12-05 | Université Libre de Bruxelles | Tools for isolating and following cardiovascular progenitor cells |
WO2011099007A1 (en) | 2010-02-10 | 2011-08-18 | Nayacure Therapeutics Ltd. | Pharmaceutical compositions and methods for the treatment and prevention of cancer |
BR112012021451B8 (en) | 2010-02-25 | 2021-05-25 | Abt Holding Co | use of cells that have a desired efficiency for expression and/or secretion of the pro-angiogenic factors vegf, cxcl5 and il8, methods to build a cell bank, to develop drug, and to increase the expression of one or more pro-angiogenic factors angiogenic in a cell performed in vitro, and composition |
WO2011106521A1 (en) | 2010-02-25 | 2011-09-01 | Abt Holding Company | Modulation of macrophage activation |
WO2011109279A2 (en) | 2010-03-01 | 2011-09-09 | Centocor Ortho Biotech Inc. | Methods for purifying cells derived from pluripotent stem cells |
US8662085B2 (en) | 2010-03-02 | 2014-03-04 | Siemens Aktiengesellschaft | Magnetic nanoparticle and group of nanoparticles |
AU2011232577C1 (en) | 2010-03-22 | 2016-07-14 | Stemina Biomarker Discovery, Inc. | Predicting human developmental toxicity of pharmaceuticals using human stem-like cells and metabolomics |
CN102906248A (en) | 2010-03-25 | 2013-01-30 | 国际干细胞公司 | Method of altering differentiative state of cell and compositions thereof |
WO2011123572A1 (en) | 2010-03-31 | 2011-10-06 | The Scripps Research Institute | Reprogramming cells |
SG184440A1 (en) | 2010-04-08 | 2012-11-29 | Univ Edinburgh | Chondrogenic progenitor cells, protocol for derivation of cells and uses thereof |
WO2011128897A1 (en) | 2010-04-12 | 2011-10-20 | Technion Research & Development Foundation Ltd. | Populations of pancreatic progenitor cells and methods of isolating and using same |
WO2011133661A2 (en) | 2010-04-21 | 2011-10-27 | Research Development Foundation | Methods and compositions related to dopaminergic neuronal cells |
JP5841322B2 (en) | 2010-04-22 | 2016-01-13 | オレゴン ヘルス アンド サイエンス ユニバーシティ | Fumaryl acetoacetate hydrolase (FAH) deficient pig and use thereof |
DK2561078T3 (en) | 2010-04-23 | 2019-01-14 | Cold Spring Harbor Laboratory | NEW STRUCTURALLY DESIGNED SHRNAs |
WO2011138687A2 (en) | 2010-05-06 | 2011-11-10 | Regenics As | Use of cellular extracts for skin rejuvenation |
CN102242146B (en) * | 2010-05-10 | 2015-11-25 | 高丽大学校产学协力团 | Composition and the method with its generation generate induced pluripotent stem cells |
EP2568991B3 (en) | 2010-05-12 | 2018-11-28 | ABT Holding Company | Modulation of splenocytes in cell therapy |
CN107338217B (en) | 2010-05-12 | 2021-02-09 | 詹森生物科技公司 | Differentiation of human embryonic stem cells |
WO2011159726A2 (en) | 2010-06-14 | 2011-12-22 | The Scripps Research Institute | Reprogramming of cells to a new fate |
WO2011158125A2 (en) | 2010-06-17 | 2011-12-22 | Katholieke Universiteit Leuven | Methods for differentiating cells into hepatic stellate cells and hepatic sinusoidal endothelial cells, cells produced by the methods, and methods for using the cells |
AU2011268056B2 (en) | 2010-06-18 | 2014-04-24 | Cellular Dynamics International, Inc. | Cardiomyocyte medium with dialyzed serum |
JP6660080B2 (en) | 2010-07-01 | 2020-03-04 | リジェネレイティブ リサーチ ファウンデーション | Undifferentiated cell culture method using sustained release composition |
WO2012006440A2 (en) | 2010-07-07 | 2012-01-12 | Cellular Dynamics International, Inc. | Endothelial cell production by programming |
EP2593117B1 (en) | 2010-07-12 | 2019-03-20 | University of Southern California | Biocompatible substrate for facilitating interconnections between stem cells and target tissues and methods for implanting same |
US9121011B2 (en) | 2010-07-21 | 2015-09-01 | Kyoto University | Method for inducing differentiation of human pluripotent stem cell into intermediate mesoderm cell |
WO2012013969A1 (en) | 2010-07-26 | 2012-02-02 | The University Of Manchester | Targeted differentiation of stem cells |
WO2012014207A2 (en) * | 2010-07-27 | 2012-02-02 | Technion Research & Development Foundation Ltd. | Method for generating induced pluripotent stem cells from keratinocytes derived from plucked hair follicles |
US9144585B2 (en) | 2010-07-27 | 2015-09-29 | Technion Research & Development Foundation Limited | Isolated mesenchymal progenitor cells and extracellular matrix produced thereby |
ES2571991T3 (en) | 2010-07-28 | 2016-05-27 | Neuralstem Inc | Procedures for treating and / or reversing diseases and / or neurodegenerative disorders |
KR20190027969A (en) | 2010-08-12 | 2019-03-15 | 얀센 바이오테크 인코포레이티드 | Treatment of diabetes with pancreatic endocrine precursor cells |
CA2838330C (en) | 2010-08-23 | 2021-01-26 | President And Fellows Of Harvard College | Optogenetic probes for measuring membrane potential |
WO2012027474A1 (en) | 2010-08-24 | 2012-03-01 | Regents Of The University Of Minnesota | Non-static suspension culture of cell aggregates |
ES2585028T3 (en) | 2010-08-31 | 2016-10-03 | Janssen Biotech, Inc. | Differentiation of pluripotent stem cells |
CA2809303A1 (en) | 2010-08-31 | 2012-03-08 | Janssen Biotech, Inc. | Differentiation of human embryonic stem cells |
JP6168991B2 (en) | 2010-08-31 | 2017-07-26 | ヤンセン バイオテツク,インコーポレーテツド | Differentiation of human embryonic stem cells |
NZ607547A (en) | 2010-09-01 | 2015-06-26 | Univ Jefferson | Composition and method for muscle repair and regeneration |
DK2614141T3 (en) | 2010-09-07 | 2019-09-09 | Technion Res & Dev Foundation | PRESENT PROCEDURES AND CULTIVATION MEDIA FOR CULTURING PLURIPOTENT STEM CELLS |
US9617511B2 (en) | 2010-09-07 | 2017-04-11 | The Board Of Regents Of The University Of Texas System | Tissue-specific differentiation matrices and uses thereof |
CN103459611B (en) | 2010-09-17 | 2016-11-02 | 哈佛大学校长及研究员协会 | The functional genomics research that effectiveness and the safety of pluripotent stem cell are characterized |
CA2812300A1 (en) | 2010-10-01 | 2012-04-05 | The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services | Manipulation of stem cell function by p53 isoforms |
US8895291B2 (en) | 2010-10-08 | 2014-11-25 | Terumo Bct, Inc. | Methods and systems of growing and harvesting cells in a hollow fiber bioreactor system with control conditions |
WO2012054896A1 (en) | 2010-10-22 | 2012-04-26 | Biotime Inc. | Methods of modifying transcriptional regulatory networks in stem cells |
CA2822638C (en) | 2010-12-22 | 2021-02-16 | Fate Therapeutics, Inc. | Cell culture platform for single cell sorting and enhanced reprogramming of ipscs |
WO2012091978A2 (en) | 2010-12-31 | 2012-07-05 | University Of Georgia Research Foundation, Inc. | Differentiation of human pluripotent stem cells to multipotent neural crest cells |
PL2667878T3 (en) | 2011-01-25 | 2016-10-31 | Compositions and methods for cell transplantation | |
US9102914B2 (en) | 2011-02-03 | 2015-08-11 | Empire Technology Development Llc | 3D trophoblast matrix for preparing organ-specific stem cells |
JP6005666B2 (en) | 2011-02-08 | 2016-10-12 | セルラー ダイナミクス インターナショナル, インコーポレイテッド | Production of hematopoietic progenitor cells by programming |
CA2827945C (en) | 2011-02-23 | 2021-10-12 | The Board Of Trustees Of The Leland Stanford Junior University | Methods of detecting aneuploidy in human embryos |
EP2678425B1 (en) | 2011-02-23 | 2017-08-23 | Kyoto University | Method for producing dendritic cells from pluripotent stem cells |
EP2681555B1 (en) | 2011-02-25 | 2020-04-08 | Benaroya Research Institute | Detection of an allergic disorder |
KR102351944B1 (en) | 2011-02-28 | 2022-01-18 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | Cell culture system |
AU2012225784B2 (en) | 2011-03-04 | 2016-03-17 | The Regents Of The University Of California | Locally released growth factors to mediate motor recovery after stroke |
US20140329314A1 (en) | 2011-03-29 | 2014-11-06 | Christopher O'Sullivan | Enriched populations of cardiomyocyte lineage cells from pluripotent stem cells |
JP5840855B2 (en) | 2011-03-30 | 2016-01-06 | 学校法人東京女子医科大学 | Method for producing myocardial sheet from embryonic stem cells |
JP6185907B2 (en) | 2011-03-30 | 2017-08-23 | セルラー ダイナミクス インターナショナル, インコーポレイテッド | Prestimulation of pluripotent stem cells for neural differentiation |
US8785190B2 (en) | 2011-04-06 | 2014-07-22 | Sanbio, Inc. | Methods and compositions for modulating peripheral immune function |
JP5745919B2 (en) * | 2011-04-28 | 2015-07-08 | 浜松ホトニクス株式会社 | Cell analysis method, cell analysis apparatus, and cell analysis program |
US8877489B2 (en) | 2011-12-05 | 2014-11-04 | California Institute Of Technology | Ultrathin parylene-C semipermeable membranes for biomedical applications |
WO2012149468A2 (en) | 2011-04-29 | 2012-11-01 | University Of Southern California | Instruments and methods for the implantation of cell-seeded substrates |
AU2012266404B2 (en) | 2011-06-06 | 2016-08-11 | ReGenesys BVBA | Expansion of stem cells in hollow fiber bioreactors |
GB201110331D0 (en) | 2011-06-16 | 2011-08-03 | Isis Innovation | Method of cryopreserving pluripotent stem cells |
US10865383B2 (en) | 2011-07-12 | 2020-12-15 | Lineage Cell Therapeutics, Inc. | Methods and formulations for orthopedic cell therapy |
WO2013010965A1 (en) | 2011-07-15 | 2013-01-24 | Universite Libre De Bruxelles | Generation of mesodermal cells from pluripotent stem cells |
CA2841985C (en) | 2011-07-29 | 2019-09-17 | Cellular Dynamics International, Inc. | Metabolic maturation in stem cell-derived tissue cells |
WO2013021389A2 (en) | 2011-08-09 | 2013-02-14 | Yeda Research And Development Co.Ltd. | Downregulation of mir-7 for promotion of beta cell differentiation and insulin production |
EP3578042A1 (en) | 2011-08-26 | 2019-12-11 | Yecuris Corporation | Fumarylacetoacetate hydrolase (fah)-deficient and immunodeficient rats and uses thereof |
WO2013049856A2 (en) | 2011-09-30 | 2013-04-04 | Hare Joshue M | Renal stem cells isolated from kidney |
GB201117469D0 (en) | 2011-10-10 | 2011-11-23 | Cell Guidance Systems Ltd | Culture media for pluripotent stem cells |
EP2770051B1 (en) | 2011-10-21 | 2017-09-27 | ARKRAY, Inc. | Method for culturing pluripotency-maintained singly dispersed cells by means of laminar flow |
GB201119335D0 (en) | 2011-11-09 | 2011-12-21 | Univ Leuven Kath | Hepatitis virus infectable stem cells |
EP2594635A1 (en) | 2011-11-18 | 2013-05-22 | Univercell Biosolutions | Method for generating primate cardiovascular progenitor cells for clinical and drug cells testing use from primate embryonic stem cells or embryonic-like state cells, and their applications |
JP5999658B2 (en) | 2011-11-25 | 2016-09-28 | 国立大学法人京都大学 | Method for culturing pluripotent stem cells |
WO2013082268A1 (en) | 2011-11-30 | 2013-06-06 | The Wistar Institute Of Anatomy And Biology | Methods and compositions for regulation of cell aging, carcinogenesis, and reprogramming |
CA2857295C (en) | 2011-12-01 | 2021-06-29 | New York Stem Cell Foundation | Automated system for producing induced pluripotent stem cells or differentiated cells |
US20150004144A1 (en) | 2011-12-02 | 2015-01-01 | The General Hospital Corporation | Differentiation into brown adipocytes |
US8497124B2 (en) | 2011-12-05 | 2013-07-30 | Factor Bioscience Inc. | Methods and products for reprogramming cells to a less differentiated state |
EP3260140B1 (en) | 2011-12-05 | 2021-02-03 | Factor Bioscience Inc. | Methods and products for transfecting cells |
US9248013B2 (en) | 2011-12-05 | 2016-02-02 | California Institute Of Technology | 3-Dimensional parylene scaffold cage |
JP6143268B2 (en) | 2011-12-19 | 2017-06-07 | 国立大学法人京都大学 | Method for inducing differentiation from human pluripotent stem cells to intermediate mesoderm cells |
KR102203056B1 (en) | 2011-12-22 | 2021-01-14 | 얀센 바이오테크 인코포레이티드 | Differentiation of human embryonic stem cells into single hormonal insulin positive cells |
RU2636220C2 (en) | 2011-12-23 | 2017-11-21 | Депуи Синтез Продактс, Инк. | Detection of cells obtained from umbilical cord tissue |
EP2615176A1 (en) | 2012-01-11 | 2013-07-17 | Paul-Ehrlich-Institut Bundesamt für Sera und Impfstoffe | Novel pseudotyped lentiviral particles and their use in the in vitro targeted transduction of undifferentiated pluripotent human embryonic stem cells and induced pluripotent stem cells |
CA2861068C (en) | 2012-01-13 | 2023-10-24 | The General Hospital Corporation | Isolated human lung progenitor cells and uses thereof |
US9775890B2 (en) | 2012-01-25 | 2017-10-03 | Université Catholique de Louvain | Factor Xa inhibitor used with liver-derived progenitor cells |
EP2809152B1 (en) | 2012-02-01 | 2019-11-20 | Nayacure Therapeutics Ltd | Method for inducing immune tolerance to organ transplants |
EP3401394A1 (en) | 2012-02-22 | 2018-11-14 | Exostem Biotec Ltd | Generation of neural stem cells |
RU2664467C2 (en) | 2012-03-07 | 2018-08-17 | Янссен Байотек, Инк. | Medium with defined composition for propagation and renewal of pluripotent stem cells |
JP6112733B2 (en) | 2012-04-06 | 2017-04-12 | 国立大学法人京都大学 | Method for inducing erythropoietin-producing cells |
SG10201809901SA (en) | 2012-04-24 | 2018-12-28 | Vcell Therapeutics Inc | Generating Pluripotent Cells De Novo |
KR20150004834A (en) | 2012-04-24 | 2015-01-13 | 인터내셔날 스템 셀 코포레이션 | Derivation of neural stem cells and dopaminergic neurons from human pluripotent stem cells |
CA2871795C (en) | 2012-04-30 | 2023-03-14 | University Health Network | Methods and compositions for generating pancreatic progenitors and functional beta cells from hpscs |
JP5432322B2 (en) | 2012-05-08 | 2014-03-05 | 株式会社大塚製薬工場 | Mammalian cell suspension for prevention of pulmonary embolism containing trehalose |
DK2800811T3 (en) | 2012-05-25 | 2017-07-17 | Univ Vienna | METHODS AND COMPOSITIONS FOR RNA DIRECTIVE TARGET DNA MODIFICATION AND FOR RNA DIRECTIVE MODULATION OF TRANSCRIPTION |
US20140017717A1 (en) | 2012-05-31 | 2014-01-16 | Auxogyn, Inc. | In vitro embryo blastocyst prediction methods |
KR102468315B1 (en) | 2012-06-08 | 2022-11-16 | 얀센 바이오테크 인코포레이티드 | Differentiation of human embryonic stem cells into pancreatic endocrine cells |
CA2882028C (en) | 2012-07-31 | 2023-11-07 | Basil M. Hantash | Human leukocyte antigen-g (hla-g) modified cells and methods |
US20150216957A1 (en) | 2012-08-20 | 2015-08-06 | Boris Markosian | Placental vaccination therapy for cancer |
EP2900808B1 (en) | 2012-09-28 | 2019-04-03 | Scripps Health | Methods of differentiating stem cells into chondrocytes |
AU2013237760A1 (en) | 2012-10-08 | 2014-04-24 | Biotime, Inc. | Differentiated progeny of clonal progenitor cell lines |
US9974885B2 (en) | 2012-10-29 | 2018-05-22 | Scripps Health | Methods of transplanting chondrocytes |
EP2912166B1 (en) | 2012-10-29 | 2019-05-01 | Scripps Health | Methods of producing pluripotent stem cells from chondrocytes |
RU2019143431A (en) | 2012-11-01 | 2020-04-28 | Фэктор Байосайенс Инк. | METHODS AND PRODUCTS FOR EXPRESSION OF PROTEINS IN CELLS |
CN104822843B (en) | 2012-11-02 | 2020-01-21 | 施特米纳生物标记研发公司 | Predicting human developmental toxicity of drugs using human stem-like cells and metabolomic ratios |
AU2013248265B2 (en) | 2012-11-08 | 2018-11-01 | Viacyte, Inc. | Scalable primate pluripotent stem cell aggregate suspension culture and differentiation thereof |
WO2014091312A2 (en) | 2012-12-10 | 2014-06-19 | Regenics As | Use of cellular extracts for skin rejuvenation |
EP2938721B1 (en) | 2012-12-28 | 2019-10-16 | Kyoto University | Method for inducing astrocytes |
DK2938723T3 (en) | 2012-12-31 | 2023-02-20 | Janssen Biotech Inc | DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS TO PANCREATIC ENDOCRINE CELLS USING HB9 REGULATORS |
CN105705634A (en) | 2012-12-31 | 2016-06-22 | 詹森生物科技公司 | Suspension and clustering of human pluripotent cells for differentiation into pancreatic endocrine cells |
CN105008518B (en) | 2012-12-31 | 2020-08-07 | 詹森生物科技公司 | Culturing human embryonic stem cells at an air-liquid interface for differentiation into pancreatic endocrine cells |
US10370644B2 (en) | 2012-12-31 | 2019-08-06 | Janssen Biotech, Inc. | Method for making human pluripotent suspension cultures and cells derived therefrom |
US10241108B2 (en) | 2013-02-01 | 2019-03-26 | Ares Trading S.A. | Abnormal syngamy phenotypes observed with time lapse imaging for early identification of embryos with lower development potential |
JP6495658B2 (en) | 2013-02-08 | 2019-04-03 | 国立大学法人京都大学 | Method for producing megakaryocytes and platelets |
WO2014127289A1 (en) | 2013-02-15 | 2014-08-21 | International Stem Cell Corporation | Use of neural cells derived from human pluripotent stem cells for the treatment of neurodegenerative diseases |
CA2901747A1 (en) | 2013-02-22 | 2014-08-28 | Cellular Dynamics International, Inc. | Hepatocyte production via forward programming by combined genetic and chemical engineering |
EP2966163B1 (en) | 2013-03-06 | 2018-01-17 | Kyoto University | Culture system for pluripotent stem cells and method for subculturing pluripotent stem cells |
WO2014143383A1 (en) | 2013-03-13 | 2014-09-18 | Agilent Technologies, Inc. | Transposome tethered to a gene delivery vehicle |
US8859286B2 (en) | 2013-03-14 | 2014-10-14 | Viacyte, Inc. | In vitro differentiation of pluripotent stem cells to pancreatic endoderm cells (PEC) and endocrine cells |
JP2016520291A (en) | 2013-03-14 | 2016-07-14 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Production of medial ganglion progenitor cells in vitro |
EP3736343A1 (en) | 2013-03-15 | 2020-11-11 | Whitehead Institute For Biomedical Research | Cellular discovery platform for neurodegenerative diseases |
JP6473077B2 (en) | 2013-03-21 | 2019-02-20 | 国立大学法人京都大学 | Pluripotent stem cells for inducing neural differentiation |
WO2014157257A1 (en) | 2013-03-25 | 2014-10-02 | 公益財団法人先端医療振興財団 | Cell sorting method |
WO2014165663A1 (en) | 2013-04-03 | 2014-10-09 | Cellular Dynamics International, Inc. | Methods and compositions for culturing endoderm progenitor cells in suspension |
KR102393715B1 (en) | 2013-04-05 | 2022-05-02 | 유니버시티 헬스 네트워크 | Methods and compositions for generating chondrocyte lineage cells and/or cartilage like tissue |
WO2014168585A1 (en) | 2013-04-10 | 2014-10-16 | Agency For Science, Technology And Research | Polycaprolactone microcarriers for stem cell culture and fabrication thereof |
CN105338989B (en) | 2013-04-12 | 2022-04-08 | 赛维里奥·拉弗朗切西卡 | Improvements in organs for transplantation |
US10961508B2 (en) | 2013-04-12 | 2021-03-30 | Kyoto University | Method for inducing alveolar epithelial progenitor cells |
WO2014174470A1 (en) | 2013-04-23 | 2014-10-30 | Yeda Research And Development Co. Ltd. | Isolated naive pluripotent stem cells and methods of generating same |
DK2992088T3 (en) | 2013-04-30 | 2019-11-11 | Univ Leuven Kath | CELL THERAPY FOR MYELODYSPLASTIC SYNDROMES |
US9822342B2 (en) | 2013-05-14 | 2017-11-21 | Kyoto University | Method of efficiently inducing cardiomyocytes |
US9758763B2 (en) | 2013-05-29 | 2017-09-12 | The Regents Of The University Of California | Methods and compositions for somatic cell proliferation and viability |
US10961531B2 (en) | 2013-06-05 | 2021-03-30 | Agex Therapeutics, Inc. | Compositions and methods for induced tissue regeneration in mammalian species |
WO2014200905A2 (en) | 2013-06-10 | 2014-12-18 | President And Fellows Of Harvard College | Early developmental genomic assay for characterizing pluripotent stem cell utility and safety |
EP3008169B1 (en) | 2013-06-10 | 2021-11-10 | Academisch Ziekenhuis Leiden | Differentiation and expansion of endothelial cells from pluripotent stem cells and the in vitro formation of vasculature like structures |
RU2692595C2 (en) | 2013-06-11 | 2019-06-25 | Президент Энд Феллоус Оф Гарвард Колледж | SC-β CELLS AND COMPOSITIONS AND METHODS FOR THEIR CREATION |
EP3020803B1 (en) | 2013-06-11 | 2020-03-11 | Kyoto University | Method for producing renal precursor cells |
JP6493881B2 (en) | 2013-06-12 | 2019-04-03 | 国立大学法人京都大学 | Method for selecting induced pluripotent stem cells and method for inducing differentiation into blood cells |
US9624471B2 (en) | 2013-06-12 | 2017-04-18 | University Of Washington Through Its Center For Commercialization | Methods for maturing cardiomyocytes and uses thereof |
AU2014300400B2 (en) | 2013-06-28 | 2019-11-28 | Otsuka Pharmaceutical Factory,Inc. | Trehalose and dextran-containing solution for transplanting mammalian cells |
US9796962B2 (en) | 2013-08-07 | 2017-10-24 | Kyoto University | Method for generating pancreatic hormone-producing cells |
KR102473199B1 (en) | 2013-08-16 | 2022-12-01 | 예일 유니버시티 | Epithelial cell differentiation of human mesenchymal stromal cells |
CA2921948C (en) | 2013-09-04 | 2019-11-19 | Otsuka Pharmaceutical Factory, Inc. | Method for preparing pluripotent stem cells |
WO2015034012A1 (en) | 2013-09-05 | 2015-03-12 | 国立大学法人京都大学 | New method for inducing dopamine-producing neural precursor cells |
CA2921081A1 (en) | 2013-09-13 | 2015-03-19 | University Health Network | Methods and compositions for generating epicardium cells |
JP6474795B2 (en) | 2013-10-01 | 2019-02-27 | カディマステム リミテッド | Directed differentiation of astrocytes from human pluripotent stem cells for use in drug screening and treatment of amyotrophic lateral sclerosis (ALS) |
WO2015064754A1 (en) | 2013-11-01 | 2015-05-07 | 国立大学法人京都大学 | Novel chondrocyte induction method |
WO2015069736A1 (en) | 2013-11-08 | 2015-05-14 | The Mclean Hospital Corporation | METHODS FOR EFFICIENT GENERATION OF GABAergic INTERNEURONS FROM PLURIPOTENT STEM CELLS |
US9932607B2 (en) | 2013-11-15 | 2018-04-03 | The Board Of Trustees Of The Leland Stanford Junior University | Site-specific integration of transgenes into human cells |
US10633625B2 (en) | 2013-11-16 | 2020-04-28 | Terumo Bct, Inc. | Expanding cells in a bioreactor |
US20160298096A1 (en) | 2013-11-18 | 2016-10-13 | Crispr Therapeutics Ag | Crispr-cas system materials and methods |
JP2017504311A (en) | 2013-12-11 | 2017-02-09 | ファイザー・リミテッドPfizer Limited | Method for generating retinal pigment epithelial cells |
WO2015089277A1 (en) | 2013-12-12 | 2015-06-18 | The Regents Of The University Of California | Methods and compositions for modifying a single stranded target nucleic acid |
EP2896688A1 (en) | 2014-01-20 | 2015-07-22 | Centre National de la Recherche Scientifique (CNRS) | A method of producing beta pancreatic cells from progenitor cells through the use of hydrogen peroxide |
WO2015112839A1 (en) | 2014-01-23 | 2015-07-30 | President And Fellows Of Harvard College | Engineered polymeric valves, tubular structures, and sheets and uses thereof |
US9770489B2 (en) | 2014-01-31 | 2017-09-26 | Factor Bioscience Inc. | Methods and products for nucleic acid production and delivery |
WO2015119642A1 (en) | 2014-02-10 | 2015-08-13 | The Johns Hopkins University | Low oxygen tension enhances endothelial fate of human pluripotent stem cells |
US11078462B2 (en) | 2014-02-18 | 2021-08-03 | ReCyte Therapeutics, Inc. | Perivascular stromal cells from primate pluripotent stem cells |
KR102340553B1 (en) | 2014-03-04 | 2021-12-21 | 페이트 세러퓨틱스, 인코포레이티드 | Improved reprogramming methods and cell culture platforms |
EP3119879B1 (en) | 2014-03-19 | 2019-12-25 | INSERM (Institut National de la Santé et de la Recherche Médicale) | A method for inducing human cholangiocyte differentiation |
BR112016021395A8 (en) | 2014-03-19 | 2021-07-13 | Vcell Therapeutics Inc | methods of generating a pluripotent cell and preparing a test cell or tissue, using a pluripotent cell, and composition |
WO2015140005A1 (en) | 2014-03-19 | 2015-09-24 | Ifom Fondazione Istituto Firc Di Oncologia Molecolare | Method of generation of pluripotent cells |
WO2015143350A1 (en) | 2014-03-20 | 2015-09-24 | Auxogyn, Inc. | Quantitative measurement of human blastocyst and morula morphology developmental kinetics |
US9694036B2 (en) | 2014-03-21 | 2017-07-04 | Cellular Dynamics International, Inc. | Production of midbrain dopaminergic neurons and methods for the use thereof |
WO2015164228A1 (en) | 2014-04-21 | 2015-10-29 | Cellular Dynamics International, Inc. | Hepatocyte production via forward programming by combined genetic and chemical engineering |
EP3954759A1 (en) | 2014-05-16 | 2022-02-16 | Janssen Biotech, Inc. | Use of small molecules to enhance mafa expression in pancreatic endocrine cells |
US20170095512A1 (en) | 2014-06-02 | 2017-04-06 | Kadimastem Ltd. | Methods of inducing myelination and maturation of oligodendrocytes |
US10240127B2 (en) | 2014-07-03 | 2019-03-26 | ReCyte Therapeutics, Inc. | Exosomes from clonal progenitor cells |
TWI719939B (en) | 2014-07-14 | 2021-03-01 | 日商中外製藥股份有限公司 | Method of identifying protein epitopes |
CN108064274A (en) | 2014-07-30 | 2018-05-22 | 耶达研究及发展有限公司 | For cultivating the culture medium of multipotential stem cell |
WO2016025510A1 (en) | 2014-08-12 | 2016-02-18 | Rappolee Daniel A | Systems and methods to detect stem cell stress and uses thereof |
EP3183337B1 (en) | 2014-08-22 | 2019-02-06 | Procella Therapeutics AB | Use of jagged 1/frizzled 4 as a cell surface marker for isolating human cardiac ventricular progenitor cells |
US10596200B2 (en) | 2014-08-22 | 2020-03-24 | Procella Therapeutics Ab | Use of LIFR or FGFR3 as a cell surface marker for isolating human cardiac ventricular progenitor cells |
EP3188763B1 (en) | 2014-09-02 | 2020-05-13 | The Regents of The University of California | Methods and compositions for rna-directed target dna modification |
US11261453B2 (en) | 2014-09-12 | 2022-03-01 | Whitehead Institute For Biomedical Research | Cells expressing apolipoprotein E and uses thereof |
KR102617137B1 (en) | 2014-09-15 | 2023-12-27 | 칠드런'즈 메디컬 센터 코포레이션 | Methods and compositions to increase somatic cell nuclear transfer (scnt) efficiency by removing histone h3-lysine trimethylation |
US9371516B2 (en) | 2014-09-19 | 2016-06-21 | Regenerative Medical Solutions, Inc. | Compositions and methods for differentiating stem cells into cell populations comprising beta-like cells |
EP3207122B1 (en) | 2014-10-14 | 2019-05-08 | FUJIFILM Cellular Dynamics, Inc. | Generation of keratinocytes from pluripotent stem cells and maintenance of keratinocyte cultures |
KR102100021B1 (en) | 2014-10-20 | 2020-04-13 | 뉴럴스템, 인크. | Stable neural stem cells comprising an exogenous polynucleotide coding for a growth factor and methods of use thereof |
WO2016063986A1 (en) | 2014-10-24 | 2016-04-28 | 大日本住友製薬株式会社 | Production method for retinal tissue |
EP3220945A2 (en) | 2014-11-17 | 2017-09-27 | Yeda Research and Development Co., Ltd. | Methods of treating diseases related to mitochondrial function |
GB2548740A (en) | 2014-11-25 | 2017-09-27 | Harvard College | Methods for generation of podocytes from pluripotent stem cells and cells produced by the same |
WO2016100035A1 (en) | 2014-12-19 | 2016-06-23 | Janssen Biotech, Inc. | Suspension culturing of pluripotent stem cells |
WO2016103269A1 (en) | 2014-12-23 | 2016-06-30 | Ramot At Tel-Aviv University Ltd. | Populations of neural progenitor cells and methods of producing and using same |
AU2015373050B2 (en) | 2014-12-30 | 2022-09-29 | Cell Cure Neurosciences Ltd. | RPE cell populations and methods of generating same |
EP3240890B1 (en) | 2014-12-30 | 2021-06-16 | Cell Cure Neurosciences Ltd. | Assessing retinal pigment epithelial cell populations |
WO2016118824A1 (en) | 2015-01-22 | 2016-07-28 | Regenerative Medical Solutions, Inc. | Markers for differentiation of stem cells into differentiated cell populations |
JP7199809B2 (en) | 2015-02-13 | 2023-01-06 | ファクター バイオサイエンス インコーポレイテッド | Nucleic acid product and its administration method |
CA2976283C (en) | 2015-02-17 | 2022-04-26 | University Health Network | Methods for making and using sinoatrial node-like pacemaker cardiomyocytes and ventricular-like cardiomyocytes |
SI3059307T2 (en) | 2015-02-20 | 2022-08-31 | Inserm(Institut National De La Sante Et De La Recherche Medicale) | Use of a laminin for differentiating pluripotent cells into hepatocyte lineage cells |
JP6738572B2 (en) | 2015-03-06 | 2020-08-12 | 国立大学法人京都大学 | Method for inducing differentiation of alveolar epithelial cells |
CN107530379A (en) | 2015-03-11 | 2018-01-02 | 佩特尼资源有限公司 | For treating the pancreatic endocrine progenitor cell therapy of obesity and diabetes B (T2D) |
WO2016185457A1 (en) | 2015-05-19 | 2016-11-24 | Yeda Research And Development Co. Ltd. | Methods of promoting lymphangiogenesis |
EP3327118B1 (en) | 2015-07-17 | 2021-12-29 | Kyoto University | Method for inducing vascular endothelial cells |
EP3328995B1 (en) | 2015-07-29 | 2021-03-31 | Hadasit Medical Research Services And Development | Large scale production of retinal pigment epithelial cells |
CA2993912A1 (en) | 2015-08-05 | 2017-02-09 | Cell Cure Neurosciences Ltd. | Preparation of retinal pigment epithelium cells |
EP3331995A1 (en) | 2015-08-05 | 2018-06-13 | Cell Cure Neurosciences Ltd. | Preparation of photoreceptors for the treatment of retinal diseases |
US11312940B2 (en) | 2015-08-31 | 2022-04-26 | University Of Louisville Research Foundation, Inc. | Progenitor cells and methods for preparing and using the same |
WO2017044488A1 (en) | 2015-09-08 | 2017-03-16 | Cellular Dynamics International, Inc. | Macs-based purification of stem cell-derived retinal pigment epithelium |
WO2017044483A1 (en) | 2015-09-08 | 2017-03-16 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Method for reproducible differentiation of clinical-grade retinal pigment epithelium cells |
US11649432B2 (en) | 2015-09-08 | 2023-05-16 | Sumitomo Pharma Co., Ltd. | Method for producing retinal pigment epithelial cells |
ES2897974T3 (en) | 2015-09-11 | 2022-03-03 | Astellas Pharma Inc | Method for producing kidney progenitor cells |
CA2998287A1 (en) | 2015-09-24 | 2017-04-20 | Crispr Therapeutics Ag | Novel family of rna-programmable endonucleases and their uses in genome editing and other applications |
AU2016338680B2 (en) | 2015-10-16 | 2022-11-17 | Fate Therapeutics, Inc. | Platform for the induction and maintenance of ground state pluripotency |
JP6800987B2 (en) | 2015-10-19 | 2020-12-16 | フジフィルム セルラー ダイナミクス,インコーポレイテッド | Production of virus-accepting pluripotent stem cell (PSC) -derived hepatocytes |
AU2016342179B2 (en) | 2015-10-20 | 2022-08-18 | FUJIFILM Cellular Dynamics, Inc. | Multi-lineage hematopoietic precursor cell production by genetic programming |
JP6987769B2 (en) | 2015-10-26 | 2022-01-05 | セル キュア ニューロサイエンシズ リミテッド | Preparation method of retinal pigment epithelial cells |
IL258899B2 (en) | 2015-10-30 | 2024-03-01 | Univ California | Methods of generating t-cells from stem cells and immunotherapeutic methods using the t-cells |
SG11201804117XA (en) | 2015-11-18 | 2018-06-28 | Univ Georgia | Neural cell extracellular vessicles |
CN108779435B (en) | 2015-12-07 | 2022-05-03 | 再生疗法有限公司 | Method for the re-derivation of different pluripotent stem cell-derived brown adipocytes |
WO2017117333A1 (en) | 2015-12-30 | 2017-07-06 | Cellular Dynamics International, Inc. | Microtissue formation using stem cell-derived human hepatocytes |
WO2017172086A1 (en) | 2016-02-19 | 2017-10-05 | Leung Chuen Yan | Genetic markers for engraftment of human cardiac ventricular progenitor cells |
EP3423158B1 (en) | 2016-02-24 | 2023-11-15 | The Rockefeller University | Embryonic cell-based therapeutic candidate screening systems, models for huntington's disease and uses thereof |
WO2017152073A1 (en) | 2016-03-04 | 2017-09-08 | University Of Louisville Research Foundation, Inc. | Methods and compositions for ex vivo expansion of very small embryonic-like stem cells (vsels) |
WO2017153982A1 (en) | 2016-03-06 | 2017-09-14 | Yeda Research And Development Co. Ltd. | Method for modulating myelination |
US11534466B2 (en) | 2016-03-09 | 2022-12-27 | Aal Scientifics, Inc. | Pancreatic stem cells and uses thereof |
US20210155895A1 (en) | 2016-04-04 | 2021-05-27 | Lineage Cell Therapeutics, Inc. | Pluripotent Stem Cell-Derived 3D Retinal Tissue and Uses Thereof |
MA45479A (en) | 2016-04-14 | 2019-02-20 | Janssen Biotech Inc | DIFFERENTIATION OF PLURIPOTENT STEM CELLS IN ENDODERMAL CELLS OF MIDDLE INTESTINE |
SG11201809279YA (en) | 2016-04-22 | 2018-11-29 | Univ Kyoto | Method for producing dopamine-producing neural precursor cells |
WO2017202814A1 (en) | 2016-05-24 | 2017-11-30 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for the treatment of neuropathological disorders characterized by a loss of cortical neurons |
CA3025057A1 (en) | 2016-05-25 | 2017-11-30 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Methods and compositions for treating cancers |
US20200325449A1 (en) | 2016-06-02 | 2020-10-15 | The Cleveland Clinic Foundation | Complement inhibition for improving cell viability |
WO2017216771A2 (en) | 2016-06-17 | 2017-12-21 | Genesis Technologies Limited | Crispr-cas system, materials and methods |
MA45502A (en) | 2016-06-21 | 2019-04-24 | Janssen Biotech Inc | GENERATION OF FUNCTIONAL BETA CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS WITH GLUCOSE-DEPENDENT MITOCHONDRIAL RESPIRATION AND TWO-PHASE INSULIN SECRETION RESPONSE |
EP3478824A1 (en) | 2016-07-01 | 2019-05-08 | Centre National de la Recherche Scientifique - CNRS | Amplifying beta cell differentiation with small molecules bet (bromodomain and extraterminal family of bromodomain-containing proteins) inhibitors |
CA3029582A1 (en) | 2016-07-01 | 2018-01-04 | Research Development Foundation | Elimination of proliferating cells from stem cell-derived grafts |
ES2901379T3 (en) | 2016-08-16 | 2022-03-22 | Fujifilm Cellular Dynamics Inc | Methods to differentiate pluripotent cells |
CN116115629A (en) | 2016-08-17 | 2023-05-16 | 菲克特生物科学股份有限公司 | Nucleic acid products and methods of administration thereof |
JP7248571B2 (en) | 2016-10-05 | 2023-03-29 | フジフィルム セルラー ダイナミクス,インコーポレイテッド | Generation of mature lineages from MeCP2-disrupted induced pluripotent stem cells |
JP2019533703A (en) | 2016-11-02 | 2019-11-21 | エーエーエル サイエンティフィックス,インコーポレイテッド | Non-mesenchymal human lung stem cells and methods for their use to treat respiratory diseases |
JP7006943B2 (en) | 2016-11-04 | 2022-01-24 | 国立大学法人 東京大学 | Mesenchymal cells and mesenchymal stem cells cryopreservation solution, frozen product, and cryopreservation method |
JP7088564B2 (en) | 2016-11-10 | 2022-06-21 | ヴィアサイト,インコーポレイテッド | PDX1 pancreatic endoderm cells and methods thereof in a cell delivery device |
CN109996873A (en) | 2016-11-25 | 2019-07-09 | 国立研究开发法人理化学研究所 | Transplanting cell mass and its manufacturing method |
US10508263B2 (en) | 2016-11-29 | 2019-12-17 | Procella Therapeutics Ab | Methods for isolating human cardiac ventricular progenitor cells |
AU2017377309B9 (en) | 2016-12-14 | 2021-06-03 | Otsuka Pharmaceutical Factory, Inc. | Mammalian cell cryopreservation liquid |
US10828330B2 (en) | 2017-02-22 | 2020-11-10 | IO Bioscience, Inc. | Nucleic acid constructs comprising gene editing multi-sites and uses thereof |
WO2018164240A1 (en) | 2017-03-08 | 2018-09-13 | 大日本住友製薬株式会社 | Method for producing retinal pigment epithelial cells |
JP2020511539A (en) | 2017-03-16 | 2020-04-16 | リネージ セル セラピューティクス インコーポレイテッド | Method to measure the therapeutic effect of retinal disease treatment |
EP3601530A1 (en) | 2017-03-20 | 2020-02-05 | IFOM Fondazione Istituto Firc di Oncologia Molecolare | Method of generating 2 cell-like stem cells |
EP3612557B1 (en) | 2017-04-18 | 2022-01-19 | FUJIFILM Cellular Dynamics, Inc. | Antigen-specific immune effector cells |
AU2018255975A1 (en) | 2017-04-20 | 2019-08-15 | Oregon Health & Science University | Human gene correction |
WO2018235583A1 (en) | 2017-06-19 | 2018-12-27 | 公益財団法人神戸医療産業都市推進機構 | Method for predicting differentiation ability of pluripotent stem cell, and reagent for same |
KR20200029479A (en) | 2017-07-20 | 2020-03-18 | 고쿠리쓰 겐큐 가이하쓰 호징 리가가쿠 겐큐소 | Methods of preservation of nerve tissue |
CN110945119A (en) | 2017-07-20 | 2020-03-31 | 国立研究开发法人理化学研究所 | Method for maturation of retinal tissue comprising continuous epithelium |
AU2018308976A1 (en) | 2017-07-31 | 2020-02-20 | Biotime, Inc. | Compositions and methods for restoring or preventing loss of vision caused by disease or traumatic injury |
EP3663393A1 (en) | 2017-08-23 | 2020-06-10 | Procella Therapeutics AB | Use of neuropilin-1 (nrp1) as a cell surface marker for isolating human cardiac ventricular progenitor cells |
SG11202001889YA (en) | 2017-09-08 | 2020-03-30 | Riken | Cell aggregate including retinal tissue and production method therefor |
WO2019054514A1 (en) | 2017-09-14 | 2019-03-21 | 国立研究開発法人理化学研究所 | Method for producing retinal tissues |
EP3683304A4 (en) | 2017-09-14 | 2021-06-09 | Riken | Method for amplifying cone photoreceptors or rod photoreceptors using dorsalization signal transmitter or ventralization signal transmitter |
US20210363521A1 (en) | 2017-11-09 | 2021-11-25 | Vertex Pharmaceuticals Incorporated | CRISPR/CAS Systems For Treatment of DMD |
US11618884B2 (en) | 2017-11-14 | 2023-04-04 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Regulatory T cells genetically modified for the lymphotoxin alpha gene and uses thereof |
US11679148B2 (en) | 2017-11-24 | 2023-06-20 | Institut National De La Santé Et De La Recherche Médicale (Inserm) | Methods and compositions for treating cancers |
KR20200088880A (en) | 2017-11-24 | 2020-07-23 | 스미또모 가가꾸 가부시끼가이샤 | A method for producing a cell mass comprising nerve cells/tissue and non-nerve epithelial tissue, and a cell mass therefrom |
AU2018373588A1 (en) | 2017-11-24 | 2020-07-02 | Sumitomo Chemical Company, Limited | Method for producing cell mass including pituitary tissue, and cell mass thereof |
EP3724332A1 (en) | 2017-12-14 | 2020-10-21 | CRISPR Therapeutics AG | Novel rna-programmable endonuclease systems and their use in genome editing and other applications |
EP4000568A1 (en) | 2017-12-29 | 2022-05-25 | Cell Cure Neurosciences Ltd. | Retinal pigment epithelium cell compositions |
EP3757208A4 (en) | 2018-02-19 | 2021-12-01 | Sumitomo Dainippon Pharma Co., Ltd. | Cell aggregate, mixture of cell aggregates, and method for preparing same |
US20210054353A1 (en) | 2018-03-19 | 2021-02-25 | Crispr Therapeutics Ag | Novel rna-programmable endonuclease systems and uses thereof |
CA3095717A1 (en) | 2018-03-30 | 2019-10-03 | Takeda Pharmaceutical Company Limited | Heterocyclic compound |
US20210009956A1 (en) | 2018-03-30 | 2021-01-14 | Kyoto University | Cardiomyocyte maturation promoter |
AU2019256723A1 (en) | 2018-04-20 | 2020-11-05 | FUJIFILM Cellular Dynamics, Inc. | Method for differentiation of ocular cells and use thereof |
WO2019213276A1 (en) | 2018-05-02 | 2019-11-07 | Novartis Ag | Regulators of human pluripotent stem cells and uses thereof |
EP3572512A1 (en) | 2018-05-24 | 2019-11-27 | B.R.A.I.N. Ag | A method for engineering a protein |
WO2020006273A1 (en) | 2018-06-27 | 2020-01-02 | Juvena Therapeutics, Inc. | Heparin-associated polypeptides and uses thereof |
EP3824912A4 (en) | 2018-07-19 | 2022-04-20 | Kyoto University | Plate-shaped cartilage derived from pluripotent stem cells and method for producing plate-shaped cartilage |
EP3828262A4 (en) | 2018-07-23 | 2022-03-30 | Kyoto University | Novel renal progenitor cell marker and method for concentrating renal progenitor cells using same |
EP3833383A1 (en) | 2018-08-06 | 2021-06-16 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for treating cancers |
EP3833365A4 (en) | 2018-08-10 | 2022-05-11 | Vertex Pharmaceuticals Incorporated | Stem cell derived islet differentiation |
CA3110464A1 (en) | 2018-08-24 | 2020-02-27 | Sumitomo Chemical Company, Limited | Cell cluster including olfactory neuron or precursor cell thereof, and method for producing same |
SG11202102876XA (en) | 2018-09-28 | 2021-04-29 | Otsuka Pharma Factory Inc | Mammal cell preserving solution containing acarbose or stachyose |
JP7437766B2 (en) | 2018-10-31 | 2024-02-26 | 国立大学法人京都大学 | Method for producing pluripotent stem cells that are free from mesendoderm differentiation resistance |
JP7410518B2 (en) | 2018-11-15 | 2024-01-10 | Jsr株式会社 | Method for producing brain organoids |
JP2022513073A (en) | 2018-11-19 | 2022-02-07 | ザ ユナイテッド ステイツ オブ アメリカ アズ リプリゼンテッド バイ ザ セクレタリー、デパートメント オブ ヘルス アンド ヒューマン サービシーズ | Biodegradable tissue replacement implants and their use |
WO2020113025A1 (en) | 2018-11-28 | 2020-06-04 | Milica Radisic | Methods for tissue generation |
JP7446242B2 (en) | 2018-12-20 | 2024-03-08 | 住友化学株式会社 | Cell population containing embryonic erythroblasts and its production method, cell culture composition, and compound testing method |
WO2020130147A1 (en) | 2018-12-21 | 2020-06-25 | 国立大学法人京都大学 | Lubricin-localized cartilage-like tissue, method for producing same and composition comprising same for treating articular cartilage damage |
WO2020138430A1 (en) | 2018-12-28 | 2020-07-02 | 国立研究開発法人理化学研究所 | Therapeutic drug for disease accompanied by disorders in retinal system cells or retinal tissue |
EP3914698A1 (en) | 2019-01-23 | 2021-12-01 | Yeda Research and Development Co. Ltd | Culture media for pluripotent stem cells |
US20220104481A1 (en) | 2019-02-13 | 2022-04-07 | Tigenix, S.A.U. | Cryopreservation of stem cells |
CA3127851A1 (en) | 2019-02-27 | 2020-09-03 | Tigenix, S.A.U. | Improved stem cell populations for allogeneic therapy |
CA3130789A1 (en) | 2019-03-07 | 2020-09-10 | The Regents Of The University Of California | Crispr-cas effector polypeptides and methods of use thereof |
WO2020209959A1 (en) | 2019-03-08 | 2020-10-15 | Crispr Therapeutics Ag | Nucleobase-editing fusion protein systems, compositions, and uses thereof |
US20220145274A1 (en) | 2019-03-12 | 2022-05-12 | Crispr Therapeutics Ag | Novel high fidelity rna-programmable endonuclease systems and uses thereof |
CN113557311A (en) | 2019-03-13 | 2021-10-26 | 大日本住友制药株式会社 | Quality evaluation method for neural retina for transplantation and neural retina sheet for transplantation |
KR20210144793A (en) | 2019-03-29 | 2021-11-30 | 고리츠다이가쿠호진 요코하마시리츠다이가쿠 | Screening Methods and Toxicity Assessment Methods |
US20220192178A1 (en) | 2019-04-26 | 2022-06-23 | Otsuka Pharmaceutical Factory, Inc. | Trehalose-containing liquid for mammalian cell preservation |
JPWO2020218480A1 (en) | 2019-04-26 | 2020-10-29 | ||
WO2020225606A1 (en) | 2019-05-08 | 2020-11-12 | Crispr Therapeutics Ag | Crispr/cas all-in-two vector systems for treatment of dmd |
JP2022534555A (en) | 2019-05-09 | 2022-08-02 | フジフィルム セルラー ダイナミクス,インコーポレイテッド | Method for producing hepatocyte |
US20220259561A1 (en) | 2019-05-14 | 2022-08-18 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Regulatory t cells targeted by lymphotoxin alpha blocking agent and uses thereof |
EP3973049A1 (en) | 2019-05-22 | 2022-03-30 | Hadasit Medical Research Services and Development Ltd. | Methods of culturing human pluripotent cells |
EP3754014A1 (en) | 2019-06-21 | 2020-12-23 | Centre d'Etude des Cellules Souches (CECS) | Automated method for preparing retinal pigment epithelium cells |
WO2020264072A1 (en) | 2019-06-25 | 2020-12-30 | Semma Therapeutics, Inc. | Enhanced differentiation of beta cells |
JP7385244B2 (en) | 2019-06-27 | 2023-11-22 | 国立大学法人 東京大学 | Method for isolating pancreatic progenitor cells |
US10501404B1 (en) | 2019-07-30 | 2019-12-10 | Factor Bioscience Inc. | Cationic lipids and transfection methods |
WO2021030424A1 (en) | 2019-08-13 | 2021-02-18 | Semma Therapeutics, Inc. | Pancreatic differentiation |
JPWO2021045217A1 (en) | 2019-09-06 | 2021-03-11 | ||
WO2021069593A1 (en) | 2019-10-09 | 2021-04-15 | INSERM (Institut National de la Santé et de la Recherche Médicale) | T cells modified to express mutated cxcr4 or partially deleted and uses thereof |
CN115175989A (en) | 2019-11-20 | 2022-10-11 | 住友制药株式会社 | Method for freezing nerve cells |
CN115003156A (en) | 2019-11-20 | 2022-09-02 | 住友制药株式会社 | Method for freezing cell aggregates |
KR20220106975A (en) | 2019-11-25 | 2022-08-01 | 고쿠리츠 다이가쿠 호진 교토 다이가쿠 | T-Cell Master Cell Bank |
WO2021105407A1 (en) | 2019-11-29 | 2021-06-03 | Novadip Biosciences | miRNA-BASED PHARMACEUTICAL COMPOSITIONS AND USES THEREOF FOR THE PREVENTION AND THE TREATMENT OF TISSUE DISORDERS |
TW202136500A (en) | 2019-11-29 | 2021-10-01 | 比利時商諾瓦迪生物科學公司 | Biomaterials for the prevention and the treatment of tissue disorders |
WO2021117886A1 (en) | 2019-12-12 | 2021-06-17 | 国立大学法人千葉大学 | Freeze-dried preparation containing megakaryocytes and platelets |
EP3875581A1 (en) | 2020-03-02 | 2021-09-08 | Centre d'Etude des Cellules Souches (CECS) | Automated method for preparing keratinocytes |
EP3875580A1 (en) | 2020-03-02 | 2021-09-08 | Centre d'Etude des Cellules Souches (CECS) | Methods for preparing keratinocytes |
CN115605581A (en) | 2020-03-13 | 2023-01-13 | 戈利弗疗法公司(Fr) | Liver stem cell-like cells for the treatment and/or prevention of fulminant liver diseases |
BR112022018640A2 (en) | 2020-03-19 | 2022-11-08 | Orizuru Therapeutics Inc | METHODS FOR PRODUCING A CELL POPULATION COMPRISING CARDIOMYOCYTES AND FOR PURIFYING CARDIOMYOCYTES, CELL POPULATION CONTAINING CARDIOMYOCYTES, AND, AGENT FOR CELL TRANSPLANTATION THERAPY |
US20230212519A1 (en) | 2020-03-19 | 2023-07-06 | Orizuru Therapeutics, Inc. | Method for purifying cardiomyocytes |
EP4132479A1 (en) | 2020-04-07 | 2023-02-15 | Ramot at Tel-Aviv University Ltd. | Cannabidiol-containing compositions and uses thereof |
MX2022015012A (en) | 2020-05-29 | 2023-03-03 | Fujifilm Cellular Dynamics Inc | Retinal pigmented epithelium and photoreceptor dual cell aggregates and methods of use thereof. |
BR112022024064A2 (en) | 2020-05-29 | 2023-01-31 | Fujifilm Cellular Dynamics Inc | RETINAL PIGMENTED EPITHELIUM BILAYER AND PHOTORECEPTORS AND USE THEREOF |
US20230235319A1 (en) | 2020-06-12 | 2023-07-27 | Bayer Aktiengesellschaft | Crispr-cas12a directed random mutagenesis agents and methods |
WO2022014604A1 (en) | 2020-07-13 | 2022-01-20 | 国立大学法人京都大学 | Skeletal muscle precursor cells and method for purifying same, composition for treating myogenic diseases, and method for producing cell group containing skeletal muscle precursor cells |
JPWO2022054925A1 (en) | 2020-09-11 | 2022-03-17 | ||
EP4201428A1 (en) | 2020-09-11 | 2023-06-28 | Sumitomo Pharma Co., Ltd. | Medium for tissue for transplantation |
WO2022063224A1 (en) | 2020-09-24 | 2022-03-31 | 中国科学院动物研究所 | Activated pluripotent stem cell, and preparation method therefor and use thereof |
US20230399622A1 (en) | 2020-10-16 | 2023-12-14 | Fundació Centre De Regulació Genòmica | Therapy for degenerative disease and tissue damage |
EP4005577A1 (en) | 2020-11-26 | 2022-06-01 | Novadip Biosciences | Cellular and/or extracellular extracts for preventing and/or treating cancer and/or inflammation |
CN112538458A (en) | 2020-11-26 | 2021-03-23 | 北京赛尔湃腾科技咨询合伙企业(有限合伙) | Method for reprogramming cells |
KR20230079442A (en) | 2020-12-23 | 2023-06-07 | 미쓰이 가가쿠 가부시키가이샤 | Culture member and use thereof |
JPWO2022191216A1 (en) | 2021-03-09 | 2022-09-15 | ||
WO2022207889A1 (en) | 2021-04-01 | 2022-10-06 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Liver organoid manufacturing methods, liver organoids obtained with the same, and uses thereof |
EP4319876A1 (en) | 2021-04-07 | 2024-02-14 | Fujifilm Cellular Dynamics, Inc. | Dopaminergic precursor cells and methods of use |
AU2022258097A1 (en) | 2021-04-11 | 2023-11-02 | President And Fellows Of Harvard College | Cardiomyocytes and compositions and methods for producing the same |
KR20240005837A (en) | 2021-05-03 | 2024-01-12 | 아스텔라스 인스티튜트 포 리제너러티브 메디슨 | Method for generating mature corneal endothelial cells |
KR20240005887A (en) | 2021-05-07 | 2024-01-12 | 아스텔라스 인스티튜트 포 리제너러티브 메디슨 | How to Generate Mature Hepatocytes |
AU2022280062A1 (en) | 2021-05-28 | 2023-11-30 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods to generate macular, central and peripheral retinal pigment epithelial cells |
AU2022282379A1 (en) | 2021-05-28 | 2023-11-30 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Biodegradable tissue scaffold with secondary matrix to host weakly adherent cells |
WO2022258511A1 (en) | 2021-06-07 | 2022-12-15 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for generating highly functional hepatocytes by differentiating hepatoblasts |
WO2022261320A1 (en) | 2021-06-09 | 2022-12-15 | Lineage Cell Therapeutics, Inc. | Methods and compositions for treating retinal diseases and conditions |
EP4101928A1 (en) | 2021-06-11 | 2022-12-14 | Bayer AG | Type v rna programmable endonuclease systems |
KR20240021218A (en) | 2021-06-11 | 2024-02-16 | 바이엘 악티엔게젤샤프트 | Novel type V RNA programmable endonuclease system |
IL309344A (en) | 2021-06-17 | 2024-02-01 | Univ Kyoto | Method for producing cerebral cortical cell preparation derived from human pluripotent stem cells |
WO2023009676A1 (en) | 2021-07-28 | 2023-02-02 | Lineage Cell Therapeutics, Inc. | Expansion of retinal pigment epithelium cells |
EP4144841A1 (en) | 2021-09-07 | 2023-03-08 | Bayer AG | Novel small rna programmable endonuclease systems with impoved pam specificity and uses thereof |
WO2023039588A1 (en) | 2021-09-13 | 2023-03-16 | FUJIFILM Cellular Dynamics, Inc. | Methods for the production of committed cardiac progenitor cells |
WO2023049826A1 (en) | 2021-09-23 | 2023-03-30 | President And Fellows Of Harvard College | Genetically encoded voltage indicators and uses thereof |
WO2023069979A1 (en) | 2021-10-20 | 2023-04-27 | University Of Rochester | Isolated glial progenitor cells for use in the competition treatment of age-related white matter loss |
US20230226116A1 (en) | 2021-10-20 | 2023-07-20 | University Of Rochester | Method for rejuvenating glial progenitor cells and rejuvenated glial progenitor cells per se |
WO2023070019A1 (en) | 2021-10-21 | 2023-04-27 | Vertex Pharmaceuticals Incorporated | Hypoimmune cells |
WO2023077140A2 (en) | 2021-11-01 | 2023-05-04 | Vertex Pharmaceuticals Incorporated | Stem cell derived pancreatic islet differentiation |
WO2023095149A1 (en) | 2021-11-29 | 2023-06-01 | Ramot At Tel-Aviv University Ltd. | Methods and compositions for treating spinal cord injury |
WO2023118068A1 (en) | 2021-12-23 | 2023-06-29 | Bayer Aktiengesellschaft | Novel small type v rna programmable endonuclease systems |
WO2023167986A1 (en) | 2022-03-02 | 2023-09-07 | Lineage Cell Therapeutics, Inc. | Methods and compositions for treating hearing loss |
WO2023178239A1 (en) | 2022-03-16 | 2023-09-21 | The Children's Medical Center Corporation | Hpsc-derived articular chondrocyte compositions, systems and methods of use thereof |
WO2023199113A1 (en) | 2022-04-15 | 2023-10-19 | Smartcella Solutions Ab | COMPOSITIONS AND METHODS FOR EXOSOME-MEDIATED DELIVERY OF mRNA AGENTS |
WO2023211857A1 (en) | 2022-04-25 | 2023-11-02 | Lineage Cell Therapeutics, Inc. | Methods and compositions for treating vision loss |
WO2023215455A1 (en) | 2022-05-05 | 2023-11-09 | University Of Rochester | Dual macroglial-microglial approach towards therapeutic cell replacement in neurodegenerative and neuropsychiatric disease |
WO2023230391A1 (en) | 2022-05-26 | 2023-11-30 | Tender Food, Inc. | Plant-based shredded meat products, and methods of producing the same |
WO2023237587A1 (en) | 2022-06-10 | 2023-12-14 | Bayer Aktiengesellschaft | Novel small type v rna programmable endonuclease systems |
US20240090531A1 (en) | 2022-09-16 | 2024-03-21 | Tender Food, Inc. | Plant and animal cell blended meat products and methods of producing the same |
Family Cites Families (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3060078A (en) * | 1960-12-02 | 1962-10-23 | Burlington Industries Inc | Bonding of polyethylene terephthalate fibers to certain rubbers |
GB1051340A (en) * | 1962-09-21 | 1900-01-01 | ||
JP2740320B2 (en) | 1988-08-04 | 1998-04-15 | アムラド・コーポレイション・リミテッド | In vitro propagation of embryonic stem cells |
US20030032178A1 (en) * | 1988-08-04 | 2003-02-13 | Williams Robert Lindsay | In vitro propagation of embryonic stem cells |
JP2750464B2 (en) * | 1990-01-30 | 1998-05-13 | 日本ゼオン株式会社 | Method for producing fiber-rubber composite |
US5061620A (en) | 1990-03-30 | 1991-10-29 | Systemix, Inc. | Human hematopoietic stem cell |
US5340740A (en) | 1992-05-15 | 1994-08-23 | North Carolina State University | Method of producing an avian embryonic stem cell culture and the avian embryonic stem cell culture produced by the process |
US5589376A (en) | 1992-07-27 | 1996-12-31 | California Institute Of Technology | Mammalian neural crest stem cells |
WO1994003585A1 (en) * | 1992-08-04 | 1994-02-17 | Commonwealth Scientific And Industrial Research Organisation | A method for maintaining embryonic stem cells and avian factor useful for same |
US5453357A (en) * | 1992-10-08 | 1995-09-26 | Vanderbilt University | Pluripotential embryonic stem cells and methods of making same |
US5690926A (en) * | 1992-10-08 | 1997-11-25 | Vanderbilt University | Pluripotential embryonic cells and methods of making same |
US5523226A (en) * | 1993-05-14 | 1996-06-04 | Biotechnology Research And Development Corp. | Transgenic swine compositions and methods |
US5591625A (en) * | 1993-11-24 | 1997-01-07 | Case Western Reserve University | Transduced mesenchymal stem cells |
US5449620A (en) * | 1994-01-25 | 1995-09-12 | Thomas Jefferson University | Apparatus and method for culturing embryonic stem cells |
US5541081A (en) * | 1994-03-22 | 1996-07-30 | President And Fellows Of Harvard College | Process for assessing oocyte and embryo quality |
JP3496292B2 (en) * | 1994-09-30 | 2004-02-09 | 日本ゼオン株式会社 | Composites of nitrile group-containing highly saturated copolymer rubber and fiber |
US5843780A (en) * | 1995-01-20 | 1998-12-01 | Wisconsin Alumni Research Foundation | Primate embryonic stem cells |
US5654099A (en) * | 1996-02-21 | 1997-08-05 | Dayco Products, Inc. | Method of improving adhesion between alkylated chlorosulfonated polyethylene (ACSM) or chlorosulfonated polyethylene (CSM), and resorcinol formaldehyde latex (RFL) treated polyester cord |
IL154159A0 (en) * | 2000-08-01 | 2003-07-31 | Yissum Res Dev Co | Directed differentiation of ebryonic cells |
US20070077654A1 (en) * | 2004-11-01 | 2007-04-05 | Thomson James A | Platelets from stem cells |
-
1996
- 1996-01-18 US US08/591,246 patent/US5843780A/en not_active Expired - Lifetime
- 1996-01-19 EP EP05024871A patent/EP1640448A3/en not_active Withdrawn
- 1996-01-19 CA CA2190528A patent/CA2190528C/en not_active Expired - Lifetime
- 1996-01-19 EP EP96903521A patent/EP0770125A4/en not_active Withdrawn
- 1996-01-19 AU AU47584/96A patent/AU4758496A/en not_active Abandoned
- 1996-01-19 WO PCT/US1996/000596 patent/WO1996022362A1/en active Application Filing
-
1998
- 1998-06-26 US US09/106,390 patent/US6200806B1/en not_active Expired - Lifetime
-
2001
- 2001-01-16 US US09/761,289 patent/US20010024825A1/en not_active Abandoned
- 2001-10-18 US US09/982,637 patent/US7029913B2/en not_active Expired - Fee Related
-
2003
- 2003-05-06 US US10/430,496 patent/US20060040383A1/en not_active Abandoned
-
2005
- 2005-01-11 US US11/033,335 patent/US20050158854A1/en not_active Abandoned
- 2005-01-14 US US11/036,245 patent/US7582479B2/en not_active Expired - Fee Related
-
2008
- 2008-03-12 US US12/047,135 patent/US7781216B2/en not_active Expired - Fee Related
-
2010
- 2010-06-23 US US12/822,004 patent/US8273569B2/en not_active Expired - Fee Related
-
2012
- 2012-08-27 US US13/595,587 patent/US20120328582A1/en not_active Abandoned
-
2014
- 2014-05-19 US US14/281,341 patent/US20150056698A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
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EP1640448A3 (en) | 2006-04-26 |
US20090011503A1 (en) | 2009-01-08 |
CA2190528A1 (en) | 1996-07-25 |
US8273569B2 (en) | 2012-09-25 |
US20110256623A1 (en) | 2011-10-20 |
US6200806B1 (en) | 2001-03-13 |
AU4758496A (en) | 1996-08-07 |
US7029913B2 (en) | 2006-04-18 |
US7781216B2 (en) | 2010-08-24 |
US20010024825A1 (en) | 2001-09-27 |
US20030008392A1 (en) | 2003-01-09 |
US20060040383A1 (en) | 2006-02-23 |
US5843780A (en) | 1998-12-01 |
EP0770125A4 (en) | 1998-01-14 |
US20150056698A1 (en) | 2015-02-26 |
WO1996022362A1 (en) | 1996-07-25 |
US20120328582A1 (en) | 2012-12-27 |
EP0770125A1 (en) | 1997-05-02 |
EP1640448A2 (en) | 2006-03-29 |
US20050158854A1 (en) | 2005-07-21 |
US20050164381A1 (en) | 2005-07-28 |
US7582479B2 (en) | 2009-09-01 |
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