EP1240318A1 - Embryonic or stem-like cells produced by cross species nuclear transplantation - Google Patents
Embryonic or stem-like cells produced by cross species nuclear transplantationInfo
- Publication number
- EP1240318A1 EP1240318A1 EP00930498A EP00930498A EP1240318A1 EP 1240318 A1 EP1240318 A1 EP 1240318A1 EP 00930498 A EP00930498 A EP 00930498A EP 00930498 A EP00930498 A EP 00930498A EP 1240318 A1 EP1240318 A1 EP 1240318A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- cell
- embryonic
- human
- oocyte
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
Definitions
- the present invention generally relates to the production of embryonic or stem-like cells by the transplantation of cell nuclei derived from animal or human cells into enucleated animal oocytes of a species different from the donor nuclei.
- the present invention more specifically relates to the production of primate or human embryonic or stem-like cells by transplantation of the nucleus of a primate or human cell into an enucleated animal oocyte, e.g., a primate or ungulate oocyte and in a preferred embodiment a bovine enucleated oocyte.
- the present invention further relates to the use of the resultant embryonic or stemlike cells, preferably primate or human embryonic or stem-like cells for therapy, for diagnostic applications, for the production of differentiated cells which may also be used for therapy or diagnosis, and for the production of transgenic embryonic or transgenic differentiated cells, cell lines, tissues and organs.
- the embryonic or stem-like cells obtained according to the present invention may themselves be used as nuclear donors in nuclear transplantation or nuclear transfer methods for the production of chimeras or clones, preferably transgenic cloned or chimeric animals. 5 BACKGROUND OF THE INVENTION
- ES cells can be passaged in an undifferentiated state, provided that a feeder layer of fibroblast cells
- ES cells have been previously reported to possess numerous applications. For example, it has been reported that ES cells can be used as an in vitro model for differentiation, especially for the study of genes which are involved in the regulation of early
- Mouse ES cells can give rise to germline chimeras when introduced into preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley et al., Nature, 309:255-256 (1984)).
- ES cells In view of their ability to transfer their genome to the next generation, ES cells have potential utility for germline manipulation of livestock animals by using ES cells
- nuclei from like preimplantation livestock embryos support the development of enucleated oocytes to term (Smith et al., Biol Reprod., 40:1027-1035 (1989); and Keefer et al., Biol. Reprod., 50:935-939 (1994)). This is in contrast to nuclei from mouse embryos which beyond the eight-cell stage after transfer reportedly do not
- ES cells from livestock animals are highly desirable because they may provide a potential source of totipotent donor nuclei, genetically manipulated or otherwise, for nuclear transfer procedures.
- LO cell lines from pig blastocysts disclose the isolation of embryonic cell lines from porcine blastocysts. These cells are stably maintained in mouse embryonic fibroblast feeder layers without the use of conditioned medium. These cells reportedly differentiate into several different cell types during culture (Gerfen et al., Id.).
- .5 pluripotent bovine primordial germ cell-derived cell lines maintained in long-term culture. These cells, after approximately seven days in culture, produced ES-like colonies which stain positive for alkaline phosphatase (AP), exhibited the ability to form embryoid bodies, and spontaneously differentiated into at least two different cell types. These cells also reportedly expressed mRNA for the transcription factors OCT4, OCT6 and HES1, a pattern of homeobox genes which is believed to be expressed by ES cells exclusively. Also recently, Campbell et al., Nature, 380:64-68 (1996) reported the production 5 of live lambs following nuclear transfer of cultured embryonic disc (ED) cells from day nine ovine embryos cultured under conditions which promote the isolation of ES cell lines in the mouse. The authors concluded based on their results that ED cells from day nine ovine embryos are totipotent by nuclear transfer and that totipotency is maintained in culture.
- ED cultured embryonic disc
- Collas et al taught the use of granulosa cells (adult somatic cells) to produce bovine nuclear transfer embryos. However, unlike the present invention, these experiments did not involve cross-species nuclear transfer. Also, unlike the present invention ES-like cell colonies were not obtained.
- primate embryonic stem cells that are (i) capable of proliferation in an in vitro culture for over one year; (ii) maintain a karyotype in which all chromosomes characteristic of the primate species are present and not noticeably altered
- the cells include the presence of all the chromosomes characteristic of the primate species and in which none of the chromosomes are altered. Further, these cells are respectfully positive for the TRA-1-60, and TRA-1-81 markers. The cells purportedly differentiate into endoderm, mesoderm and ectoderm cells when injected into a SCID mouse. Also, purported embryonic stem cell lines derived from human or primate blastocysts are
- Parkinson's disease is caused by degeneration of dopaminergic neurons in the substantia nigra.
- Standard treatment for Parkinson's involves administration of L-DOPA, which temporarily ameliorates the loss of dopamine,
- a different approach to treating Parkinson's which promises to have broad applicability to treatment of many brain diseases and central nervous system injury, involves transplantation of cells or tissues from fetal or neonatal animals into the adult brain. Fetal neurons from a variety of brain regions can be incorporated into the adult brain.
- L 5 grafts have been shown to alleviate experimentally induced behavioral deficits, including complex cognitive functions, in laboratory animals. Initial test results from human clinical trials have also been promising. However, supplies of human fetal cells or tissue obtained from miscarriages is very limited. Moreover, obtaining cells or tissues from aborted fetuses is highly controversial.
- L5 nuclear transfer by introducing cytoplasm from one or more immature oocytes which cytoplasm is compatible (same or closely related species) as the donor cell or nucleus.
- Such immature oocytes optionally may be matured in vitro and/or activated in vitro prior to collection and introduction of cytoplasm into the recipient enucleated oocyte.
- an enucleated somatic cell e.g., an enucleated human somatic cell
- a nuclear transfer by fusing an enucleated somatic cell (e.g., an enucleated human somatic cell) (karyoplast) with an activated or non-activated, enucleated or non-enucleated oocyte of a different species, e.g., bovine, or by fusion with an activated or unactivated cross- species NT unit which may be cleaved or uncleaved.
- an enucleated somatic cell e.g., an enucleated human somatic cell
- a different species e.g., bovine
- an activated or unactivated cross- species NT unit which may be cleaved or uncleaved.
- It is another object of the invention to provide a novel method for producing lineage-defective non-human primate or human embryonic or stem-like cells which 5 involves transplantation of the nucleus of a non-human primate or human cell, e.g., a human adult cell into an enucleated non-human primate or human oocyte, wherein such cell has been genetically engineered to be incapable of differentiation into a specific cell lineage or has been modified such that the cells are "mortal", and thereby do not give rise to a viable offspring, e.g., by engineering expression of anti-sense or ribozyme telomerase
- L5 of the MHC I family and in particular Ped genes such as Q7 and/or Q9.
- a DNA construct that provides for the expression of genes that inhibit 5 apoptosis, e.g., Bcl-2 or Bcl-2 family members and/or by the expression of antisense ribozymes specific to genes that induce apoptosis during early embryonic development.
- a detectable marker e.g., one that encodes a visualizable (e.g., fluorescent tag) marker protein.
- an enucleated animal oocyte e.g., a human, primate or ungulate enucle- ated oocyte.
- Such therapies include by way of example freatment of diseases and injuries including Parkinson's, Huntington's, Alzheimer's, ALS, spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver diseases, heart disease, cartilage replacement, burns, vascular diseases, urinary fract diseases, as well as for the treatment of immune defects, bone marrow transplantation, cancer, among other diseases.
- transgenic or genetically engineered embryonic or stem-like cells produced according to the invention for gene therapy, in particular for the treatment and/or prevention of the diseases and injuries identified, supra. It is another object of the invention to use the embryonic or stem-like cells produced according to the invention or transgenic or genetically engineered embryonic or stem-like cells produced according to the invention as nuclear donors for nuclear transplantation.
- transgenic animals e.g., non- human primates, rodents, ungulates, etc.
- transgenic animals e.g., non- human primates, rodents, ungulates, etc.
- transgenic animals can be used to produce, e.g., animal models for study of human diseases, or for the production of desired polypeptides, e.g., therapeutics or nutripharmaceuticals.
- FIGURES Figure 1 is a photograph of a nuclear transfer (NT) unit produced by transfer of an adult human cell into an enucleated bovine oocyte.
- Figures 2 to 5 are photographs of embryonic stem-like cells derived from a NT unit such as is depicted in Figure 1.
- the present invention provides a novel method for producing embryonic or stemlike cells, and more specifically non-human primate or human embryonic or stem-like cells by nuclear transfer or nuclear transplantation.
- nuclear transfer or nuclear fransplantation or NT are used interchangeably.
- human embryonic or stem-like cells and cell colonies may be obtained by fransplantation of the nucleus of a human cell, e.g., an adult differentiated human cell, into an enucleated animal oocyte, which is used to produce nuclear transfer (NT) units, the cells of which upon culturing give rise to human embryonic or stem-like cells and cell colonies.
- a human cell e.g., an adult differentiated human cell
- NT nuclear transfer
- the recipient oocyte will be transplanted with a combination of (i) the donor cell or nucleus and (ii) at least one of compatible mitochondrial DNA or compatible cytoplasm.
- “Compatible” in the present application means that the mitochondrial DNA or cytoplasm is derived from a cell of the same species as the donor cell or nucleus, or a species that is very closely related to the donor cell or nucleus. For instance, if the donor cell or nucleus is a human cell or nucleus, the mitochondrial DNA or cytoplasm will be of human origin or that of a higher primate, e.g., chimpanzee, gorilla, or baboon.
- compatible cytoplasm will be of a closely related ungulate, e.g., buffalo.
- the compatible mitochondrial DNA and/or cytoplasm will be derived from a cell of the same species as donor and most preferably will be autologous to the donor cell or nucleus.
- the cytoplasm will be derived from immature oocytes or blastomeres, wherein such immature oocytes optionally may be matured in vitro and/or activated in vitro prior to collection of cytoplasm therefrom.
- immature oocytes optionally may be matured in vitro and/or activated in vitro prior to collection of cytoplasm therefrom.
- the NT units used to produce ES-like cells will be cultured to a size of at least 2 to 400 cells, preferably 4 to 128 cells, and most preferably to a size of at least about 50 cells.
- embryonic or stem-like cells refer to cells produced according to the present invention.
- the present application refers to such cells as stemlike cells rather than stem cells because of the manner in which they are typically produced, i.e., by cross-species nuclear transfer. While these cells are expected to possess similar differentiation capacity as normal stem cells they may possess some insignificant differences because of the manner they are produced. For example, because these stemlike cells may possess the mitochondria of the oocytes used for nuclear fransfer, and they may not behave or be mo ⁇ hogenetically identical to conventional embryonic stem cells.
- the present discovery was made based on the observation that nuclear transplantation of the nucleus of an adult human cell, specifically a human epithelial cell obtained from the oral cavity of a human donor, when transferred into an enucleated bovine oocyte, resulted in the formation of nuclear transfer units, the cells of which upon culturing gave rise to human stem-like or embryonic cells and human embryonic or stemlike cell colonies.
- This result has recently been reproduced by transplantation of keratinocytes from an adult human into an enucleated bovine oocyte with the successful production of a blastocyst and ES cell line.
- bovine oocytes and human oocytes and likely mammals in general must undergo maturation processes during embryonic development which are sufficiently similar or conserved so as to permit the bovine oocyte to function as an effective substitute or surrogate for a human oocyte.
- oocytes in general comprise factors, likely proteinaceous or nucleic acid in nature, that induce embryonic development under 5 appropriate conditions, and these functions that are the same or very similar in different species. These factors may comprise material RNAs and/or telomerase.
- human cell nuclei can be effectively fransplanted into bovine oocytes, it is reasonable to expect that human cells may be transplanted into oocytes of other non-related species, e.g., other ungulates as well as other animals.
- other non-related species e.g., other ungulates as well as other animals.
- L 0 other ungulate oocytes should be suitable, e.g. pigs, sheep, horses, goats, etc.
- oocytes from other sources should be suitable, e.g. oocytes derived from other primates, such as chimpanzees, macaques, baboons, gorilla, Rhesus monkey, amphibians, rodents, rabbits, guinea pigs, etc.
- the present invention involves the transplantation of an animal or human cell nucleus or animal or human cell into an oocyte (preferably enucleated) of an animal species different from the donor nuclei, by injection or fusion, and to optionally further inject into said recipient oocyte compatible
- NT unit containing cells which may be used to obtain embryonic or stem-like cells and or cell cultures.
- Enucleation may be effected before or after nuclear transfer.
- the invention may involve the fransplantation of an ungulate cell nucleus or ungulate cell into an enucleated
- NT units 25 oocyte of another species, e.g., another ungulate or non-ungulate, by injection or fusion, which cells and/or nuclei are combined to produce NT units which are then cultured under conditions suitable to obtain multicellular NT units, preferably comprising at least about 2 to 400 cells, more preferably 4 to 128 cells, and most preferably at least about 50 cells.
- the cells of such NT units may be used to produce embryonic or stem-like cells or cell colonies upon culturing.
- the preferred embodiment of the invention comprises the production of non- 5 human primate or human embryonic or stem-like cells by transplantation of the nucleus of a donor human cell or a human cell into an enucleated human, primate, or non-primate animal oocyte, e.g., an ungulate oocyte, and in a preferred embodiment a bovine enucleated oocyte.
- the enucleated oocyte will also be injected with human cytoplasm (e.g., from at least one immature or mature oocyte or blastomere), or fused L 0 with a karyoplast????? (enucleated human oocyte or blastomere, or that of a higher primate) and/or human mitochondrial DNA.
- the embryonic or stem-like cells will be produced by a nuclear fransfer process comprising the following steps:
- oocytes from a suitable source, e.g. a mammal and most preferably a primate or an ungulate source, e.g. bovine,
- steps (iii) and (iv) may be effected in either order;
- the nuclear transfer process preferably will also include the introduction of compatible mitochondrial DNA and/or cytoplasm, i.e., derived from the same species or closely related species to the donor cell or nucleus. This may be effected before, concurrent, or after introduction of the donor cell or nucleus into the recipient oocyte.
- the compatible cytoplasm and/or mitochondrial DNA will be infroduced proximate to infroduction of the donor cell or nucleus into the recipient oocyte, i.e., within about twenty-four hours of such introduction, more preferably within about six hours of such introduction, and most preferably within about two to four hours of such introduction.
- Sources of compatible cytoplasm include in particular immature oocytes which can be obtained in large numbers from oocytes.
- oocytes can be obtained from ovaries of consenting donors, e.g., women undergoing hysterectomies.
- Such immature oocytes optionally will be matured in vitro prior to collection of cytoplasm therefrom.
- Methods of effecting in vitro maturation of oocytes are described in U.S. Patent 5,945,577, incorporated by reference in its entirety herein. Additionally, such immature oocytes can further optionally be activated in vitro prior to collection of cytoplasm therefrom.
- Methods for parthenogenetically activating oocytes, including human oocytes are well known.
- Exemplary methods for effecting in vitro activation of oocytes are described in U.S. Serial No. 08/888,057, which is incorporated by reference in its entirety herein.
- Preferred methods for effecting in vitro activation include the use of DMAP and ionomycin, and the use of cycloheximide and ionomycin.
- the amount of cytoplasm infroduced into the recipient oocyte preferably will be an amount that increases its cell volume by at most five- fold, preferably no more than about two-fold, and most preferably no more than about one and a half-fold. This may be effected by injecting cytoplasm from compatible oocytes into the recipient oocyte or alternatively by fusing the recipient oocyte of compatible ooplasts (enucleated oocytes of the same or very similar species as the recipient oocyte.) Optionally, all or part of the cytoplasm of the recipient oocyte may be removed to provide more cell volume for the infroduced compatible cytoplasm. With respect thereto, it has been suggested in the literature that maintaining appropriate nucleus/cytoplasm ratios and cell volume is importance to blastocyst formation and development. (Karyikova et al, Reprod, Nutr.
- nucleic acid sequences and/or proteins that are present in the cytoplasm of oocytes which facilitate embryogenesis while such factors are believed to be somewhat conserved across phylogenetically diverse species
- L5 compatible mitochondrial DNA i.e., of the same or very similar species as the donor cell or nucleus. This can be effected before, concurrent, or after introduction of the donor cell or nucleus.
- human mitochondrial DNA will be derived from cells of the particular donor, e.g., liver cells and tissue.
- tissue culture or from tissue.
- the particular cells or tissue will depend upon the particular species of the donor cell.
- Examples of cells or tissues that may be used as sources of mitochondria include fibroblasts, epithelium, liver, lung, keratinocyte, stomach, heart, bladder, pancreas, esophageal, lymphocytes, monocytes, mononuclear cells, cumulus cells, uterine cells, placental cells, intestinal cells, hematopoietic cells, oocytes, and tissues containing such cells.
- mitochondria can be isolated from tissue culture cells and rat liver. 5 It is anticipated that the same or similar procedures may be used to isolate mitochondria from other cells and tissues, i.e., human cells and tissues. As noted above, preferred source of mitochondria comprises human liver tissue because such cells contain a large number of mitochondria. Those skilled in the art will be able to modify the procedure as necessary, dependent upon the particular cell line or tissue.
- the isolated DNA can also be used to isolate mitochondria from other cells and tissues.
- L 0 be further purified, if desired, known methods, e.g., density gradient centrifugation.
- the resultant isolated mitochondrial DNA will preferably be injected into the recipient oocyte.
- the entire mitochondrial DNA isolated from one or several cells will be infroduced to ensure that the recipient oocyte has a full complement of compatible mitochondrial DNA.
- the mitochondrial DNA will be derived from
- L5 an autologous source, i.e., cells or tissues obtained from the human subject who is the source of the donor cell or nucleus, or a person genetically compatible therewith, e.g., close relative.
- compatible mitochondria can be derived from immature oocytes and introduced together with the compatible cytoplasm.
- Human or animal cells may be obtained and cultured by well known methods.
- Human and animal cells useful in the present invention include, by way of example, epithelial, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), other immune cells, erythrocytes, macrophages, melanocytes, monocytes, mononuclear cells, stem cells, fibroblasts, cardiac muscle cells, and other muscle cells, etc.
- the human cells used for nuclear fransfer may be obtained from different organs, e.g., skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, etc. These are just examples of suitable donor cells.
- Suitable donor cells may be obtained from any cell or organ of the body. This includes all somatic and germ cells.
- the donor cells or nucleus will comprise an actively dividing, i.e., non-quiescent (G l5 S or M cell cycle), cells as this has been reported to enhance cloning efficacy. Still more preferably, such donor cells will be in the Gl cell cycle.
- quiescent donor cells G 0
- Quiescent cells may be obtained, e.g., appropriate culture conditions, by serum starvation.
- the resultant blastocysts may be used to obtain embryonic stem cell lines according to the culturing methods reported by Thomson et al., Science, 282: 1145-1147
- the resultant blastocysts having a discernible inner cell mass can be cultured according to the method disclosed in U.S. Patent No. 5,905,042, inco ⁇ orated by reference in its entirety herein.
- This method essentially comprises culturing of the entire inner cell mass or a portion thereof on a feeder layer, wherein contact with the feeder layer is maintained throughout culturing.
- This culturing method further comprises selective removal of a portion of the cultured inner cell mass as culturing proceeds, i.e., the outermost portion of the cultured inner cell mass that comprises cells possessing an ES-like appearance.
- This outermost portion of the cell colony is then introduced onto another feeder layer, typically fetal fibroblasts.
- This method has been found to provide 5 for the maintenance of non-differentiated (embryonic stem) cells in tissue culture, i.e., cells which exhibit an ES-like mo ⁇ hology which, upon removal from the feeder layer, differentiate into other cell types.
- the cells used as donors for nuclear transfer were epithelial cells derived from the oral cavity of a human donor and adult human
- L 0 keratinocytes L 0 keratinocytes.
- the disclosed method is applicable to other human cells or nuclei.
- the cell nuclei may be obtained from both human somatic and germ cells.
- Trichostatin-A has been shown to inhibit histone deacetylase in a reversible manner (Adenot et al, "Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos.” Development (Nov. 1997) 124(22): 4615-4625; Yoshida et al. "Trichostatin
- demethylation of DNA is thought to be a requirement for proper access of transcription factors to DNA regulatory sequences.
- Global demethylation of DNA from the eight-cell stage to the blastocyst stage in preimplantation embryos has previously been described (Stein et al., Mol. Reprod. & Dev., 47(4): 421-429).
- Jaenisch et al. (1997) have reported that 5-azacytidine can be used to reduce the level of DNA methylation in cells, potentially leading to increased access of transcription factors to DNA regulatory sequences. Accordingly, donor cells may be exposed to 5-azacytidine (5-Aza) previous to fusion, or 5-Aza may be added to the culture medium from the 8 cell stage to blastocyst. Alternatively, other known methods for effecting DNA demethylation may be used.
- the oocytes used as the recipient for nuclear transfer or for recovery of compatible cytoplasm may be obtained from animals including mammals and amphibians. Suitable mammalian sources for oocytes include sheep, bovines, ovines, pigs, horses, rabbits, goats, guinea pigs, mice, hamsters, rats, primates, humans, etc. In the preferred embodiments, the oocytes will be obtained from primates or ungulates, e.g., a bovine. In the case of human oocytes, a suitable source comprises ovaries obtained from consenting women, e.g., those undergoing hysterectomies. Methods for isolation of oocytes are well known in the art. Essentially, this will comprise isolating oocytes from the ovaries or reproductive tract of a mammal or amphibian, e.g., a bovine. A readily available source of bovine oocytes is slaughterhouse materials.
- oocytes preferably are matured in vitro before these cells can be used as recipient cells for nuclear fransfer. This process generally requires collecting immature
- (prophase I) oocytes from animal ovaries, e.g., bovine ovaries obtained at a slaughterhouse and maturing the oocytes in a maturation medium prior to fertilization or enucleation until the oocyte attains the metaphase II stage, which in the case of bovine oocytes generally occurs about 18-24 hours post-aspiration.
- this period of time is known as the "maturation period.”
- “aspiration” refers to aspiration of the immature oocyte from ovarian follicles.
- metaphase II stage oocytes which have been matured in vivo have been successfully used in nuclear fransfer techniques.
- mature metaphase II oocytes can be collected surgically from either non-superovulated or superovulated cows or heifers 35 to 48 hours past the onset of estras or past the injection of human chorionic gonadofropin (hCG) or similar hormone.
- hCG human chorionic gonadofropin
- in vivo matured oocytes can be collected from other species, e.g., sheep, horse, goat pig, et al.
- the oocyte activation period generally ranges from about 16-52 hours, preferably about
- immature oocytes may be washed in HEPES buffered hamster embryo culture medium (HECM) as described in Seshagine et al., Biol. Reprod., 40, 544- 606, 1989, and then placed into drops of maturation medium consisting of 50 microliters of tissue culture medium (TCM) 199 containing 10% fetal calf serum which contains 5 appropriate gonadotropins such as luteinizing hormone (LH) and follicle stimulating hormone (FSH), and esfradiol under a layer of lightweight paraffin or silicon at 39°C.
- TCM tissue culture medium
- FSH follicle stimulating hormone
- the oocytes will preferably be enucleated. Prior to enucleation the oocytes will preferably be
- L 0 removed and placed in HECM containing 1 milligram per miUiliter of hyaluronidase prior to removal of cumulus cells. This may be effected by repeated pipetting through very fine bore pipettes or by vortexing briefly.
- the stripped oocytes are then screened for polar bodies, and the selected metaphase II oocytes, as determined by the presence of polar bodies, are then used for nuclear transfer. Enucleation is typically effected at this
- enucleation may be effected before or after infroduction of donor cell or nucleus because the donor nucleus is readily discernible from endogenous nucleus.
- Enucleation may be effected by known methods, such as described in U.S. Patent No. 4,994,384 which is inco ⁇ orated by reference herein.
- metaphase II oocytes are either placed in HECM, optionally containing 7.5 micrograms per miUiliter
- cytochalasin B for immediate enucleation, or may be placed in a suitable medium, for example CRlaa, plus 10% estrus cow serum, and then enucleated later, preferably not more than 24 hours later, and more preferably 16-18 hours later.
- a suitable medium for example CRlaa, plus 10% estrus cow serum
- Enucleation may be accomplished microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm. The oocytes may then be screened to identify
- This screening may be effected by staining the oocytes with 1 microgram per miUiliter 33342 Hoechst dye in HECM, and then viewing the oocytes under ultraviolet irradiation for less than 10 seconds.
- the oocytes that have been successfully enucleated can then be placed in a suitable culture medium.
- the recipient oocytes will typically be enucleated at a time ranging from about 10 hours to about 40 hours after the initiation of in vitro maturation, more preferably from about 16 hours to about 24 hours after initiation of in vitro maturation, and most preferably about 16-18 hours after initiation of in vitro maturation.
- Enucleation may be effected before, simultaneous or after nuclear fransfer. Also, enucleation may be effected before, after or simultaneous to activation.
- a single animal or human cell or nucleus derived therefrom which is typically heterologous to the enucleated oocyte will then be transferred into the perivitelline space of the oocyte, typically enucleated, used to produce the NT unit.
- removal of endogenous nucleus may alternatively be effected after nuclear fransfer.
- the animal or human cell or nucleus and the enucleated oocyte will be used to produce NT units according to methods known in the art.
- the cells may be fused by elecfro- fusion. Elecfrofusion is accomplished by providing a pulse of electricity that is sufficient to cause a fransient break down of the plasma membrane. This breakdown of the plasma membrane is very short because the membrane reforms rapidly.
- nucleus in some cases (e.g. with small donor nuclei) it may be preferable to inject the nucleus directly into the oocyte rather than using electroporation fusion.
- electroporation fusion Such techniques are disclosed in Collas and Barnes, Mol. Reprod. Dev., 38:264-267 (1994), and inco ⁇ orated by reference in its entirety herein.
- the human or animal cell and oocyte are elecfrofused in a 500 ⁇ m chamber by application of an electrical pulse of 90- 120V for about 15 ⁇ sec, about 24 hours after initiation of oocyte maturation.
- the resultant fused NT units are preferably placed in a suitable medium until activation, e.g., one identified infra.
- activation will be effected shortly thereafter, typically less than 24 hours later, and preferably about 4-9 hours later.
- activation may be effected from about twelve hours prior to nuclear transfer to about twenty-four hours after nuclear fransfer. More typically, activation is effected simultaneous or shortly after nuclear transfer, e.g., about four to nine hours later.
- the NT unit may be activated by known methods. Such methods include, e.g., culturing the NT unit at sub-physiological temperature, in essence by applying a cold, or actually cool temperature shock to the NT unit. This may be most conveniently done by culturing the NT unit at room temperature, which is cold relative to the physiological temperature conditions to which embryos are normally exposed.
- activation may be achieved by application of known activation agents.
- penetration of oocytes by sperm during fertilization has been shown to activate prefusion oocytes to yield greater numbers of viable pregnancies and multiple genetically identical calves after nuclear transfer.
- treatments such as electrical and chemical shock or cycloheximide freatment may also be used to activate NT embryos after fusion.
- Suitable oocyte activation methods are the subject of U.S. Patent No. 5,496,720, to Susko-Parrish et al., which is herein inco ⁇ orated by reference.
- oocyte activation may be effected by simultaneously or sequentially: (i) increasing levels of divalent cations in the oocyte, and (ii) reducing phosphorylation of cellular proteins in the oocyte.
- divalent cations into the oocyte cytoplasm, e.g., magnesium, strontium, barium or calcium, e.g., in the form of an iono- phore.
- divalent cations include the use of electric 5 shock, freatment with ethanol and treatment with caged chelators.
- Phosphorylation may be reduced by known methods, e.g., by the addition of kinase inhibitors, e.g., serine-threonine kinase inhibitors, such as 6-dimethylaminopurine, staurosporine, 2-aminopurine, and sphingosine.
- kinase inhibitors e.g., serine-threonine kinase inhibitors, such as 6-dimethylaminopurine, staurosporine, 2-aminopurine, and sphingosine.
- phosphorylation of cellular proteins may be inhibited by L o infroduction of a phosphatase into the oocyte, e.g., phosphatase 2A and phosphatase 2B.
- oocytes with 10 to 12 picoliters of sperm factor isolated, e.g., from primates, pigs, bovine, sheep, goats, horses, mice, rats, rabbits or hamsters;
- oocytes or NT units typically about 22 to 28 hours post maturation in about 2 mM DMAP for about one hour, followed by incubation for about two to twelve hours, preferably about eight hours, in 5 ⁇ g/ml of cytochalasin B and 20 ⁇ g/ml cycloheximide.
- the above activation protocols are exemplary of protocols used for nuclear transfer procedures, e.g., those including the use of primate or human donor cells or oocytes.
- the above activation protocols may be used when either or both the donor cell and nucleus is of ungulate origin, e.g., a sheep, buffalo, horse, goat, bovine, pig and/or wherein the oocyte is of ungulate origin, e.g., sheet, pig, buffalo, horse, goat, bovine, etc., L 0 as well as for other species.
- ungulate origin e.g., a sheep, buffalo, horse, goat, bovine, pig and/or wherein the oocyte is of ungulate origin, e.g., sheet, pig, buffalo, horse, goat, bovine, etc., L 0 as well as for other species.
- activation may be effected before, simultaneous, or after nuclear fransfer. In general, activation will be effected about 40 hours prior to nuclear transfer and fusion to about 40 hours after nuclear transfer and fusion, more preferably about 24 hours before to about 24 hours after nuclear transfer and fusion, and most preferably from L5 about 4 to 9 hours before nuclear fransfer and fusion to about 4 to 9 hours after nuclear transfer and fusion. Activation is preferably effected after or proximate to in vitro or in vivo maturation of the oocyte, e.g., approximately simultaneous or within about 40 hours of maturation, more preferably within about 24 hours of maturation.
- Activated NT units may be cultured in a suitable in vitro culture medium until the 20 generation of embryonic or stem-like cells and cell colonies.
- Culture media suitable for culturing and maturation of embryos are well known in the art. Examples of known media, which may be used for bovine embryo culture and maintenance, include Ham's F-10 + 10% fetal calf serum (FCS), Tissue Culture Medium-199 (TCM-199) + 10% fetal calf serum, Tyrodes-Albumin-Lactate-Pyravate (TALP), Dulbecco's Phosphate Buffered 25 Saline (PBS), Eagle's and Whitten's media.
- TCM-199 One of the most common media used for the collection and maturation of oocytes is TCM-199, and 1 to 20% serum supplement including fetal calf serum, newborn serum, estrual cow serum, lamb serum or steer serum.
- a preferred maintenance medium includes TCM-199 with Earl salts, 10% fetal calf serum, 0.2 Ma pyruvate and 50 ⁇ g/ml gentamicin sulphate. Any of the above may also involve co-culture with a variety of cell types such as granulosa cells, oviduct cells, BRL cells and uterine cells and STO cells. 5
- human epithelial cells of the endometrium secrete leukemia inhibitory factor (LIF) during the preimplantation and implantation period. Therefore, the addition of LIF to the culture medium could be of importance in enhancing the in vitro development of the reconstructed embryos.
- LIF leukemia inhibitory factor
- CR1 contains the nutritional substances necessary to support an embryo.
- CR1 contains hemicalcium L-lactate in amounts ranging from 1.0 mM to 10 mM, preferably 1.0 mM
- Hemicalcium L-lactate is L-lactate with a hemicalcium salt inco ⁇ orated therein.
- NT units may be cultured in biological
- fluids such as peritoneal fluid, amniotic fluid, vitreous/aqueous humor and lymph fluid.
- the cultured NT unit or units are preferably washed and then placed in a suitable media, e.g., the media identified above, such as CRIaa medium, Ham's F-10,
- Tissue Culture Media -199 TCM-199
- PBS Phosphate Buffered Saline
- Eagle's or Whitten's preferably containing
- Suitable feeder layers include, by way of example, fibroblasts and epithelial cells, e.g., fibroblasts and uterine epithelial cells derived from ungulates, chicken fibroblasts, murine (e.g., mouse or rat) fibroblasts, STO and SI-m220 feeder cell lines, and BRL cells.
- the feeder cells will comprise mouse embryonic fibroblasts.
- Means for preparation of a suitable fibroblast feeder layer are described in 5 the example which follows and is well within the skill of the ordinary artisan.
- the NT units are cultured on the feeder layer until the NT units reach a size suitable for obtaining cells which may be used to produce embryonic stem-like cells or cell colonies.
- these NT units will be cultured until they reach a size of at least about 2 to 400 cells, more preferably about 4 to 128 cells, and most preferably at least L 0 about 50 cells.
- the culturing will be effected under suitable conditions, i.e., about 38.5 °C and 5% C0 2 , with the culture medium changed in order to optimize growth typically about every 2-5 days, preferably about every 3 days.
- Examples of a culture media suitable for human embryo culture include the medium reported in Jones et al, Human Reprod., 13(1):169-177 (1998), the Pl-catalog #99242 25 medium, and the P- 1 catalog #99292 medium, both available from Irvine Scientific, Santa
- Another preferred medium comprises ACM + uridine + glucose + 1000 IU of LIF.
- suitable media include naturally occurring biological fluids such as peritoneal fluid, vitreous /aqueous humor, amniotic fluid, and lymph fluid.
- the cells used in the present invention will preferably 5 comprise mammalian somatic cells, most preferably cells derived from an actively proliferating (non-quiescent) mammalian cell culture.
- the donor cell will be genetically modified by the addition, deletion or substitution of a desired DNA sequence.
- the donor cell e.g., a keratinocyte or fibroblast, e.g., of human, primate or bovine origin, may be transfected or transformed
- L0 with a DNA construct that provides for the expression of a desired gene product, e.g., therapeutic polypeptide.
- a desired gene product e.g., therapeutic polypeptide.
- lymphokines e.g., IGF-I, IGF-II, interferons, colony stimulating factors, connective tissue polypeptides such as collagens, genetic factors, clotting factors, enzymes, enzyme inhibitors, etc.
- desired genes may be "knocked in” or “knocked out” by homologous recombination.
- the donor cells may be modified prior to nuclear transfer, e.g., to effect impaired cell lineage development, enhanced embryonic development and/or inhibition of apoptosis. Examples of desirable modifications are discussed further below.
- One aspect of the invention will involve genetic modification of the donor cell
- a human cell such that it is lineage deficient and therefore when it is used for nuclear fransfer it will be unable to give rise to a viable offspring.
- This is desirable especially in the context of human nuclear transfer embryos, wherein for ethical reasons, production of a viable embryo may be an unwanted outcome.
- This can be effected by genetically engineering a human cell such that it is incapable of differentiating into specific cell
- cells may be genetically modified such that when used as nuclear transfer donors the resultant "embryos" do not contain or substantially lack at least one of mesoderm, endoderm or ectoderm tissue. This can be accomplished by, e.g., knocking out or impairing the expression of one or more mesoderm, endoderm or ectoderm specific genes. Examples thereof include:
- Endoderm GATA-6, GATA-4; Ectoderm: RNA helicase A, H beta 58.
- a desired somatic cell e.g., a human keratinocyte, epithelial cell or fibroblast
- a desired somatic cell will be genetically engineered such that one or more genes specific to particular cell lineages are “knocked out” and/or the expression of such genes significantly impaired. This may be effected by known methods, e.g., homologous recombination.
- a preferred genetic system for effecting "knock-out" of desired genes is disclosed by Capecchi et al, U.S. Patents 5,631,153 and 5,464,764, which reports positive-negative selection (PNS) vectors that enable targeted modification of DNA sequences in a desired mammalian genome.
- PPS positive-negative selection
- Such genetic modification will result in a cell that is incapable of differentiating into a particular cell lineage when used as a nuclear transfer donor.
- This genetically modified cell will be used to produce a lineage-defective nuclear transfer embryo, i.e., that does not develop at least one of a functional mesoderm, endoderm or ectoderm. Thereby, the resultant embryos, even if implanted, e.g., into a human uterus, would not give rise to a viable offspring.
- the ES cells that result from such nuclear transfer will still be useful in that they will produce cells of the one or two remaining non-impaired lineage.
- an ectoderm deficient human nuclear transfer embryo will still give rise to mesoderm and endoderm derived differentiated cells.
- An ectoderm deficient cell can be produced by deletion and/or impairment of one or both of RNA helicase A or H beta 58 genes.
- These lineage deficient donor cells may also be genetically modified to express another desired DNA sequence.
- the genetically modified donor cell will give rise to a lineage-deficient blastocyst which, when plated, will differentiate into at most two of the embryonic germ layers.
- the donor cell can be modified such that it is "immortal". This can be achieved by expressing anti-sense or ribozyme telomerase genes. This can be effected by known genetic methods that will provide for expression of antisense DNA or ribozymes, or by gene knockout. These "immortal" cells, when used for nuclear transfer, will not be capable of differentiating into viable offspring.
- Another preferred embodiment of the present invention is the production of nuclear transfer embryos that grow more efficiently in tissue culture. This is advantageous in that it should reduce the requisite time and necessary fusions to produce ES cells and/or offspring (if the blastocysts are to be implanted into a female surrogate). This is desirable also because it has been observed that blastocysts and ES cells resulting from nuclear transfer may have impaired development potential. While these problems may often be alleviated by alteration of tissue culture conditions, an alternative solution is to enhance embryonic development by enhancing expression of genes involved in embryonic development. For example, it has been reported that the gene products of the Ped type, which are members of the MHC I family, are of significant importance to embryonic development.
- a DNA construct containing the Q7 and/or Q9 gene will be infroduced into donor somatic cells prior to nuclear transfer.
- an expression construct can be constructed containing a strong constitutive mammalian promoter operably linked to the Q7 and/or Q9 genes, an IRES, one or more suitable selectable markers, e.g,. neomycin, ADA, DHFR, and a poly-A sequence, e.g., bGH polyA sequence.
- Still another aspect of the invention involves the construction of donor cells that are resistant to apoptosis, i.e., programmed cell death. It has been reported in the literature that cell death related genes are present in preimplantation stage embryos.
- genes reported to induce apoptosis include, e.g., Bad, Bok, BH3, Bik, Hrk, BNIP3, Bim L , Bad, Bid, and EGL-1.
- genes that reportedly protect cells from programmed cell death include, by way of example, BcL-XL, Bcl-w, Mcl-1, Al, Nr-13, BHRF-1, LMW5-HL, ORF16, Ks-Bel-2, E1B-19K, and CED-9.
- donor cells can be constructed wherein genes that induce apoptosis are "knocked out” or wherein the expression of genes that protect the cells from apoptosis is enhanced or turned on during embryonic development.
- this can be effected by introducing a DNA construct that provides for regulated expression of such protective genes, e.g., Bcl-2 or related genes during embryonic development.
- the gene can be "turned on” by culturing the embryo under specific growth conditions.
- it can be linked to a constitutive promoter.
- a DNA construct containing a Bcl-2 gene operably linked to a regulatable or constitutive promoter e.g., PGK, SV40, CMV, ubiquitin, or beta-actin, an IRES, a suitable selectable marker, and a poly-A sequence
- a regulatable or constitutive promoter e.g., PGK, SV40, CMV, ubiquitin, or beta-actin, an IRES, a suitable selectable marker, and a poly-A sequence
- a desired donor mammalian cell e.g., human keratinocyte or fibroblast.
- donor cells when used to produce nuclear fransfer embryos, should be resistant to apoptosis and thereby differentiate more efficiently in tissue culture. Thereby, the speed and/or number of suitable preimplantation embryos produced by nuclear transfer can be increased. Another means of accomplishing the same result is to impair the expression of one or more genes that induce apoptosis. This will be effected by knock-out or by the use of antisense or ribozymes against genes that are expressed in and which induce apoptosis early on in embryonic development. Examples thereof are identified above. Cell death genes that may be expressed in the antisense orientation include BAX, Apaf-1, and capsases.
- a transgene may be infroduced that encodes for methylase or demethylase in the sense or antisense orientation.
- DNAs that encode methylase and demethylase enzymes are well known in the art.
- donor cells may be constructed containing both modifications, i.e., impairment of apoptosis-inducing genes and enhanced expression of genes that impede or prevent apoptosis.
- the construction and selection of genes that affect apoptosis, and cell lines that express such genes is disclosed in U.S. Patent No. 5,646,008, which patent is inco ⁇ orated by reference herein. Many DNAs that promote or inhibit apoptosis have been reported and are the subject of numerous patents.
- Another means of enhancing cloning efficiency is to select cells of a particular cell cycle stage as the donor cell. It has been reported that this can have significant effects on nuclear transfer efficiency. (Barnes et al, Mol. Reprod. Devel, 36(1):33-41 (1993). Different methods for selecting cells of a particular cell cycle stage have been reported and include serum starvation (Campbell et al, Nature, 380:64-66 (1996); Wilmut et al, Nature, 385:810-813 (1997), and chemical synchronization (Urbani et al, Exp. Cell Res., 219(1): 159-168 (1995).
- a particular cyclin DNA may be operably linked to a regulatory sequence, together with a detectable marker, e.g., green fluorescent protein (GFP), followed by the cyclin destruction box, and optionally insulation sequences to enhance cyclin and marker protein expression.
- a detectable marker e.g., green fluorescent protein (GFP)
- GFP green fluorescent protein
- cells of a desired cell cycle can be easily visually detected and selected for use as a nuclear transfer donor.
- An example thereof is the cyclin Dl gene in order to select for cells that are in Gl.
- any cyclin gene should be suitable for use in the claimed invention. (See, e.g., King et al, Mol. Biol. Cell, Vol. 7(9): 1343-1357 (1996)).
- donor cells will be constructed that express one or more of such cyclin genes under easily detectable conditions, preferably visualizable, e.g., by the use of a fluorescent label.
- a particular cyclin DNA may be operably linked to a regulatory sequence, together with a detectable marker, e.g., green fluorescent protein (GFP), followed by the cyclin destruction box, and optionally insulation sequences to enhance cyclin and/or marker protein expression.
- GFP green fluorescent protein
- cells of a desired cell cycle can be easily visually detected and selected for use as a nuclear transfer donor.
- An example thereof is the cyclin Dl gene which can be used to select for cells that are in Gl .
- any cyclin gene should be suitable for use in the claimed invention.
- the present invention provides different methods for enhancing nuclear transfer efficiency, preferably a cross-species nuclear transfer process. While the present inventors have demonstrated that nuclei or cells of one species when inserted or fused with an enucleated oocyte of a different species can give rise to nuclear transfer embryos that produce blastocysts, which embryos can give rise to ES cell lines, the efficiency of such process is quite low. Therefore, many fusions typically need to be effected to produce a blastocyst the cells of which may be cultured to produce ES cells and ES cell lines.
- Yet another means for enhancing the development of nuclear transfer embryos in vitro is by optimizing culture conditions.
- One means of achieving this result will be to culture NT embryos under conditions impede apoptosis.
- proteases such as capsases can cause oocyte death by apoptosis similar to other cell types.
- blastocyst development will be enhanced by including in culture media used for nuclear fransfer and to maintain blastocysts or culture preimplantation stage embryos one or more capsase inhibitors.
- Such inhibitors include by way of example capsase-4 inhibitor I, capsase-3 inhibitor I, capsase-6 inhibitor II, capsase-9 inhibitor II, and capsase- 1 inhibitor I.
- the amount thereof will be an amount effective to inhibit apoptosis, e.g., 0.00001 to 5.0% by weight of medium; more preferably 0.01% to 1.0% by weight of medium.
- the foregoing methods may be used to increase the efficiency of nuclear transfer by enhancing subsequent blastocyst and embryo development in tissue culture. After NT units of the desired size are obtained, the cells are mechanically removed from the zone and are then used to produce embryonic or stem-like cells and cell lines.
- NT unit which typically will contain at least about 50 cells
- washing such cells and plating the cells onto a feeder layer, e.g., irradiated fibroblast cells.
- a feeder layer e.g., irradiated fibroblast cells.
- the cells used to obtain the stem-like cells or cell colonies will be obtained from the inner most portion of the cultured NT unit which is preferably at least 50 cells in size.
- NT units of smaller or greater cell numbers as well as cells from other portions of the NT unit may also be used to obtain ES-like cells and cell colonies.
- donor cell DNA to the oocyte 's cytosol may facilitate the dedifferentiation process. This can be accomplished by re- cloning, i.e., by taking blastomeres from a reconstructed embryo and fusing them with a new enucleated oocyte. Alternatively, the donor cell may be fused with an enucleated oocyte and four to six hours later, without activation, chromosomes removed and fused with a younger oocyte. Activation would occur thereafter.
- the cells are maintained in the feeder layer in a suitable growth medium, e.g., alpha MEM supplemented with 10% FCS and 0.1 mM beta-mercaptoethanol (Sigma) and L-glutamine.
- a suitable growth medium e.g., alpha MEM supplemented with 10% FCS and 0.1 mM beta-mercaptoethanol (Sigma) and L-glutamine.
- the growth medium is changed as often as necessary to optimize growth, e.g., about every 2-3 days.
- This culturing process results in the formation of embryonic or stem-like cells or cell lines.
- colonies are observed by about the second day of culturing in the alpha MEM medium.
- this time may vary dependent upon the particular nuclear donor cell, specific oocyte and culturing conditions.
- One skilled in the art can vary the culturing conditions as desired to optimize growth of the particular embryonic or stem-like cells.
- Other suitable media are disclosed herein.
- the embryonic or stem-like cells and cell colonies obtained will typically exhibit an appearance similar to embryonic or stem-like cells of the species used as the nuclear cell donor rather than the species of the donor oocyte.
- the cells exhibit a mo ⁇ hology more similar to mouse embryonic stem cells than bovine ES-like cells.
- the individual cells of the human ES-line cell colony are not well defined, and the perimeter of the colony is refractive and smooth in appearance. Further, the cell colony has a longer cell doubling time, about twice that of mouse ES cells. Also, unlike bovine and porcine derived ES cells, the colony does not possess an epithelial-like appearance.
- primate stem cells are SSEA-1 (-), SSEA-4 (+), TRA-1-60 (+), TRA-1-81 (+) and alkaline phosphatase (+). It is anticipated that human and primate ES cells produced according to the present methods will exhibit similar or identical marker expression. Alternatively, that such cells are actual human or primate embryonic stem cells will be confirmed based on their capability of giving rise to all of mesoderm, ectoderm and endoderm tissues. This will be demonsfrated by culturing ES cells produced according to the invention under appropriate conditions, e.g., as disclosed by Thomsen,
- the resultant embryonic or stem-like cells and cell lines preferably human embryonic or stem-like cells and cell lines, have numerous therapeutic and diagnostic applications. Most especially, such embryonic or stem-like cells may be used for cell transplantation therapies. Human embryonic or stem-like cells have application in the freatment of numerous disease conditions.
- mouse embryonic stem (ES) cells are capable of differentiating into almost any cell type, e.g., hematopoietic stem cells. Therefore, human embryonic or stem-like cells produced according to the invention should possess similar differentiation capacity.
- the embryonic or stem-like cells according to the invention will be induced to differentiate to obtain the desired cell types according to known methods.
- the subject human embryonic or stem-like cells may be induced to differentiate into hematopoietic stem cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary fract cells, etc., by culturing such cells in differentiation medium and under conditions which provide for cell differentiation.
- hematopoietic stem cells from an embryonic cell line by subjecting stem cells to an induction procedure comprising initially culturing aggregates of such cells in a suspension culture medium lacking retinoic acid followed by culturing in the same medium containing retinoic acid, followed by transferral of cell aggregates to a substrate which provides for cell attachment.
- one skilled in the art may culture the subject embryonic or stem-like cells to obtain desired differentiated cell types, e.g., neural cells, muscle cells, hematopoietic cells, etc.
- desired differentiated cell types e.g., neural cells, muscle cells, hematopoietic cells, etc.
- Bcl-2 or Bcl-xl might be useful for enhancing in vitro development of specific cell lineages.
- Bcl-2 prevents many, but not all, forms of apoptotic cell death that occur during lymphoid and neural development.
- a thorough discussion of how Bcl-2 expression might be used to inhibit apoptosis of relevant cell lineages following transfection of donor cells is disclosed in U.S. Patent No. 5,646,008, which is herein inco ⁇ orated by reference.
- the subject embryonic or stem-like cells may be used to obtain any desired differentiated cell type. Therapeutic usages of such differentiated human cells are unparalleled.
- human hematopoietic stem cells may be used in medical treatments requiring bone marrow transplantation. Such procedures are used to treat many diseases, e.g., late stage cancers such as ovarian cancer and leukemia, as well as diseases that compromise the immune system, such as AIDS.
- Hematopoietic stem cells can be obtained, e.g., by fusing adult somatic cells of a cancer or AIDS patient, e.g., epithelial cells or lymphocytes with an enucleated oocyte, e.g., bovine oocyte, obtaining embryonic or stem-like cells as described above, and culturing such cells under conditions which favor differentiation, until hematopoietic stem cells are obtained.
- oocyte e.g., bovine oocyte
- Such hematopoietic cells may be used in the treatment of diseases including cancer and AIDS.
- adult somatic cells from a patient with a neurological disorder may be fused with an enucleated animal oocyte, e.g., a primate or bovine oocyte, human embryonic or stem-like cells obtained therefrom, and such cells cultured under differentiation conditions to produce neural cell lines.
- an enucleated animal oocyte e.g., a primate or bovine oocyte, human embryonic or stem-like cells obtained therefrom, and such cells cultured under differentiation conditions to produce neural cell lines.
- oocyte e.g., a primate or bovine oocyte, human embryonic or stem-like cells obtained therefrom, and such cells cultured under differentiation conditions to produce neural cell lines.
- Specific diseases treatable by transplantation of such human neural cells include, by way of example, Parkinson's
- Parkinson's disease it has been demonstrated that transplanted fetal brain neural cells make the proper connections with surrounding cells and produce dopamine. This can result in long-term reversal of Parkinson's disease symptoms.
- donor cells may be any suitable cells.
- CD34-neo may be used for selection of hematopoietic cells, Pwl-neo for muscle cells, Mash-1-neo for sympathetic neurons, Mal-neo for human CNS neurons of the grey matter of the cerebral cortex, etc.
- matrices to facilitate tissue may be used for selection of hematopoietic cells, Pwl-neo for muscle cells, Mash-1-neo for sympathetic neurons, Mal-neo for human CNS neurons of the grey matter of the cerebral cortex, etc.
- the great advantage of the subject invention is that it provides an essentially limitless supply of isogenic or synegenic human cells suitable for transplantation. Therefore, it will obviate the significant problem associated with current transplantation methods, i.e., rejection of the transplanted tissue which may occur because of host- vs-
- the present invention should eliminate, or at least greatly reduce, the need for anti-rejection drugs, such as cyclosporine, imulan, FK-506, glucocorticoids, rapamycin, and derivatives thereof.
- diseases and conditions treatable by isogenic cell therapy include, by way of example, spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver diseases, i.e., hypercholesterolemia, heart diseases, cartilage replacement, burns, foot ulcers, gastrointestinal diseases, vascular diseases, kidney disease, urinary tract disease, and aging related diseases and conditions.
- human embryonic or stem-like cells produced according to the invention may be used to produce genetically engineered or transgenic human differentiated cells.
- this will be effected by introducing a desired gene or genes, which may be heterologous, or removing all or part of an endogenous gene or genes of human embryonic or stem-like cells produced according to the invention, and allowing such cells to differentiate into the desired cell type.
- a preferred method for achieving such modification is by homologous recombination because such technique can be used to insert, delete or modify a gene or genes at a specific site or sites in the stem-like cell genome.
- This methodology can be used to replace defective genes, e.g., defective immune system genes, cystic fibrosis genes, or to introduce genes which result in the expression of therapeutically beneficial proteins such as growth factors, lymphokines, cytokines, enzymes, etc.
- the gene encoding brain derived growth factor may be infroduced into human embryonic or stem-like cells, the cells differentiated into neural cells and the cells fransplanted into a Parkinson's patient to retard the loss of neural cells during such disease.
- cell types fransfected with BDNF varied from primary cells to immortalized cell lines, either neural or non-neural (myoblast and fibroblast) derived cells.
- astrocytes have been fransfected with BDNF gene using retro viral vectors, and the cells grafted into a rat model of Parkinson's disease (Yoshimoto et al.,
- desired genes may be introduced into the subject human embryonic or stem-like cells, and the cells differentiated into desired cell types, e.g., hematopoietic cells, neural cells, pancreatic cells, cartilage cells, etc.
- Genes which may be introduced into the subject embryonic or stem-like cells include, by way of example, epidermal growth factor, basic fibroblast growth factor, glial derived neurotrophic growth factor, insulin-like growth factor (I and II), neurofrophin-3, neurotro ⁇ hin-4/5, ciliary neurotrophic factor, AFT-1, cytokine genes (interleukins, interferons, colony stimulating factors, tumor necrosis factors (alpha and beta), etc.), genes encoding therapeutic enzymes, collagen, human serum albumin, etc.
- TK thymidine kinase
- the subject embryonic or stem-like cells preferably human cells, also may be used as an in vitro model of differentiation, in particular for the study of genes which are involved in the regulation of early development.
- differentiated cell tissues and organs using the subject embryonic or stem- L 5 like cells may be used in drug studies.
- subject cells may be used to express recombinant DNAs.
- subject embryonic or stem-like cells may be used as nuclear donors for the production of other embryonic or stem-like cells and cell colonies.
- cultured inner cell mass, or stem cells, produced according to the invention 20 may be infroduced into animals, e.g., SCID mice, cows, pigs, e.g., under the renal capsule or intramuscularly and used to produce a teratoma therein.
- This teratoma can be used to derive different tissue types.
- the inner cell mass produced by X-species nuclear fransfer may be infroduced together with a biodegradable, biocompatible polymer matrix that provides for the formation of 3-dimensional tissues. After tissue formation, the 25 polymer degrades, ideally just leaving the donor tissue, e.g., cardiac, pancreatic, neural, lung, liver. In some instances, it may be advantageous to include growth factors and proteins that promote angiogenesis.
- the formation of tissues can be effected totally in vitro, with appropriate culture media and conditions, growth factors, and biodegradable polymer matrices.
- Epithelial cells were lightly scraped from the inside of the mouth of a consenting adult with a standard glass slide. The cells were washed off the slide into a petri dish
- the bovine oocyte cytoplasm and the donor nucleus are fused together using elecfrofusion techniques.
- One fusion pulse consisting of 90 V for 15 ⁇ sec was applied to the NT unit. This occurred at 24 hours post-initiation of maturation (hpm) of the oocytes.
- the NT units were placed in CRlaa medium until 28 hpm.
- NT unit activation was at 28 hpm.
- a brief description of the activation procedure is as follows: NT units were exposed for four min to ionomycin (5 ⁇ M; CalBiochem, La Jolla, CA) in TL-HEPES supplemented with 1 mg/ml BSA and then washed for five min in TL- HEPES supplemented with 30 mg/ml BSA. The NT units were then transferred into a microdrop of CRlaa culture medium containing 0.2 mM DMAP (Sigma) and cultured at 38.5 °C 5% C0 for four to five hours.
- the NT units were washed and then placed in a 5 CRlaa medium plus 10% FCS and 6 mg/ml BSA in four well plates containing a confluent feeder layer of mouse embryonic fibroblasts (described below).
- the NT units were cultured for three more days at 38.5 °C and 5% C0 2 .
- the culture medium was changed every three days until day 12 after the time of activation. At this time NT units reaching the desired cell number, i.e., about 50 cell number, were mechanically removed
- Fibroblast cells were plated in tissue culture flasks and cultured in alpha-MEM medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen, UT), penicillin (100 IU/ml) and streptomycin (50 ⁇ l/ml).
- FCS fetal calf serum
- the irradiated fibroblasts were grown and maintained in a humidified atmosphere with 5% C0 2 in air at 37 °C.
- the culture plates which had a uniform monolayer of cells were then used to culture embryonic cell lines. Production of embryonic cell line.
- NT unit cells obtained as described above were washed and plated directly onto irradiated feeder fibroblast cells. These cells included those of the inner portion of the NT unit.
- the cells were maintained in a growth medium consisting of alpha MEM supplemented with 10% FCS and 0.1 mM beta-mercaptoethanol (Sigma). Growth medium was exchanged every two to three days. The initial colony was observed by the second day of culture. The colony was propagated and exhibits a similar mo ⁇ hology to previously disclosed mouse embryonic stem (ES) cells. Individual cells within the colony are not well defined and the perimeter of the colony is retractile and smooth in appearance. The cell colony appears to have a slower cell doubling time than mouse ES cells. Also, unlike bovine and porcine derived ES cells, the colony does not have an epithelial appearance thus far.
- Figures 2 through 5 are photographs of ES-like cell colonies obtained as described, supra.
- the human embryonic cells obtained are transferred to a differentiation medium and cultured until differentiated human cell types are obtained.
- the one NT unit that developed a structure having greater than 16 cells was plated down onto a fibroblast feeder layer. This structure was attached to the feeder layer and started to propagate forming a colony with a ES cell-like mo ⁇ hology (See, e.g., Figure 2).
- ES cell-like mo ⁇ hology See, e.g., Figure 2.
- 4 to 16 cell stage structures were not used to try and produce 5 an ES cell colony, it has been previously shown that this stage is capable of producing ES or ES-like cell lines (mouse, Eistetter et al., Devel Growth and Differ., 31:275-282 (1989); Bovine, Stice et al., 1996)). Therefore, it is expected that 4 - 16 cell stage NT units should also give rise to embryonic or stem-like cells and cell colonies.
- L 0 line with an enucleated bovine oocyte which was cultured in media comprising ACM, uridine, glucose, and 1000 IU of LIF.
- media comprising ACM, uridine, glucose, and 1000 IU of LIF.
- This Example relates to isolation of mitochondria and use thereof to enhance the efficiency of cross-species nuclear transfer.
- the number of mitochondria per cell varies from cell line to cell line.
- mouse L cells contain - 100 mitochondria per cell, whereas there are at least twice that number in HeLa cells.
- the cells are swollen in 20 a hypotonic buffer and ruptured with a few strokes in a Dounce homogenizer using a tight-fitting pestle, and the mitochondria are isolated by differential centrifugation.
- the solutions, tubes, and homogenizer should be pre-chilled on ice. All centrifugation steps are at 40 °C. This protocol is based on starting with a washed cell pellet of 1-2 ml. The cell pellet is resuspended in 11 ml of ice-cold RSB and fransferred 25 to a 16 ml Dounce homogenizer.
- RSB Buffer RSB (A hypotonic buffer for swelling the tissue culture cells) 10 mM NaCl 1.5 mM MgCl 2 MgCl 2 5 The cells are allowed to swell for five to ten minutes. The progress of the swelling is maintained using a phase contrast microscope. The swollen cells are replaced, preferably by several strokes with a pestle. Immediately after, 8 ml of 2.5x MS buffer are added to give a final concentration of lx MS. The top of the homogenizer is then covered with Parafilm and mixed by inverting a couple of times. L O 2.5x MS Buffer
- MS Buffer is an iso-osmotic buffer to maintain the tonicity of the organelles and prevent agglutination. Thereafter, the homogenate is transferred to a centrifuge tube for differential centrifugation. The homogenizer is rinsed with a small amount of MS buffer and added to the homogenate. The volume is brought to 30 ml with MS buffer. The homogenate 25 is then centrifuged at 1300 g for five minutes to remove nuclei, unbroken cells, and large membrane fragments. The supernatant is then poured into a clean centrifuge tube. The nuclear spin-down is repeated twice.
- the supernatant is then transferred to a clean centrifuge tube and a pellet containing the mitochondria is centrifuged at 17,000 g for 15 minutes. The supernatant is discarded and the inside of the tube wiped with a Kimwipe. The mitochondria is washed by re-suspending the pellet in IX MS and repeating the 17,000 g sedimentation. The supernatant is discarded and the pellet is resuspended in a buffer. Mitochondria can be stored at -80 °C for prolonged periods, e.g., up to a year, but preferably will be used shortly thereafter for NT.
- This basic protocol can be modified.
- the cells may be harvested in stationary growth phase when the fewest cells are actively dividing, and CaCl 2 substituted for MgCl 2 in the RSB to stabilize the nuclear membrane.
- the washing of the mitochondrial pellet is omitted as is the density gradient purification. Instead, the mitochondrial pellet is simply resuspended and lysed, and the mitochondrial DNA purified from any remaining nuclear DNA.
- suitable methods for purifying mitachondria and mitochondrial DNA are well known in the art.
- the homogenization buffer should be optimized for the tissue, and the optimal way to homogenize the tissue utilized. Suitable methods are well known in the art. Rat liver is the most frequently used tissue for mitochondrial preparations because it is readily available, is easy to homogenize, and the cells contain a large number of mitochondria (1000-2000 per cell). For example, a motor-driven, Teflon and glass Potter- Elvehjem homogenizer can be used homogenize rat liver. Alternatively, if the tissue is soft enough, a Dounce homogenizer with a loose pestle can be used. The yield and purity of the mitochondrial preparation is influenced by the method of preparation, speed of preparation, and the age and physiological condition of the animal. As noted, methods of purifying mitochondria are well known.
- the buffer, tubes, and homogenizer will be pre-chilled. Pre-chilling a glass and Teflon type homogenizer creates the proper gap between the tube and pestle.
- the centrifugation steps are preferably effected at 40 °C.
- the process will comprise removal of the liver, taking care not to rupture the gall bladder. This is placed in a beaker on ice and any connective tissue is removed. The tissue is recognized and returned to the beaker, e.g., using very sha ⁇ scissors, a scalpel, or razor blade, mince it into 1-2 slices. The pieces are then rinsed, preferably twice, with homogenization buffer (IX MS) to remove most of the blood, and the tissue transferred to the homogenizer tube. Enough homogenization buffer if added to prepare a 1:10 (w/v) homogenate.
- IX MS homogenization buffer
- Mitochondria isolated by the above or other known procedures are inco ⁇ orated, typically by injection, into any of the following (in the case of human donor cell/bovine oocyte nuclear fransfer):
- non-activated, non-enucleated bovine oocytes (i) non-activated, non-enucleated bovine oocytes; (ii) non-activated, enucleated bovine oocytes; (iii) activated, enucleated bovine oocytes;
- enucleated somatic cells typically human (of the same species as donor cell or nucleus)
- Enucleation Procedures 5 Methods for the large-scale enucleation of cells with cytochalasin B are well known in the art. Enucleation is preferably effected using the monolayer technique. This method uses small numbers of cells attached to the growth surface of a culture disc and is ideal if limited numbers of donor cells are available. Another suitable procedure, the gradient technique, requires centrifugation of cells through Ficoll gradients and is best L 0 suited for enucleation of large number (>10 7 ) of cells.
- the monolayer technique is ideal for virtually any cell which grows attached to the growth surface.
- Polycarbonate or polypropylene 250-ml wide-mouth centrifuge bottles with screw- top caps are sterilized by autoclaving.
- the caps preferably are autoclaved separately from L5 the bottle to prevent damage to the centrifuge bottle.
- the bottle are prepared for the enucleation procedure by the sterile addition of 30 ml DMEM, 2 ml bovine serum, and 0.32 ml cytochalasin B (1 mg/ml) to each.
- the caps are placed on the bottles, and the bottles are maintained at 37° prior to use.
- the cells to be enucleated are seeded on a 20 culture dish (35 x 15 mm; Nunc Inc., Naperville, IL). Typically, the cells are grown for at least twenty- four hours on the dishes to promote maximal attachment to the growth surface. Preferably, the cells are prevented from becoming confluent.
- the culture dish is prepared for centrifugation by wiping the outside of the bottom half of the dish (containing the cells) with 70% (v/v) ethanol for the pu ⁇ ose of sterilization. 25 Alternatively, the dish can be kept sterile during cell culturing by maintaining it within a larger, sterile culture dish.
- the medium is removed from the dish and the dish (without top) is placed upside down within the centrifuge bottle.
- the rotor (GSA, DuPont, Wilmington, DE) and centrifuge are preferably pre- warmed to 37° by centrifugation for 30-45 minutes at 8000 ⁇ m.
- the HS-4 swinging- bucket rotor (DuPont) can alternatively be used.
- the optimal time and speed of centrifugation varies for each cell type. For myoblasts and fibroblasts, the centrifuge 5 bottle with the culture dish is placed in the pre-warmed rotor and centrifuged for approximately 20 minutes (interval between the time when the rotor reaches the desired speed and the time when the centrifuge is turned off). Preferably, speeds of 6500 to 7200 ⁇ m are used.
- the centrifuge bottle After centrifugation, the centrifuge bottle is removed from the rotor, and the
- L0 culture plate is removed from the bottles with forceps. A small amount of medium is maintained in the plate to keep the cells moist in order to maintain cell viability.
- the outside of the dish, including the top edge, is wiped with a sterile wiper, then moistened with 95% (v/v) ethanol, to remove any medium and to dry it. A sterile top is placed onto the dish. If the enucleated cells are not going to be used immediately, complete culture
- L 5 medium medium supplemented with the appropriate concentration of serum
- the resultant enucleated cells are fused with any of (i) - (viii) above.
Abstract
Description
Claims
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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 |
WO2001000650A1 (en) * | 1999-06-30 | 2001-01-04 | Advanced Cell Technology, Inc. | Cytoplasmic transfer to de-differentiate recipient cells |
BR0013999A (en) * | 1999-09-14 | 2002-05-21 | Univ Massachusetts Public Inst | Embryonic or stem cell-like cell lines produced by cross-species nuclear transplantation and methods to improve embryonic development by genetic alteration of donor cells or by tissue culture conditions |
AUPR247901A0 (en) * | 2001-01-10 | 2001-02-01 | Garelag Pty Ltd | Activation of nuclear transfer embryos |
AU2002313817A1 (en) | 2001-08-27 | 2003-03-10 | Advanced Cell Technology, Inc. | Trans-differentiation and re-differentiation of somatic cells and production of cells for cell therapies |
EP1486563A4 (en) * | 2002-02-27 | 2008-01-09 | Yasumitsu Nagao | Embryonically modified animal and method of constructing the same |
EP1633768A2 (en) * | 2003-05-13 | 2006-03-15 | The Regents Of The University Of Colorado | Diagnostic and therapeutic treatments related to mitochondrial disorders |
EP2479256A1 (en) | 2004-11-04 | 2012-07-25 | Advanced Cell Technology, Inc. | 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 |
EP2302034B1 (en) | 2005-08-03 | 2020-10-14 | Astellas Institute for Regenerative Medicine | Improved methods of reprogramming animal somatic cells |
US8372644B2 (en) * | 2006-02-06 | 2013-02-12 | Transplantation Limited | Method for growing adult cells |
CN101679942A (en) | 2007-02-23 | 2010-03-24 | 先进细胞技术公司 | Highly efficient methods for reprogramming differentiated cells and for generating animals and embryonic stem cells from reprogrammed cells |
US10865383B2 (en) | 2011-07-12 | 2020-12-15 | Lineage Cell Therapeutics, Inc. | Methods and formulations for orthopedic cell therapy |
CA2980580C (en) | 2015-03-23 | 2024-01-23 | Astellas Institute For Regenerative Medicine | Improved assays for potency of human retinal pigment epithelium (rpe) cells and photoreceptor progenitors |
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WO1999005266A2 (en) * | 1997-07-26 | 1999-02-04 | Wisconsin Alumni Research Foundation | Trans-species nuclear transfer |
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