WO2006100490A1 - Culture medium containing kinase inhibitor, and use thereof - Google Patents
Culture medium containing kinase inhibitor, and use thereof Download PDFInfo
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- WO2006100490A1 WO2006100490A1 PCT/GB2006/001064 GB2006001064W WO2006100490A1 WO 2006100490 A1 WO2006100490 A1 WO 2006100490A1 GB 2006001064 W GB2006001064 W GB 2006001064W WO 2006100490 A1 WO2006100490 A1 WO 2006100490A1
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- Prior art keywords
- cells
- gsk3
- inhibitor
- culture medium
- medium
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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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- 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]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
- C12N2501/727—Kinases (EC 2.7.)
Definitions
- the present invention relates to maintenance of a self renewing phenotype in pluripotent stem cells.
- the methods and compositions provided are suitable for culturing and isolating pluripotent stem cells such as embryonic stem (ES) cells, especially mammalian, including mouse and human, stem cells.
- ES embryonic stem
- this invention relates to self-renewing cultures of mouse and human ES cells and to methods and compositions therefor.
- pluripotent stem cell cultures in the presence of medium containing serum and Leukaemia Inhibitory Factor (LIF) is well known (Smith et al. (1988) Nature 336: 688-90).
- LIF Leukaemia Inhibitory Factor
- Such methods have been used to maintain pluripotent embryonic stem (ES) cells from strains of "permissive" mice over many passages.
- ES embryonic stem
- feeder cells or extracts thereof usually mouse fibroblast cells. Under such conditions it is possible to maintain human ES cells in a pluripotent state over many passages in culture.
- ES cells can only be maintained, or are best maintained, using medium that contains serum or serum extract, and hence is undefined, or using cell culture conditions that require the presence of other cells, such as the fibroblast feeder cells used to maintain human ES cells.
- any undefined component whether in the medium or produced by e.g. the feeder cells, potentially interferes with or hinders research into ES cell propagation and differentiation. This prevents development of good manufacturing practices for therapeutic and other applications of ES cells and their progeny.
- Some defined ES cell media are known but alternative and preferably improved defined media are needed.
- pluripotent stem cells such as ES cells
- serum- free media comprising (1) agonists of gpl30 (e.g. LIF) and (2) agonists of the TGF- ⁇ superfamily (e.g. BMP4) or Id signalling pathways
- gpl30 e.g. LIF
- TGF- ⁇ superfamily e.g. BMP4
- Id signalling pathways Id signalling pathways
- cytokines the purity of which is compromised by the need to manufacture them in cellular systems and then remove potential contaminants from the production broth.
- Another problem with some cytokines is that they have a narrow range of concentration over which they are effective and non-toxic. Media components which have a broader range and/or are less toxic at higher concentrations would be highly useful. Cytokines can also have limited stability in storage, and more stable media components are sought.
- An object of the invention is to overcome or at least ameliorate problems in the art, preferably to provide alternative, more preferably improved, methods of culturing and culture media suitable for pluripotent stem cells, which are capable of supporting self-renewal of said stem cells for many passages.
- a further object of the invention is to provide an alternative culturing system that permits maintenance of a pluripotent stem cell culture in vitro until differentiation of the cells can be induced in a controlled manner.
- a still further object of the invention is to provide methods and compositions that enhance the derivation and isolation of pluripotent stem cells and facilitate their derivation and isolation from organisms refractory to ES cell isolation or from which pluripotent stem cells have not yet been isolated.
- inhibition of GSK3 in a pluripotent cell in the presence of gpl30 signalling is used to promote self-renewal of the cell.
- pluripotent stem cells such as ES cells
- medium preferably serum-free
- an agonist of the gpl30 signalling pathway e.g. LIF
- GSK3 e.g. using a small molecule GSK3 inhibitor
- the invention has a number of applications.
- a combination of gpl30 signalling and inhibition of GSK3 can be used to grow pluripotent cells, especially ES cells, and, where 2006/001064
- a method of expanding stem cells in culture comprises culturing the cells in the presence of an inhibitor of GSK3.
- Culture medium can be prepared containing one or more GSK3 inhibitors.
- ES cells can be derived using GSK3 inhibitors and gp 130 agonists.
- GSK3 inhibition refers to inhibition of one or more GSK3 enzymes.
- GSK3- ⁇ is inhibited.
- GSK3- ⁇ inhibitors are also suitable, and in general inhibitors for use in the invention inhibit both.
- a wide range of GSK3 inhibitors are known and, by way of example, the inhibitors CHIR 98014, CHIR 99021, AR-AO144-18, SB216763 and SB415286 have been used to promote self renewal of ES cells.
- Other inhibitors are known and useful in the invention.
- the inhibitors of certain embodiments are specific for GSK3- ⁇ and GSK3- ⁇ , substantially do not inhibit erk2 and substantially do not inhibit cdc2.
- the inhibitors have at least 100 fold, more preferably at least 200 fold, very preferably at least 400 fold selectivity for human GSK3 over mouse erk2 and/or human cdc2, measured as ratio OfIC 50 values; here, reference to GSK3 IC 50 values refers to the mean values for human GSK3- ⁇ and GSK3- ⁇ . Good results have been obtained with CHIR 99021 and CHIR 98014, which both are specific for GSK3. Examples of GSK3 inhibitors are described in Bennett C, et al, J. Biological Chemistry, vol. 277, no. 34, August 23 2002, pp30998-31004 and in Ring DB, et al, Diabetes, vol. 52, March 2003, p ⁇ 588- 595. Suitable concentrations for use of CHIR 99021 are in the range 0.01 to 100, preferably 0.1 to 10, more preferably 0.3 to 3 micromolar.
- a method of promoting self-renewal of mouse pluripotent cells in culture comprises (i) inhibiting GSK3, and (ii) activating gpl30 downstream signalling.
- Activation of one or more gpl30 downstream signalling pathways can be achieved by use of a cytokine acting through gpl30, for example a cytokine or other agonist of the
- Cytokines capable of acting through gpl30, and thus of activating gpl30 signal transduction include LIF, CNTF, cardiotrophin, oncostatin M, IL-6 plus sIL-6 receptor and hyper IL-6.
- Suitable cytokines include mimetics, fusion proteins or chimaeras that can bind to and/or activate signalling though gpl30. The role of cytokines acting through gpl30 in the presence of serum is well established, but the capacity of those cytokines to sustain undifferentiated cells in the absence of serum is limited.
- An advantage of the invention is that in the presence of GSK3 inhibitor and gpl30 agonist pluripotent cells can be grown in defined medium.
- the present invention therefore enables alternative and/or improved culture of ES cells in medium that is free of serum, serum extract, feeder cells and feeder cell extract.
- Embryonic stem cells have been reported from a number of mammalian sources including mouse (Bradley et al (1984) Nature 309: 255-56), American mink (MoI
- a second aspect of the invention provides a method of culture of pluripotent cells, especially ES cells, so as to promote self renewal, comprising maintaining the cells in medium containing:-
- Methods of the invention can be used generally for growing pluripotent cells, including growing ES cells in medium which is free of serum and free of serum extract, which cells have previously been passaged in the presence of serum or serum extract. Preferably, such methods are also carried out in the absence of feeder cells and/or feeder cell extracts.
- culture of ES cells can be carried out comprising the steps of:-
- the present invention also provides a method of obtaining a transfected population of ES cells, comprising:-
- the selectable marker may encode antibiotic resistance, a cell surface marker or another selectable marker as described e.g. in EP-A-0695351, and preferably comprises a nucleotide sequence encoding the selectable marker operatively linked to a promoter which preferentially expresses the selectable marker in desired cells.
- the present invention provides a method of culture of pluripotent, especially ES, cells, comprising the steps of transferring an individual cell to a culture vessel, such as an individual well on a plate, and culturing the cell in the presence of a GSK3 inhibitor and an activator of gpl30 downstream signalling pathways, so as to obtain a clonal population of pluripotent, especially ES, cells, all of which are progeny of a single cell.
- the culture conditions can be altered to direct differentiation of the cells into one or more cell types selected from ectodermal, mesodermal or endodermal cell fates. Addition of, or withdrawal of cytokines and signalling factors, can enable the derivation of specific differentiated cell populations at high efficiency. Differentiation of an ES cell towards a non- neuroectodermal fate may be achieved by maintaining the ES cell in the presence of a cytokine acting through gpl30 and a GSK3 inhibitor and then withdrawing the cytokine whilst maintaining the GSK3 inhibitor and/or adding a further signalling molecule capable of directing differentiation.
- the methods described above all optionally includes the step of obtaining and/or isolating a differentiated cell which is the product of the process.
- One medium is for self-renewal of pluripotent, especially ES, cells, the medium comprising an inhibitor of GSK3 and an activator of a gpl30 downstream signalling pathway.
- Another medium of the invention is a stem cell culture medium, comprising an inhibitor of GSK3.
- the invention provides medium that is free of serum and serum extract.
- One such medium comprises :-
- Preferred medium for human pluripotent stem cells may be different in that it may be free of gpl30 agonists; it hence comprises a GS K3 inhibitor, and an agonist of the FGF receptor, optionally supplemented with an activator of gpl30 downstream signalling pathways.
- a specific human pluripotent stem cell medium comprises (a) a GSK3 inhibitor, and (b) FGF, and optionally (c) LIF or hyper IL-6.
- Preferred medium for pluripotent stem cells other than human stem cells, such as but not limited to medium for mouse cells comprises a GSK3 inhibitor, an activator of gpl30 downstream signaling pathways and an inhibitor of ES cell differentiation.
- a specific medium for non-human pluripotent stem cells comprises (a) LIF, (b) a GSK3 inhibitor and (c) optionally an inhibitor of FGF. Substitutions of media components can be made as described herein.
- Basal medium is medium that supplies essential sources of carbon and/or vitamins and/or minerals for the cells.
- the basal medium is generally free of protein and incapable on its own of supporting self-renewal of cells.
- the iron transporter provides a source of iron or provides ability to take up iron from the culture medium. Suitable iron transporters include transferrin and apotransferrin. It is preferred that the medium further comprises one or more of insulin or insulin-like growth factor and albumin (preferably recombinant) or albumin substitute, and is free of feeder cells and feeder cell extract.
- a particular medium of the invention comprises LIF, GSK3 inhibitor, insulin, albumin and transferrin, with or without additional basal medium.
- LIF can be substituted by other activators of gpl30 signalling, though preferred medium comprises the gpl30 receptor binding cytokine, LIF, suitable concentrations of which are generally between lOU/ml and 1000U/ml, more preferably between 50U/ml and 500U/ml, even more preferably in the region of 100 U/ml.
- the GSK3 inhibitor is preferably as described herein in more detail.
- the invention further provides a method of deriving a pluripotent cell from a blastocyst, comprising: -
- the method comprises culturing the blastocyst in LIF 5 more preferably for a period of from 2 to 4 days.
- the isolated cell or cells are preferably cultured in serum free medium. Typically, the cells are replated as clumps.
- the blastocyst is also preferably cultured in serum free medium, optionally in the absence of an agonist of the BMP receptor.
- culture of cells is carried out in an adherent culture, which may be promoted by the inclusion of a cell adhesion protein on culture substrate. It is also preferred to culture pluripotent cells according to the invention in monolayer culture, though it is optional for cells to be grown in suspension culture or as pre-cell aggregates; cells can also be grown on beads or on other suitable scaffolds such as membranes or other 3-dimensional structures.
- a further component of medium for culture of pluripotent cells according to the invention, and which is preferred to be present, is a factor promoting survival and/or metabolism of the cells.
- cells are cultured in the presence of insulin.
- An alternative factor is insulin-like growth factor and other such survival and/or metabolism promoting factors may alternatively be used.
- Culture medium used in the examples of the invention preferably also comprises serum albumin.
- serum albumin This can be used in purified or preferably recombinant form, and if in a recombinant form this has the advantage of absence of potential contaminating factors, cytokines etc.
- the culture medium does not need to contain serum albumin and this component can be omitted or replaced by another bulk protein or by a synthetic polymer (polyvinyl alcohol) as described by Wiles et al. 2006/001064
- a particularly preferred medium of the invention is one that is fully defined.
- This medium does not contain any components which are undefined, that is to say components whose content is unknown or which may contain undefined or varying factors that are unspecified.
- An advantage of using a fully defined medium is that efficient and consistent protocols for culture and subsequent manipulation of pluripotent cells can be derived. Further, it is found that maintenance of cells in a pluripotent state is achievable with higher efficiency and greater predictability and that when differentiation is induced in cells cultured using a defined medium the response to the differentiation signal is more homogenous than when undefined medium is used.
- the invention also provides concentrates which can be used as additives for culture medium, and kits of components, for preparation of culture medium, the resultant medium being in accordance with the invention.
- One kit of the invention comprises first and second containers, the first containing a gpl30 agonist and the second containing a GSK3 inhibitor.
- the kits are preferably formulated so that the contents of each container can be added to culture medium so as to obtain a culture medium of the invention.
- the kits preferably contain concentrated stock solutions of their respective components.
- Methods of the invention also include a method of obtaining a differentiated cell comprising culturing a pluripotent cell as described and allowing or causing the cell to differentiate, wherein the cell contains a selectable marker which is capable of differential expression in the desired differentiated cell compared with other cell- types, including pluripotent stem cells, whereby differential expression of the selectable marker results in preferential isolation and/or survival and/or division of the desired differentiated cells.
- the differentiated cell can be a tissue stem or progenitor cell, and may be a terminally differentiated cell.
- the invention extends to a cell obtained by following any of the methods of the invention described herein.
- Cells of the invention can be used in assays for drug discovery.
- Cells of the invention may also be used for cell therapy, and thus a method of the invention comprises using a combination of gpl30 signalling and inhibition of GSK3 to derive and/or maintain pluripotent cells, deriving cells for cell therapy therefrom and using those cells in cell therapy.
- a method of culturing pluripotent cells comprises maintaining the cells in medium comprising an agonist of the FGF receptor and a GSK3 inhibitor.
- the FGF receptor agonist is preferably bFGF.
- the GSK3 inhibitor is preferably as described herein in relation to other aspects of the invention. The method is particularly suited to human pluripotent cells.
- Another such aspect comprises expressing an Eras gene in a pluripotent cell, especially a human cell, and culturing that cell in the presence of a GSK3 inhibitor.
- GSK3 inhibitor is used to promote self renewal of a cell in which Eras are otherwise activated or in which there is an equivalent signal, e.g. expression of an Eras gene on a transgene, induction of Eras expression, overexpression of a PB kinase or expression of a PI3 kinase on a transgene.
- Culture medium for these aspects of the invention comprises an agonist of a FGF receptor and a GSK3 inhibitor.
- Cell culture components may be identified which are relatively non-toxic and cell permeable.
- the GSK3 inhibitors used in the invention can be purified easily, especially compared to, say, purification of protein cytokines.
- Recombinant proteins can be expensive to make and the small molecule medium components may be more cheaply produced and more stable in storage, with a wider effective concentration range.
- Specific embodiments set out below used a combination of CHIR 99021 and LIF in a serum-free, fully defined medium and gave improved self renewal of mouse ES cells with very little differentiation. It is occasionally reported when culturing ES cells in the presence of BMP that there is some neurogenesis. This was not seen in the examples of the invention.
- Fig. 1 shows E14.1A ES cells weaned off feeder cells and grown in LIF+CHIR99021 with serum;
- Fig. 2 shows the same ES cells in crisis in LIF and serum without CHIR99021 when they were weaned off feeder cells;
- Fig. 3 shows El 4. IA mouse ES cells in serum-free medium
- Fig. 4 shows mouse ES cells grown in defined DMEM/F12+N2 medium
- Fig. 5 shows hES 181 grown in N2B27 medium with bFGF, LIF and BMP4;
- Fig. 6 shows human ES cells (hES 181) stably expressing eGFP.
- GSK-3 ⁇ inhibitors Eras, culture medium and ES cell self-renewal
- Mouse and human ES cells were grown under various conditions, using N2B27 medium unless otherwise stated and in the presence or absence of the GSK-3 ⁇ inhibitors CHIR99021, AR-AO144-18, SB216763 and SB415286.
- ImI apo-transferrin (final concentration lOmg/ml), 0.67ml BSA (final concentration 5mg/ml), 33 ⁇ l progesterone (final concentration 2 ⁇ g/ml), lOO ⁇ l putrescine (final concentration 1.6mg/ml), lO ⁇ l sodium selenite (final concentration 3 ⁇ M) and 7.187ml DMEM/F12. Store at 4 0 C and use within 1 month.
- DMEM/F12-N2 medium to 100ml of DMEM/F12, add ImI of N2 10Ox stock solution.
- the final concentration of each component of N2 in the DMEM/F12 medium is: insulin, 25 ⁇ g/ml; apo-transferrin, lOO ⁇ g/ml; progesterone, 6ng/ml; putrescine, 16 ⁇ g/ml; sodium selenite, 3OnM; BSA 50 ⁇ g/ml. Store at 4 0 C and use within 1 month.
- Neurolbasal/B27 medium to 100ml of NeurolbasalTM Medium, add 2ml of B27 and 0.5- ImI of 20OmM L-glutamine. Store at 4 0 C and use within 1 month.
- N2B27 medium mix DMEM/F12-N2 medium with Neurolbasal/B27 medium in the ratio of 1 :1. Add ⁇ -mercaptoethanol to a final concentration of 0.ImM from the 0.1 M stock. Store at 4 0 C and use within 1 month.
- LIF plus GSK-3 ⁇ inhibitors prevented feeder-dependent ES cells going into crisis when they are "weaned off' feeder cells, which ES cells were then successfully propagated in the presence of LIF plus GSK-3 ⁇ inhibitors.
- Human ES cells grew in feeder-free and conditioned medium-free conditions after forced expression of an Eras gene:- a) in N2B27 medium with bFGF, grown on laminin, fibronectin, vitronectin or matrigel; b) the same as a) but adding BMP4 (5ng/ml) as well as bFGF; and c) the same as a) but adding a GSK-3 ⁇ inhibitor as well as bFGF.
- ES cells are maintained in a combination of a GSK3 inhibitor and gpl30 signalling, or a combination of Eras signalling and GSK3 inhibitor and the invention also provides culture methods and media therefor.
Abstract
Description
Claims
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GB0505970D0 (en) | 2005-04-27 |
US20090130759A1 (en) | 2009-05-21 |
EP1863904A1 (en) | 2007-12-12 |
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