Compositions and methods for establishing and maintaining stem cells in an undiffferentiated state   

Abstract: The present invention embraces compositions and methods for establishing and maintaining stem cells and inhibiting stem cell differentiation using a selective Protein Kinase C (PKC) inhibitor.

This application is a divisional of U.S. Ser. No. 12/813,688 filed Jun. 11, 2010, which claims benefit of priority to U.S. Provisional Application Ser. Nos. 61/186,485, filed Jun. 12, 2009, and 61/249,722, filed Oct. 8, 2009, the contents of which are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Embryonic stem cells, referred to as ES cells, are derived from the inner cell mass (ICM) of embryos in the blastocyst phase, and can be cultured and maintained in vitro while being kept in an undifferentiated state. ES cells are pluripotent, possessing the capability of developing into any organ or tissue type or, at least potentially, into a complete embryo. For example, ES cells can differentiate and give rise to a succession of mature differentiated cells. Differentiation has been shown in tissue culture and in vivo.

An important application of human ES cells is their use in regenerative medicine, tissue engineering, and cell therapy: the treatment of symptoms, diseases, conditions, and disabilities with ES cell-derived replacement cells and tissues. Many diseases and disorders result from disruption of cellular function or destruction of tissues of the body. A wide spectrum of diseases may be treated based upon both the possession of a population of cells having multi-lineage potential and an understanding of the mechanisms that regulate embryonic cell development. Pluripotent stem cells that are stimulated in vitro to develop into specialized cells offer the possibility of a renewable source of replacement cells and tissue to treat numerous diseases, conditions, and disabilities.

ES cells have been derived from mouse (Evans & Kaufman (1981) Nature 292:154-156; Martin (1981) Proc. Natl. Acad. Sci. USA 78:7634-7639), hamster (Doetschmann, et al. (1999) Dev. Biol. 127:224-227), sheep (Handyside, et al. (1987) Roux\'s Arch. Dev. Biol. 198:48-55; Notarianni, et al. (1991) J. Reprod. Fertil. 43:255-260), cow (Evans, et al. (1990) Theriogenology 33:125-128), rabbit (Giles, et al. (1993) Mol. Reprod. Dev. 36:130-138), mink (Sukoyan, et al. (1993) Mol. Reprod. Dev. 36:148-158) and pig (Piedrahita, et al. (1988) Theriogenology 29:286; Evans, et al. (1990) supra; Notarianni, et al. (1990) J. Reprod. Fertil. Suppl. 41:51-56). The derivation of human ES cells has also been reported (Thomson, et al. (1998) Science 282:1145-1147; Shamblott, et al. (1998) Proc. Natl. Acad. Sci. USA 95:13726-13731).

Various methods have been described for maintaining ES cell pluripotency and to derive new ES and induced pluripotent stem (iPS) cells (Evans & Kaufman (1981) Nature 292:154-156; Niwa, et al. (1998) Genes Dev. 12:2048-2060; Sato, et al. (2004) Nature Med. 10:55-63; Takahashi & Yamanaka (2006) Cell 126:663-676; Ying, et al. (2003) Cell 115:281-292; Ying, et al. (2008) Nature 453:519-523). For example, screens for molecules that increase cloning efficiency have been described (U.S. Patent Application No. 2008/0171385). In addition, it has been shown that mouse ES cells can remain undifferentiated indefinitely in the presence of an embryonic fibroblast feeder layer. Similarly, it is reported that a feeder layer composed of mitotically inactivated mouse embryonic fibroblasts (MEFs) or other fibroblasts is required for human ES cells to remain in an undifferentiated state (see, e.g., U.S. Pat. No. 6,200,806; Amit, et al. (2000) Dev. Biol. 227:271-78; Odorico, et al. (2001) Stem Cells 19:193-204). However, while mouse ES cells will also remain undifferentiated in the absence of an embryonic fibroblast feeder layer so long as the medium is supplemented with leukemia inhibitory factor (LIF) (Smith, et al. (1988) Nature 336:688-690; Williams, et al. (1988) Nature 336:684-687), human ES cells differentiate or die in the absence of a fibroblast feeder layer, even when the medium is supplemented with LIF (Thomson, et al. (1998) supra).

SUMMARY

The present invention features methods for establishing and maintaining stem cells and for inhibiting stem cell differentiation using a selective Protein Kinase C (PKC) inhibitor. According to particular embodiments of the invention, the PKC inhibitor inhibits at least the zeta isoform of PKC. In other embodiments, the PKC inhibitor further inhibits the alpha and delta isoforms of PKC. In specific embodiments, the inhibitor is used in the range of 100 nm to 5 μM. In particular embodiments, the PKC inhibitor has a structure as set forth in Formulae I-IV as described herein. Stem cell lines which can be established and maintained in accordance with the methods of the invention include those isolated from mouse, rat or human. In particular embodiments, the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or cancer stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that Gö6983 treated ES cells maintain the expression of pluripotency markers (FIG. 1A) without induction of differentiation markers (FIG. 1B), even in the absence of serum, LIF, and BMP4 (FIG. 1C) and in the presence of collagen IV (FIG. 1D). The plots show quantitative RT-PCR analysis of markers (mean±standard error). Data presented in FIG. 1D is RT-PCR analysis of lineage-specific marker expression in E14 cells upon withdrawal of Gö6983 after five passages on collagen IV. FIG. 1E, Differentiation potential of collagen IV was determined by measuring mRNA expression of pluripotency and lineage markers. mRNA levels were measured after culturing on collagen IV for 5 days in the absence and presence of Gö6983 (mean±standard error; three independent experiments).

FIG. 2 shows the expression of PKC isoforms (FIG. 2A) upon specific knock-down of PKC ζ and the resulting effect on the expression of pluripotency markers (FIGS. 2B and 2C). Expression of PKC isoforms and pluripotency markers was determined by RT-PCR analysis (mean±standard error). Knock-down cells were grown in the absence of LIF (FIG. 2B) and cultured on collagen IV for 5 days in the presence and absence of Gö6983 (FIG. 2C).

FIG. 3 shows the results of RT-PCR analysis of NF-κB target gene expression in E14 ES cells, cultured with or without LIF and Gö6983, and in PKCζkd cells, with or without ectopic expression of RNAi-immune PKCζ cDNA (FIG. 3A) as well as PKCζkd cells cultured at different culture conditions on collagen IV (FIG. 3B). Data is presented as mean±standard error of three independent experiments. Plaur; plasminogen activator, urokinase receptor, Vim; vimentin, and Igfpb2; insulin-like growth factor binding protein 2.

FIG. 4 shows the time course of iPSC colony formation (FIG. 4A) and relative number of iPSC colonies that were derived in the presence of LIF and Gö6983 (FIG. 4B).

FIG. 5 shows that human ES cells treated with Gö6983 exhibited levels of SSEA4 expression comparable to cultures grown in TeSR1 medium or conditioned medium containing bFGF, which maintain human ES cell pluripotency.

DETAILED DESCRIPTION

Highly orchestrated signaling mechanisms and gene expression patterns endow embryonic stem (ES) cells with the capacity to maintain pluripotency or to differentiate into other cell types of an organism. It has now been found that pharmacological inhibition of Protein Kinase C (PKC) isoforms by a selective PKC inhibitor maintains the undifferentiated phenotype of multiple ES cell lines in the absence of leukemia inhibitory factor (LIF) and mouse embryonic fibroblast (MEF) feeder cells. Inhibition of PKC function also strongly inhibits differentiation of stem cells under strong differentiation cues like culturing on Collagen IV or treatment with retinoic acid (RA), which strongly induce mesodermal and ectodermal differentiation, respectively (Nishikawa, et al. (1998) Development 125:1747; Lee, et al. (2007) Stem Cells 25:2191). Stem cells maintained for multiple passages with PKC inhibitor generate chimeric mice when injected into blastocyst. In addition, new stem cell lines can be efficiently derived, and propagated in the presence of the PKC inhibitor. Inhibition of stem cell differentiation is functionally reversible as withdrawal of the inhibitor leads to a multidifferentiation program in stem cells, i.e., the cells can be induced to differentiate into one more lineages. Mechanistic analysis revealed that PKC inhibition of ES cell differentiation is associated with the continuous presence of polycomb repressor complex 2 (PRC2) at the developmental genes. These results indicate that PKC signaling is an important pathway to dictate maintenance of stem-ness vs. differentiation of mammalian embryonic stem cells and also indicate that the use of PKC inhibitors like Gö6983 are useful for establishing new mammalian stem cells for regenerative medicine purposes.

Accordingly, the present invention embraces methods for establishing and maintaining stem cells in an undifferentiated state by exposing the cells to a selective Protein Kinase C inhibitor. As is conventional in the art, a “stem cell” is a cell characterized by the ability to renew itself through mitotic cell division and differentiate into a diverse range of specialized cell types. In this respect, a stem cell of the invention possesses pluripotency and self-renewal. As used herein, the term “pluripotent” or “pluripotency” refers to the ability of a cell to develop into one of ectodermal, endodermal and mesodermal cell fate or lineage. Stem cells embraced by the present invention include, but are not limited to embryonic stem cells, adult stem cells, induced pluripotent stem cells, and cancer stem cells. “Embryonic stem cell” include cells obtained from embryos or fetuses. Adult stem cells tissue-specific stem cells such as hematopoietic stem cells. In adult organisms, tissue-specific stem cells and progenitor cells replenish specialized cells, and also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues. Induced pluripotent stem cells are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing “forced” expression of certain genes (Takahashi & Yamanaka (2006) Cell 126:663). Cancer stem cells are cancer cells (i.e., found within tumors or hematological cancers) that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. The term “cell” as used herein refers to individual cells, cell lines, or cultures derived from such cells. A “cell line” refers to a composition comprising isolated cells of the same type.

In accordance with the present invention, a pluripotent stem cell line is established by culturing cells, such as embryonic cells or adult cells, with a selective Protein Kinase C inhibitor. Embryonic cells, such as blastocytes or cells isolated from a blastocyst (e.g., fibroblasts) can be isolated from any mammal including, but not limited to, mice, rats, pigs, humans and the like. The establishment of iPSCs is also embraced by this method, wherein the cells being contacted with the PKC inhibitor are adult cells that express one or more reprogramming factors (e.g., Oct4, Sox2, Klf4 and/or c-Myc). As described herein, the cells can be plated directly on gelatin-coated plates containing the PKC inhibitor to establish a pluripotent stem cell line. By “isolated” herein is meant free from at least some of the constituents with which a component, such as a cell, is found in its natural state. More specifically, isolated can mean free from 70%, 80%, 90%, or 95% of the constituents with which a component is found in its natural state.

When passaged or cultured in the presence of a PKC inhibitor, stem cells maintain their undifferentiated phenotype or pluripotency. As such, the present invention also embraces a method for maintaining the undifferentiated phenotype of a stem cell by culturing or contacting an isolated stem cell with a selective Protein Kinase C inhibitor. The terms “maintaining” and “maintenance” refer to the stable preservation of the characteristics or phenotypes of the stem cells when cultured under specific culture conditions. Such phenotypes can include the cell morphology and gene expression profiles of the stem cells, which can be determined using the techniques described herein. For example, stem cells maintained or established in accordance with the present invention express pluripotency markers including Oct4, Nanog, Sox2 and Rex-1. The term “maintain” can also encompass the propagation of stem cells, or an increase in the number of stem cells being cultured. The invention contemplates culture conditions that permit the stem cells to remain pluripotent, while the stem cells may or may not continue to divide and increase in number.

As used herein, the terms “develop”, “differentiate” and “mature” all refer to the progression of a cell from the stage of having the potential to differentiate into at least two different cellular lineages to becoming a specialized and terminally differentiated cell. As such, the term “undifferentiated” is intended to mean a cell that has not progressed to a specialized and terminally differentiated stage.

According to particular embodiments of the invention, the undifferentiated phenotype of the embryonic stem cells is maintained in the absence of a feeder cell or leukemia inhibitory factor (LIF). The term “feeder cell” refers to a culture of cells that grows in vitro and secretes at least one factor into the culture medium, and that can be used to support the growth of another cell of interest in culture.

As demonstrated herein, culturing of stem cells in the presence of a selective PKC inhibitor inhibits differentiation of the stem cell, even under differentiation conditions, e.g., absence of LIF or presence of differentiation factors such as collagen IV. Accordingly, the present invention also embraces a method of inhibiting differentiation of a stem cell by contacting the stem cell with a selective PKC inhibitor. For the purposes of the present invention, differentiation can be inhibited partially or completely depending on the PKC inhibitor selected. Partial inhibition is intended to mean that 20%, 30%, 40%, 50%, 60%, 70% or 80% of the cells in the culture exhibit a differentiated phenotype, whereas complete inhibition is intended to mean that 95%, 99%, or 100% of the cells in the culture are undifferentiated.

The selection of the selective PKC inhibitor to be used in accordance with the present invention will be dependent upon the effect to be achieved, i.e., partial or complete inhibition of differentiation or partial or complete maintenance of the undifferentiated phenotype. For example, inhibition of PKC zeta alone can provide partial inhibition of differentiation, whereas a combination of inhibitors, or an inhibitor that inhibits multiple PKC isoforms, can provide complete inhibition of differentiation. Thus, the methods of this invention can employ any PKC inhibitor known in the art including non-specific PKC inhibitors and specific PKC inhibitors of different isoforms. However, in particular embodiments, the inhibitor of the invention is selective in that it inhibits the activity of one or more PKC isoforms and does not inhibit other protein kinases, e.g., protein kinase A, casein kinase I, protein kinase G or rho-associated kinase II. Information about selective PKC inhibitors, and methods for their preparation are readily available in the art. For example, various PKC inhibitors and their preparation are described in U.S. Pat. Nos. 5,621,101; 5,621,098; 5,616,577; 5,578,590; 5,545,636; 5,491,242; 5,488,167; 5,481,003; 5,461,146; 5,270,310; 5,216,014; 5,204,370; 5,141,957; 4,990,519; and 4,937,232, all of which are incorporated herein by reference. Commercial sources of selective protein kinase C inhibitors include Calbiochem, Sigma, and Tocris Biosciences. By way of illustration, Table 1 lists PKC inhibitors with selectivity for one or more PKC isoforms.

TABLE 1 Isoform Specificity* Inhibitor α β γ δ ζ ε Chelerythrine chloride C X X X X X Gö6983 X X X X X Gö6976 X X2 Ro-31-8425 X X1,2 X X Rottlerin X Bisindolylmaleimide I X X1 X X X ISSI-3521 X Midostaurin X X1,2 X Calphostin C X X1,2 X X X Ro-31-8220 X X1,2 X *Isoforms not listed may also be inhibited. 1PKC β1. 2PKC β2.

Preferably, the present invention employs those Protein Kinase C inhibitors that effectively inhibit at least the ζ isozyme. In certain embodiments, one or more PKC inhibitors are employed wherein said inhibitors effectively inhibit at least the ζ, α and δ isoforms. In particular embodiments, one or more PKC inhibitors are employed wherein said inhibitors effectively inhibit at least the α, β, γ, δ and ζ isoforms of PKC. In certain embodiments, the inhibitor employed in the instant methods is a bisindolylmaleimide. In so far as 2-(6-Phenyl-1H-indazol-3-yl)-1H-benzo[d]imidazoles have also been shown to be potent and isoform selective PKC ζ inhibitors (Trujillo, et al. (2009) Bioorg. Med. Chem. Lett. 19:908-911), the present invention also embraces the use of benzoimidazoles in the instant methods.

Examples of bisindolylmaleimides include Gö6983 (3-[1-[3-(dimethylamino)propyl]5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione) and bisindolylmaleimide I (GF109203X).

Moreover, PKC inhibitors can be derived from the structure of bisindolylmaleimides and/or benzoimidazoles. PKC inhibitors particularly embraced by the invention are as set forth in formulae I, II, III, IV, and V,

or a pharmaceutically acceptable salt, hydrate, or prodrug thereof.

In any of formula I, II, III, IV, or V, each of R1 and R2 is independently selected from —H, halogen, haloalkoxy, —CN, —NO2, —OR3, —N(R3)R3, —S(O)O-2R3, —N(R3)C(═O)N(R3)R3, —N(R3)C(═O)C(═O)N(R3)R3, —SO2N(R3)R3), —CO2R3, —C(═O)N(R3)R3, —C(═NR5)N(R3)R3, —C(═NR5)NR3, —N(R3) SO2R3, —N(R3)C(O)R3, —NCO2R3, —N(R3)C(O)2R3 (—N(R3)C(O)2R3), —C(═O)R3, optionally substituted alkoxy, optionally substituted lower alkyl, optionally substituted aryl, optionally substituted lower arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, or optionally substituted lower heterocyclylalkyl;

Y is selected from O, or HH;

each of m and n is independently 1 to 5;

each of A and B is independently selected from a five- to ten-membered aryl of heteroaryl;

R3 is selected from —H, optionally substituted lower alkyl, optionally substituted aryl, optionally substituted lower arylalkyl, optionally substituted heterocyclyl, or optionally substituted lower heterocycylalkyl;

two of R3, together with nitrogen to which they are attached, can combine to form an optionally substituted heterocycyl containing between one and three additional heteroatoms;

R4 is selected from —H and optionally substituted lower alkyl;

each R5 is independently selected from —H, —CN, —NO2, —OR3, —S(O)O-2R3, —CO2R3, optionally substituted lower alkyl, optionally substituted lower alkenyl, or optionally substituted lower alkynyl; and

each of L1 and L2 is independently selected from absent and —C(R3)(R3)—, —C(R3)(R3)C(R3)(R3)—, —C(R3)(R3)C(R3)(R3)C(R3)(R3)—, —C(R3)(R3)C(R3)(R3)C(R3)(R3)C(R3)(R3)—.

Examples of “halogen” groups include fluorine, chlorine, bromine and iodine.

“Haloalkoxy” means a group of the formula —OR, wherein R is a haloalkyl group. Examples of haloalkoxy moieties include, but are not limited to, trifluoromethoxy, difluoromethoxy, 2,2,2-trifluoroethoxy, and the like.

“Alkoxy” means a moiety of the formula —OR, wherein R is an alkyl moiety. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropoxy, tert-butoxy and the like.

“Lower alkyl” refers to a linear or branched alkyl group of one to six carbon atoms, i.e., C1-C6 alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, sec-butyl, tert-butyl, pentyl, n-hexyl, octyl, dodecyl, and the like.

“Lower alkenyl” refers to an alkenyl group containing from two to eight carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain or cyclic and include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like.

“Lower alkynyl” refers to an alkynyl group containing from two to eight carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like.

The term “aryl” means an aromatic radical having 4 to 18 carbon atoms and includes heteroaromatic radicals. Examples include monocyclic groups, as well as fused groups such as bicyclic groups and tricyclic groups. Some examples include phenyl group, indenyl group, 1-naphthyl group, 2-naphthyl group, azulenyl group, heptalenyl group, biphenyl group, indacenyl group, acenaphthyl group, fluorenyl group, phenalenyl group, phenanthrenyl group, anthracenyl group, cyclopentacyclooctenyl group, and benzocyclooctenyl group, pyridyl group, pyrrolyl group, pyridazinyl group, pyrimidinyl group, pyrazinyl group, triazolyl group, tetrazolyl group, benzotriazolyl group, pyrazolyl group, imidazolyl group, benzimidazolyl group, indolyl group, isoindolyl group, indolizinyl group, purinyl group, indazolyl group, furyl group, pyranyl group, benzofuryl group, isobenzofuryl group, thienyl group, thiazolyl group, isothiazolyl group, benzothiazolyl group, oxazolyl group, and isoxazolyl group.

“Lower arylalkyl” refers to an arylalkyl where the “alkyl” portion of the group has one to six carbons; this can also be referred to as C1-6 arylalkyl.

A “cycloalkyl” group refers to a group with three to ten carbons, and it may, for example, be a monocyclic group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cyclooctyl, or a condensed polycyclic group. Further, the secondary substituent in these substitutable groups may, for example, be halogen, alkyl, alkoxy or hydroxy.

“Cycloalkylalkyl” means a group of the formula —R′—R″, where R′ is alkylene (e.g., a linear saturated divalent hydrocarbon radical of one to six carbon atoms or a branched saturated divalent hydrocarbon radical of three to six carbon atoms), and R″ is cycloalkyl as defined herein.

“Heteroaryl” means a monocyclic, bicyclic or tricyclic radical of 5 to 12 ring atoms having at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, or S, the remaining ring atoms being C, with the understanding that the attachment point of the heteroaryl radical will be on an aromatic ring. The heteroaryl ring may be optionally substituted. Examples of heteroaryl moieties include, but are not limited to, optionally substituted imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyrazinyl, pyridazinyl, thiophenyl, furanyl, pyranyl, pyridinyl, pyrrolyl, pyrazolyl, pyrimidyl, quinolinyl, isoquinolinyl, quinazolinyl, benzofuranyl, benzothiophenyl, benzothiopyranyl, benzimidazolyl, benzoxazolyl, benzooxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzopyranyl, indolyl, isoindolyl, indazolyl, triazolyl, triazinyl, quinoxalinyl, purinyl, quinazolinyl, quinolizinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl and the like.

“Heteroarylalkyl” refers to a radical-RaRb where Ra is an alkylene group and Rb is a heteroaryl group as described herein.

“Heterocyclyl” means a monovalent saturated moiety composed of one to three rings, incorporating one, two, three, or four heteroatoms (chosen from nitrogen, oxygen or sulfur). The heterocyclyl ring may also be optionally substituted. Examples of heterocyclyl moieties include, but are not limited to, optionally substituted piperidinyl, piperazinyl, homopiperazinyl, azepanyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, thiadiazolylidinyl, benzothiazolidinyl, benzoazolylidinyl, dihydrofuranyl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinylsulfone, dihydroquinolinyl, dihydrisoquinolinyl, tetrahydroquinolinyl, tetrahydrisoquinolinyl, and the like. Preferred heterocyclyl include tetrahydropyranyl, tetrahydrofuranyl, piperidinyl, piperazinyl and pyrrolidinyl.

The term “lower heterocyclylalkyl” refers to lower alkyl groups as defined herein wherein at least one of the hydrogen atoms of the lower alkyl group is replaced by a heterocyclyl group as described herein.

The term “optionally substituted” means, in reference to the optionally substituted group, the group may have one or more substituents including hydroxy, alkyl, alkoxy, thiol, alkylthio, arylthio, aralkylthio, halogen, amino, carboxylic acid, and carboxylate alkyl ester.

Exemplary PKC inhibitors included within the scope of the present invention include, but are not limited to, the following compounds:

PKC inhibitors disclosed herein can be prepared using synthetic approaches routinely practiced in the art based upon the synthesis of structurally similar compounds.

As demonstrated herein, inhibitor concentrations of ≧2.5 μM were sufficient to establish and maintain stem cells in the undifferentiated state. Accordingly, particular embodiments of the invention embrace use of the selective PKC inhibitor in the range of 100 nm to 5 μM, or more desirably in the range of 1 μM to 3 μM.

Stem cells established and maintained in accordance with the present invention find application in experimental, therapeutic and prophylactic treatment of various diseases or conditions in a human or animal. Such diseases or conditions include, but are not limited to, Parkinson\'s, Alzheimer\'s, Multiple Sclerosis, spinal cord injuries, stroke, macular degeneration, burns, liver failure, heart disease, diabetes, Duchenne\'s muscular dystrophy, osteogenesis imperfecta, osteoarthritis, rheumatoid arthritis, anemia, leukemia, breast cancer, solid tumors, and AIDS.

Once established, propagated and expanded, stem cells of the invention can be exposed to a variety of soluble factors to induce differentiation. For example, IL-3 directs cells to become macrophages, mast cells or neutrophils (Wiles & Keller (1991) Development 111:259-267); IL-6 directs cells to the erythroid lineage (Biesecker & Emerson (1993) Exp. Hematol. 21:774-778); retinoic acid induces neuron formation (Slager, et al. (1993) Dev. Genet. 14:212-224; Bain, et al. (1995) Dev. Biol. 168:342-357); transforming growth factor (TGF-beta 1 induces myogenesis (Rohwedel, et al. (1994) Dev. Biol. 164:87-101), TGF-beta 1 and activin-A induce differentiation of muscle cells; retinoic acid, bFGF, BMP-4, and EGF induce differentiation of ectodermal and mesodermal cells; while NGF and HGF allow differentiation of cells from all three germ layer lineages (Schuldiner, et al. (2000) Proc. Natl. Acad. Sci. USA 97(21):11307-12).

In so far as the instant methods of establishing and maintaining stem cells are easy and cost effective, it is contemplated that the present invention can be used to maintain mammalian ES and iPS cells for regular research purposes, generate new stem cell lines from mice and other mammalian species for stem cell banking, and efficiently generate, propagate, and maintain iPS cells from patients for regenerative medicine and research purposes. Indeed, the instant methods will be useful in stem cell banking and in the generation of patient-specific iPS cells.

The invention is described in greater detail by the following non-limiting examples.

Inhibitors.

Gö6983 was purchased from three different companies (Sigma, St. Louis, Mo.; EMD Chemicals Inc.; and Tocris Biosciences) for validation. In all experiments except the concentration profile, Gö6983 was used at 5 μM final concentration. Gö6976 (0.1-2 μM) was purchased from Sigma. Rottlerin (0.1-5 μM), and RO-318425 (0.1-5 μM) were purchased from EMD Chemicals. Jak Inhibitor I (10 μM), PD0325901 (1 μM) and AKT inhibitor IX (10 μM) were from Calbiochem and CHIR99021 (3 μM) was purchased from Stemgent.

ES Cell Cultures.

E14, R1, Stat3−/−, Ezh2−/−, and Eed−/− ES cells were used in this study. Cells maintained for 5 days under different culture conditions were used for immunofluorescence study while for other purposes, they were analyzed under steady state condition, i.e., within 8-hours of LIF and other inhibitor treatment. Detailed experimental protocol for ES cell culture under different experimental conditions is described herein.

E14 ES Cell Cultures.

Cells were grown in ES-IMDM media (Lonza, Walkersville, Md.) in a feeder-free condition. ES-IMDM was supplemented with 15% serum, 105 U/100 ml of LIF (ESGRO, Millipore, Calif.) and 0.0124% monothioglycerol (MTG; Sigma Aldrich). Cells were grown for 3-5 days with change of medium every day. For inducing differentiation in monolayer culture and to determine the effect of Gö6983 in preventing differentiation, E14 cells were cultured on gelatin-coated plastics for 8 days without LIF. Details of experiments performed at clonal-density are described herein. To maintain E14 cells with Gö6983, serum supplemented ES-IMDM was used in the presence of 5 μM Gö6983 and was passed after every 3-5 days. For all assays involving elucidation of different signaling mechanism responsible for maintenance of pluripotency, cells from a ˜70% confluent ES culture plate were washed two times with 1× phosphate-buffered saline (PBS), trypsinized and plated on a 6-well tissue culture plate and treated with or without LIF or Gö6983 or different inhibitors for ˜10 hours followed by preparation of protein lysates and RNA. The PKCζ knocked-down E14 cells that were generated in this study were maintained in serum supplemented ES-IMDM on gelatin-coated plates in the absence of Gö6983 or LIF or any other inhibitor and passed after 3-5, days. For the experiments herein, cells were continuously cultured at least for 5 consecutive passages (>18 days) on gelatin-coated plates without significant differentiation.

Quantitative Clonal Assay.

ES and iPS cells were dissociated into single cells using 0.05% trypsin/EDTA and 2-20 cells were plated on each well of a 96-well culture plate. The cells were cultured for 6-7 days, colonies were stained for Nanog, and the number of Nanog positive colonies was counted. For determining maintenance of self-renewal for multiple passages at clonal density with Gö6983, E14 cells were cultured at clonal density in 96 well plates with Gö6983; cells from undifferentiated colonies were trypsizined after day 6, and again plated at clonal density with Gö6983. This procedure was repeated for five consecutive passages (>30 days).

Embryoid Body (EB) Formation.

To generate EBs, ES cells were grown in absence of LIF in ES-IMDM differentiation media containing 15% FBS, selected for endothelial cell differentiation (Stem Cell Technologies, Vancouver, BC), 1% L-glutamine, 1% ascorbic acid (Stem Cell Technologies, Ocala, Fla.), and 3 μl/ml MTG. Cells were trypsinized and were made into single cell suspensions, and washed to completely remove LIF. To generate day 4.5 EBs, 4000 cells/ml were added to ES-IMDM differentiation media.

ES Cell Differentiation on Collagen-IV and with Retinoic Acid.

To differentiate ES cells in monolayer culture on collagen IV, around 3×104 cells per well were transferred to collagen IV-coated 6-well plates (BD Biosciences, Franklin Lakes, N.J.) cultured for 5 days in ES differentiation medium containing DMEM (Invitrogen), 15% FBS (selected for endothelial differentiation, Stem Cell Technologies, Vancouver, BC), sodium pyruvate and L-glutamine with or without LIF and Gö6983, and were recovered by cell dissociation buffer (BD Biosciences, Franklin Lakes, N.J.). For culturing multiple passages with Gö6983 on collagen-IV, the recovered cells were again plated at a density of ˜3×104 cells per well and cultured again for 5 days. For the experiments herein, E14 cells were continuously cultured with Gö6983 (without LIF) up to consecutive passages on collagen IV plates without any noticeable differentiation. Besides, E14 cells maintained on collagen IV with Gö6983 for 7 consecutive passages efficiently generated chimeras. For RA-induced differentiation on monolayer culture, ES cells were treated with all-trans-retinoic acid (Sigma, St. Louis, Mo.) in ethanol at a concentration of 1 μM with or without LIF and Gö6983 for 6 days followed by study of expression of pluripotency markers (by immunofluorescence or western blot or RT-PCR analysis).

Growth Under Serum Free Condition.

E14 cells were maintained in serum-free N2B27 medium containing DMEM/F12 (Invitrogen), Neurobasal media (Invitrogen), B27 supplement (Invitrogen), N2 supplement (Invitrogen), BSA fraction V (Invitrogen), 2-mercaptoethanol (Sigma) and LIF/BMP4 (R&D Systems, Minneapolis, Minn.) with change of medium on alternate days and passed in every 2-4 days. For experiments with Gö6983, ˜5×104 E14 cells were plated on each well of a gelatin-coated 6-well plate having N2B27 medium without LIF/BMP4 and with or without 5 μM Gö6983 and cultured for 3-4 days before passing. Cells were analyzed for expression of different markers (by RT-PCR or immunofluorescence study). For experiments at clonal density, initially, E14 cells were cultured at high density in N2B27 medium with Gö6983 alone for 4 passages, then ˜200 cells were plated in each wells of a 96-well plate with Gö6983 in N2B27 and cultured for 7 more days. After 7 days, colonies were analyzed for Oct4 staining.

R1 ES Cells.

R1 Cells were maintained on MEF feeder in ES-IMDM media supplemented with 15% ES-cell quality serum, 105 U/100 ml of LIF. Cells were grown for 3-5 days with change of medium every day. For inducing differentiation in monolayer culture and to determine the effect of Gö6983, cells were cultured at feeder-free condition and without LIF for 7 days.

Ezh2−/− ES Cells. Ezh2−/− cells, passage 6, were maintained on irradiated MEF feeders in standard ES medium (DMEM; Dulbecco\'s modified Eagle\'s medium) supplemented with 15% heat-inactivated fetal calf serum, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 0.1 mM non-essential amino acid, 1% of nucleoside mix (100× stock, Sigma), 1000 U/ml LIF and 50 U/ml Penicillin/Streptomycin. For experiments involving Gö6983, Ezh2−/− cells were passed once without feeders and used for subsequent analysis. For experiments on collagen IV, ˜3×104 Ezh2−/− cells were plated in each well of a six-well tissue culture plate in the presence or absence of LIF and Gö6983.

Eed−/− ES Cells.

Eed−/− ES cells, passage 4, were maintained in same medium as mentioned for Ezh2−/− cells but supplemented with extra LIF (2000 U/ml) to suppress spontaneous differentiation. For experiments with Gö6983, Eed−/− cells were passed one time without feeders and treated similar to Ezh2−/− cells as mentioned above.