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 OF THE INVENTION
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 OF THE INVENTION
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.
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