CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the filing date of U.S. Provisional

Application No. 61/233,418 filed August 12, 2009, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates in general to stem cells. More specifically, the invention provides methods of forming induced pluripotent stem cells from somatic cells.

BACKGROUND

Embryonic stem cells (ESC) are pluripotent cells capable of differentiation into tissue types from all three embryonic germ layers (endoderm, mesoderm, and ectoderm) and are capable of growing indefinitely. These cells have a wide variety of potential applications including the study of human developmental biology, drug discovery and testing, and regenerative medicine. Thus, human embryonic stem cells (hESC) can provide an unlimited and renewable source of therapeutic transplantable material.

There are over 4 million live births in the US alone per year. Amniotic epithelial (AE) cells can be derived from full term human (or other species) placenta (1). The placenta is discarded after a live birth alleviating essentially all ethical, religious and political concerns. Unlike other parts of the placenta, AE cells are derived from the pluripotent epiblast at day 8 in humans. AE cells express several stem cell antigens (e.g. SSEA3, SSEA4, TRA 1-60 etc) and can differentiate into all three germ layers in vitro. However, unlike ES cells, AE cells cannot be passaged indefinitely and undergo replicative senescence after 6-8 passages. Although AE cells do express the key pluripotency transcription factors Oct4, Sox2, KLF4 and c-Myc there level of expression is quite different than true ESCs. Nevertheless, they are quite close in character to ESCs and reprogramming them should be significantly easier than reprogramming more differentiated somatic cells.

While use of hESC in regenerative medicine holds promise for medical therapies, significant challenges remain. Along with ethical controversies, another obstacle is the generation of patient-specific embryonic stem cells, which is critical to avoiding immunological rejection in transplantation. One approach to overcoming these obstacles is to reprogram somatic cells into pluripotent stem cells, enabling patient-specific cells to possibly serve as a source of therapeutically transplantable material. Current approaches to deliver the

reprogramming factors to somatic cells rely upon viral delivery systems to achieve high efficiency necessary to promote formation of induced pluripotent stem cells. However, the use of viruses prevents the later application of these cells for therapeutic purposes, due to the risk of tumorigenesis and carcinoma formation. Thus, there is a need in the art for non-viral approaches to induce pluripotent stem cell formation from somatic cells.

In hESC self-renewal and differentiation, the β-catenin protein and co-activators CBP and p300 play important roles. One example of a signaling pathway involving β-catenin, CBP, and p300 is the Wnt signaling pathway, which has been demonstrated to maintain pluripotency in stem cells under certain conditions (2, 3) and is critical for the expansion of progenitors (4). Wnt signaling has also been demonstrated to be important for the maintenance of pluripotency in both mouse and human embryonic stem cells in culture (5). Expression of multiple components of the Wnt pathway is evident in the P19 human embryonal carcinoma cell lines, as well as in embryonic stem cells (6).

Recent work has suggested that β-catenin/CBP is critical for cell proliferation without differentiation, whereas a switch to β-catenin/p300 is critical to initiate differentiation and limits proliferation (7). The Wnt/β-catenin pathway normally regulates expression of a range of genes involved in promoting both proliferation and differentiation. Activation of the Wnt pathway allows β-catenin to accumulate in the nucleus, bind to members of the T-CeIl Factor (TCF) family of transcription factors, and form a transcriptionally active complex, by recruiting either the transcriptional coactivator CBP or its closely related homolog, p300.

Small molecules, such as IQ-I reported by Miyabashi and colleagues (7), bind to the PR

72/130 subunit of the serine/threonine phosphatase PP2A. The binding of IQ-I to PR72/130 leads to decreased phosphorylation of the coactivator protein p300 at Ser-89. Since the phosphorylation of p300 at Ser-89 enhances the binding affinity of β-catenin to p300, inhibitors or small molecule Wnt signaling modulators, such as IQ-I thereby diminishes the β-catenin/p300 interaction and prevents β-catenin coactivator switching from CBP to p300. The invention is

-?- based on the premise that an increase in β-catenin/CBP mediated transcription at the expense of the β-catenin/p300 interaction is critical for the maintenance of pluripotency.

Methods of reprogramming somatic cells to pluripotency rely upon the expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc (8). Additional studies confirmed that mouse and human somatic cells can be reprogrammed to the pluripotent state via viral transduction with the same or similar sets of reprogramming factors (9, 10, 11, 12, 13, 14). The therapeutic potential of iPSCs has been demonstrated in animal models of sickle cell anemia and Parkinson's disease (15,13). SUMMARY OF THE INVENTION

In one embodiment, the invention includes a method of generating induced pluripotent stem (iPS) cells, comprising: providing a quantity of cells; and culturing the quantity of cells in a composition comprising a Wnt3a purified protein and a p300/catenin antagonist, whereby iPS cells are generated from the quantity of cells. The p300/catenin antagonist may be selected from the group consisting of ID-8, IQ-I, ICG-427, a small molecule structurally related to ID-8, IQ-I or ICG-427, a pharmaceutical equivalent, analog, derivative, salt or prodrug of any of the foregoing, and a molecule that modulates the interaction between β-catenin and the coactivator proteins CBP and p300 to promote proliferation, pluripotency and/or dedifferentiation of pluripotent stem cells. The composition may further comprise valproic acid or other related histine deacetylase (HDAC) inhibitors, 5-azacytidine or other inhibitors of DNA methyl transferases (DNMT), a p53 antagonist such as pifithrin, and/or L-ascorbic acid. The quantity of cells may be somatic cells or amniotic epithelial cells. The quantity of cells may be human cells or mouse cells. Culturing the quantity of cells may further comprise culturing the cells as a floating culture. The quantity of cells may be placed in a low or non-adherent culture vessel. Culturing the quantity of cells may further comprise using a culture medium comprising 15% fetal calf serum (FCS) media, Ix non-essential amino acids (NEAA), Ix L-Glutamax (L-GIn), 100 μM beta mercapto-ethanol, and/or LIF.

In another embodiment, the invention includes a composition comprising a Wnt3a purified protein and a p300/catenin antagonist. The composition may further comprise valproic acid, 5-azacytidine, a p53 antagonist and/or L-ascorbic acid. The p53 antagonist may be pifithrin. In another embodiment, the invention includes, in combination, a quantity of cells and a composition comprising a Wnt3a purified protein and a p300/catenin antagonist. The composition may further comprise valproic acid, 5-azacytidine, a p53 antagonist and/or L- ascorbic acid. The p53 antagonist may be pifithrin. The quantity of cells may be of a type selected from the group consisting of somatic cells, amniotic epithelial cells, and induced pluripotent stem cells.

In another embodiment, the invention includes a quantity of induced pluripotent stem (iPS) cells generated by a method, comprising: providing a quantity of cells; and culturing the quantity of cells in a composition comprising a Wnt3a purified protein and a p300/catenin antagonist, whereby the quantity of iPS cells are generated from the quantity of cells. The p300/catenin antagonist may be selected from the group consisting of ID-8, IQ-I, ICG-427, a small molecule structurally related to ID-8, IQ-I or ICG-427, a pharmaceutical equivalent, analog, derivative, salt or prodrug of any of the foregoing, and a molecule that modulates the interaction between β-catenin and the coactivator proteins CBP and p300 to promote

proliferation, pluripotency and/or dedifferentiation of pluripotent stem cells. The composition may further comprise valproic acid, 5-azacytidine, a p53 antagonist such as pifithrin, and/or L- ascorbic acid. The quantity of cells may be somatic cells or amniotic epithelial cells. The quantity of cells may be human cells or mouse cells.

The above-mentioned and other features of this invention and the manner of obtaining and using them will become more apparent, and will be best understood, by reference to the following description, taken in conjunction with the accompanying drawings. The drawings depict only typical embodiments of the invention and do not therefore limit its scope.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in the referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

Figure 1. Structures of p300/catenin antagonist.

Figure 2. Schematic of induced pluripotent stem cell colonies in the presence of lentivirus and/or chemical additives.

Figure 3. Schematic of the chemical reprogramming of somatic cells into iPS cells. Figure 4. Representation of iPS colonies following the chemical reprogramming of somatic cells.

Figure 5. Pluripotency gene expression of Sox2, Klf4, Nanog and c-Myc in iPS cells. Figure 6. Comparison of Oct4 and GFP expression levels of iPS cells.

DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides for advances in the field of Regenerative Medicine, such that access to a source of defined, immunopriveledged (i.e. self/autologous) derived raw material, including pluripotent stem cells or differentiated cell types derived from pluripotent stem cells. Similar to the present day use of cord blood banking, AE cells derived from the placenta can be chemically reprogrammed to generate personalized induced pluripotent cells (iPS) cells, which can be expanded, frozen down, and banked. These personalized lines can be available for regenerative medical applications at anytime during the individual's life either as pluripotent cells or via differentiation to a particular cell type.

Current methods of forming iPS cells make use of multiple viral vector integrations, which make them unsuitable for human clinical trials. The use of genome-integrating viruses could cause insertional mutagenesis and unpredictable genetic dysfunction (10). The inventors have developed a reprogramming system using chemical additives to avoid the use of viral delivery and its associated drawbacks. Furthermore, these chemical additives target the Wnt and p300/catening signaling pathways to achieve reprogramming, in contrast to the use of reprogramming factors such as Oct4, Sox2, Klf4, and c-Myc. More specifically, the inventors reprogram AE cells to generate iPS cells by activating the Wnt signaling cascade with a Wnt3a or GSK3 inhibitor and simultaneously blocking the p300/catenin interaction with IQ-I with mouse and ID8 with human AE cells.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

The term "stem cell" refers to an undifferentiated cell which is capable of self-renewal, i.e., proliferation to give rise to more stem cells, and may give rise to lineage committed progenitors which are capable of differentiation and expansion into a specific lineage. The stem cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues.

As used herein, the term "human embryonic stem cells (hESCs)" refers generally to embryonic, of human, origin stem cells . hESCs can be expanded into all three embryonic germ layers including gut epithelium (endoderm), cartilage, bone, smooth muscle, and striated muscle (mesoderm), and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). Embryonic stem cells are derived from the inner cell mass of preimplantation embryos.

"Amniotic epithelial cells (AE)" as used herein refers to a form of stem cells extracted from the placenta. AE cells have the potential to differentiate into all three germ layers

(endoderm, mesoderm, and ectoderm). AE cells have not shown a propensity for developing into teratomas and other cancer-like tumors upon injection into living tissue, like embryonic stem cells. Organs engineered from AE cells obtained from the placenta associated with a particular person's birth would not be rejected by that person; such organs would have the same genotype as that person and thus be fully compatible with that person's immune system.

"Pluripotent" as used herein refers to stem cells that have the potential to differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm. Pluripotent stem cells can give rise to any fetal or adult cell type cells that are capable of indefinite proliferation in vitro while remaining undifferentiated and maintaining a normal karyotype throughout long-term culture.

"Induced pluripotent stem" cells (iPS cells) are a type of pluripotent stem cell artificially derived from non-pluripotent cells, such as an adult somatic cell, by inducing a "forced" expression of certain genes.

The term "undifferentiated" as used herein refers to pluripotent embryonic stem cells which have not developed a characteristic of a more specialized cell. As will be recognized by one of skill in the art, the terms "undifferentiated" and "differentiated" are relative with respect to each other. An embryonic cell which is "differentiated" has a characteristic of a more specialized cell. Differentiated and undifferentiated cells are distinguished from each other by several well-established criteria, including morphological characteristics such as relative size and shape, ratio of nuclear volume to cytoplasmic volume; and expression characteristics such as detectable presence of known markers of differentiation.

Any of various types of differentiated cells may be obtained by inducing differentiation of a substantially homogeneous population of iPS cells produced according to methods of the present invention. Examples of such differentiated cells obtained according to methods of embodiments of the present invention illustratively include committed neuronal precursors, neurons, committed pancreatic beta cell precursors and pancreatic beta cells, bone cell precursors, bone cells, liver cell precursors, liver cells, muscle cell precursors, muscle cells, cardiac muscle precursors, cardiac muscle cells, skin cell precursors, skin cells, kidney cell precursors, kidney cells, vascular endothelial cell precursors, vascular endothelial cells, blood cell precursors, blood cells, adipose cell precursors, and adipose cells.

"p300/catenin antagonist" as used herein refers to agents that modulate the interaction between β-catenin and the coactivator proteins CBP and p300 to promote proliferation, pluripotency and/or dedifferentiation of pluripotent stem cells. p300/catenin antagonists that can be used in accordance with various embodiments of the present invention may include, but are not limited to, for example, ID-8, IQ-I , ICG-427, small molecules that are structurally related to ID-8, IQ-I or ICG-427 (Figure 1), pharmaceutical equivalents, analogs, derivatives, salts or prodrugs of any of the foregoing, or any molecule that modulates the interaction between β- catenin and the coactivator proteins CBP and p300 to promote proliferation, pluripotency and/or dedifferentiation of pluripotent stem cells.

The present invention relates to methods of generating iPS using chemicals and iPS cells produced through such methods. In one aspect, a culture (e.g., a floating culture) of cells (e.g., somatic cells) is established in culture media containing a Wnt3a purified protein and a p300/catenin antagonist. Valproic acid r other related histine deacetylase (HDAC) inhibitors, 5- Azacytidine or other inhibitors of DNA methyl transferases (DNMT), and/or a p53 antagonist (e.g., pifithrin) can be added to the culture media to induce the formation of iPS colonies.

In one embodiment, the cells cultured using this method are human AE or other somatic cells. In another embodiment, the somatic cells that are cultured using this method are mouse AE or mouse somatic cells.

In one embodiment, the p300/catenin antagonist binds directly to the PR72/130 subunit of the PP2A serine/threonine phosphatase. In another embodiment, the p300/catenin antagonist decreases the binding of β-catenin to p300 by inhibiting the phosphorylation of Ser 89 of p300. In another embodiment the p300/catenin antagonist blocks the activity of the Dyrk kinase family.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1

Floating culture with chemical additives

Mouse embryonic fibroblasts (MEFs) from B6;CBA-Tg(Pou5fl-EGFP)2Mnn mice with a GFP reporter knocked into the Oct4 locus to establish conditions supporting the formation of induced pluripotent stem cells are cultured on Ultra Low attachment culture plates (Costar 3473). On day 1, 5xlO ⁵ MEFs/w were cultured for 5 days in 15% fetal calf serum (FCS) media, Ix nonessential amino acids (NEAA), Ix L-Glutamax (L-GIn), 100 μM beta mercapto-ethanol and 1000 U/ml LIF with the following chemicals added to the wells: purified Wnt3a (50ng/ml), IQ-I (lOuM), valproic acid (VPA) (1OmM), and 5-Azacytidine (2uM). The cells were cultured in this manner for 5 days.

Example 2

Chemical Reprogramming of somatic cells into iPS cells

At day 6, the floating colonies from Example 1 are transferred to 0.1% gelatin (SIGMA G1890) coated 6-well plates. Plates are coated with 0.1% gelatin at 37 ⁰ C for at least an hour.

These cells are maintained in media containing 15% FCS,, Ix NEAA, Ix L-GIn, 100 μM beta mercapto-ethanol and 1000 U/ml LIF with the same chemical additives (i.e., Wnt3a, IQ-I, VPA, and 5-Azacytidine) for 10 days. On day 16, cells are cultured in medium containing in 15% FCS media, Ix NEAA, Ix L-Glutamax, and lOOμM beta- mercapto-ethanol with the following chemicals added to the wells: purified Wnt3a (50ng/ml), IQ-I (lOuM), VPA (2mM), 5-

Azacytidine (2uM), L- Ascorbic Acid (50μg/ml) and the p53 inhibitor Pifithrin-a (lOμM). On day 21 (Figure 4), cells are then cultured in 15% FCS media, Ix NEAA, Ix L-Glutamax, and lOOμM beta mercapto-ethanol with the following chemicals added to the wells: purified Wnt3a (50ng/ml) and IQ-I (lOuM) for 10 days. Cells are grown in 5% CO ₂ and 6% O ₂ chambers (Figure 3). iPS colonies formed through the use of chemical additives have a compact, bright, and round cell morphology, express GFP, and appear identical to colonies formed through use of chemicals and lentivirus delivery of reprogramming factors (Figure 2).

Example 3

Pluripotency gene expression of iPS cells

To determine whether the iPS and mouse embryonic cells expressed the key pluripotency transcription factors Nanog, Sox2, Klf4, and c-Myc, the presence of the genes was determined by qPCR. Oct4 expression was not compared as the EGFP kockin is within the Oct4 locus. The results compare the gene expression of the transcription factors under varying oxygen conditions (Figure 5). There was significant expression of Nanog and Sox2. However, there was a reduction in Klf4, and c-Myc expression.

Example 4

iPS cells were generated from Oct4 GFP mouse embryonic fibroblasts (MEFs) infected with 4 factor Lentivirus (4F). qPCR was done to compare Oct4 and GFP expression in the 4F iPS celss and D3 mouse embryonic stem (mES) cells. There is a significant reduction in GFP expression in the 4F iPS cells as compared to the D3 mES cells (Figure 6). However, OCT4 expression in both the 4F iPS cells and the D3 mES cells were comparable.

While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention.

Many modifications and variations of the invention as hereinbefore set forth can be made without departing from the spirit and scope thereof and therefore only such limitations should be imposed as are indicated by the appended claims. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

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