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US 2016O177271A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0177271 A1 LEE et al. (43) Pub. Date: Jun. 23, 2016

(54) COMBINATIONAL USE OF MECHANICAL Publication Classification MANIPULATION AND PROGRAMIN TO GENERATE PLURPOTENT STEM CELLS (51) Int. Cl. FROMISOMATIC CELLS CI2N5/074 (2006.01) (52) U.S. Cl. (71) Applicant: The Chinese University of Hong Kong, CPC ...... CI2N5/0696 (2013.01); C12N2506/09 Shatin (CN) (2013.01); C12N 2506/025 (2013.01); C12N 2501/999 (2013.01); C12N 2527/00 (2013.01) (72) Inventors: Kenneth Ka Ho LEE, Tai Po (CN); Tommy Lok Man LO, Shatin (CN): Hoi (57) ABSTRACT Hung CHEUNG, Tsz Wan Shan (CN); The present invention provides methods and compositions for Wai Yee CHAN, Ma On Shan (CN) inducing pluripotency in differentiated mammalian cells. In particular, the methods include mechanically aggregating the (21) Appl. No.: 14/579,784 cells into discrete masses or embryoid-like bodies and treated them with a compound. Provided herein are the compositions of the compounds which are derived from (22) Filed: Dec. 22, 2014 programin (e.g., reversine). Patent Application Publication Jun. 23, 2016 Sheet 1 of 10 US 2016/017.7271 A1

Patent Application Publication Jun. 23, 2016 Sheet 2 of 10 US 2016/017.7271 A1

F.G. 2A FIG. 2B

Š S Š y Š S. S. Sis

FIG. 2D Patent Application Publication Jun. 23, 2016 Sheet 3 of 10 US 2016/017.7271 A1

FIG. 3A FIG. 3B

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FIG. 4 Patent Application Publication Jun. 23, 2016 Sheet 5 of 10 US 2016/017.7271 A1

F.G. 5 Patent Application Publication Jun. 23, 2016 Sheet 6 of 10 US 2016/017.7271 A1

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Patent Application Publication Jun. 23, 2016 Sheet 7 of 10 US 2016/017.7271 A1

Šiš Ši-S:

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FIG. 7 Patent Application Publication Jun. 23, 2016 Sheet 8 of 10 US 2016/017.7271 A1

FIG. 8A FIG. 8B FIG. 8C

OCT4 Nanog 3. 6. S. Š 1OOO 590. iii. 8. 3. 3000s 400* s. - IS 2OOON - I - 2000s"" 1 2 3 4 1 2 3 4 1 2 3 4

FIG. 8D FIG. 8E

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O - - - O - 1 2 3 4 1 2 3 4

1 monolayerl DMSO monolayerl programin -likel DMSO embryoid body-likel programin Patent Application Publication Jun. 23, 2016 Sheet 9 of 10 US 2016/017.7271 A1

& FIG. 9C FIG. 9D Patent Application Publication Jun. 23, 2016 Sheet 10 of 10 US 2016/017.7271 A1

FIG 10A FIG 10B

FIG 10C FIG 10D US 2016/0177271 A1 Jun. 23, 2016

COMBINATIONAL USE OF MECHANICAL MANIPULATION AND PROGRAMINTO (II) GENERATE PLURPOTENT STEM CELLS OH FROMISOMATIC CELLS N 21 FIELD OF THE INVENTION N OM N H 0001. The present invention relates to the use of mechani (III) cal aggregation of Somatic cells to form embryoid-like cell S bodies as well as the treatment of these aggregates with pro gramin or a derivative thereof to reprogram the Somatic cells and produce pluripotent stem cells. Such reprogrammed cells HN are useful in the field of regenerative medicine. HN J-2s N BACKGROUND OF THE INVENTION (IV) S 0002 Stem cells have been trumpeted as the solution for H alleviating the serious shortage of tissue and organs for trans HN N plantation. To date, numerous types of naturally occurring and artificially produced stem cells have been identified or s NX. created. However, every one of them has their own shortcom ings. Induced pluripotent stem cells (iPSCs), produced from (V) of somatic cells such as skin (Ta NH2 kahashi and Yamanaka, Cell, 126(4):663-76 (2006)), is cur H rently regarded as the best candidate for use in regenerative N21 N medicine. However, iPSC generation remains a very slow (~4 weeks) and inefficient (<0.01%) process that results in a X heterogeneous population of cells, and requires specific ls-- expertise in human pluripotent (Takahashi et al., (VI) Cell, 131, 861-72 (2007); Yu et al., Science, 318, 1917-20 (2007)). An alternative approach is to use a small chemical ( \, molecule that can directly activate the pluripotent genes Oct4. N1 Sox2 and Nanog. N N 0003 Programin was first synthesized by Chen et al. (J. Am. Chem. Soc., 2004 126: 410-411). It was used to transdif 2 ferentiate C2C12 myogenic cells into osteocytes and adipo C ul N IXN cytes. Subsequently, it was shown that the polycomb genes in C2C12 were probably involved in the (VII) process using comparative proteomic techniques (Shen et al., Proteomics, 2007, 7(23): 4303-4316). Furthermore, it was / \ reported that treating fibroblasts with reversine could induce N them to transdifferentiate into skeletal muscle cells (Luigi et N al., 2007, Cell Death and Differentiation, 13(12): 2042-2051: Chiara et al., 2009, Electrophoresis, 30(12): 2193-2206). N N Mandal et al. (Biochip Journal, 2013, 7(3): 278-287) exam 2 ined the transcriptome profile of reversine-mediated transdif C ul N IXN ferentiation of NIH3T3 fibroblasts into osteoblasts. The study did not show that reversine activated the expression of pluri (VIII) potency genes Such as Oct4, Sox2 and Nanog. NH2 0004. There is a need for simple and efficient methods based on the use of Small molecule compounds to generate iPSCs. The present invention meets this and other needs. r F u?s N

BRIEF SUMMARY OF THE INVENTION HO O 0005. In one aspect, the present invention provides a OH, or method for producing a pluripotent from differen tiated cells. The method includes (a) culturing the differenti ated cells in a vessel; (b) mechanically agitating the differen tiated cells to form a cellular aggregate; (c) exposing the cellular aggregate to an effective amount of any one of the compounds of Formula II-IXhaving the following structures: US 2016/0177271 A1 Jun. 23, 2016

-continued method includes (a) culturing the differentiated cells at a (IX) density of about 1x10 to about 1x10° cells/ml in a vessel; (b) mechanically agitating the differentiated cells to form the cellular aggregate; and (c) culturing the cellular aggregate under conditions to produce a cell that expresses at least one pluripotent . 0013. In some embodiments, the method also includes step (d) comprising exposing the cellular aggregate to an effective amount of a compound of Formula II-IX having any ON one of the following structures: N21 (II) C lsN N OH

21 (d) detecting the expression level of Oct4, Sox2 or Nanog in N the cells of the aggregate; and (e) determining any cell is a N O.M pluripotent stem cell if the expression level of Oct4, Sox2 or N H Nanog in the cell of the aggregate is higher than the corre (III) sponding expression level in a differentiated cell; and (f) S isolating the pluripotent stem cell. In some embodiments, step (b) and (c) are performed concurrently. In other embodi H ments, step (b) and (c) are performed Subsequently. For HN N example, step (c) can be performed about 24 hours to about 96 hours or more, e.g., about 24 hours, about 36 hours, about 48 HN J.s J-2N hours, about 60 hours, about 72 hours, about 84 hours, about (IV) 96 hours or more, after step (b). S 0006. The differentiated cells of step (a) can be obtained H from a Subject or a cell line. In some embodiments, the Subject N is a mammalian Subject. The cell line can be a mammaliancell line. In some embodiments, the differentiated cells are fibro " X blasts or the differentiated cells are progenitor cells. The N N differentiated cells of step (a) can be at a density of about (V) 1x10-1x10° cells/cm. NH2 0007. The cellular aggregate does not adhere to an inner Surface of the vessel. In some embodiments, the inner Surface Na of the vessel is coated with a non-adherent Substrate (e.g., a substrate that prevents or hinders cells from adhering to it). X 0008. In some embodiments, mechanically agitating ls-- includes titurating, stirring or rocking the differentiated cells (VI) by way, e.g., shaking or rotating the culture vessel in which | \ the cells are maintained. For example, the cells can be pipet N ted using a wide bore pipet tip such that mechanical force is N applied to the cells or cell aggregates to prevent or hinder them from attaching to the culture vessel. 0009. In some embodiments, step (d) of the method y described above includes an amplification-based assay, a hybridization-based assay, an immunoassay or a flow cytom --- N etry assay. Subsequent to step (e), the method can also include (VII) expanding (proliferating) the pluripotent stem cell. Addition ally, Subsequent to step (e), the method can include inducing the pluripotent stem cell to undergo differentiation. ( \, 0010. In some instances, the higher expression level of N1 Octa, Sox2 or Nanog is at least about 10 fold or about 20-fold to about 200-fold higher compared to the differentiated cell. 0011. In another aspect of the present invention, provided herein is a pluripotent cell or cell line obtained by any one of y the exemplary embodiments of the method described above. --- N 0012. In yet another aspect, the present invention provides a method for producing a cellular aggregate from differenti ated cells, wherein the cellular aggregate comprise a cell that expresses at least one pluripotent stem cell marker. The US 2016/0177271 A1 Jun. 23, 2016

-continued -continued (VIII) (V) NH2 NH2 N21 N21 N N F ul-2N N (VI) HO O OH, or HG (IX)

(VII)

ON Na

(VIII)

0014. The method can also include, subsequent to step (c), expanding the cell that expresses at least one pluripotent stem cell marker. Optionally, Subsequent to step (c) the method can include isolating the cell that expresses at least one pluripo tent stem cell marker. The at least one pluripotent stem cell marker can be Oct4, Sox2 or Nanog. OH, or 0015. In another aspect, the present invention provides a compound of Formula II-IX having the any one of the fol lowing structures: (IX)

(II) OH

N 21 N Y.M N H (III) S

0016 Optionally, the compound is present in a mixture es. comprising differentiated cells. For instance, a compound of (IV) Formula II, III, IV, V,VI,VII,VIII or IX and the differentiated S cells can be admixed together to form a mixture. In some embodiments, the differentiated cells of the mixture are present in aggregates. 0017. In another aspect, provided herein is a pharmaceu I? tical composition for producing a pluripotent stem cell from differentiated cells, the composition comprising a compound described above and pharmaceutically acceptable salt. US 2016/0177271 A1 Jun. 23, 2016

0018. The inventors have surprising discovered that fibro FIG. 10B shows the cells treated with B27 supplement. FIGS. blasts if mechanically formed into embryoid-like bodies and 10C and 10D show neurite outgrowths from the differentiated maintained on non-adhesive culture dishes over a period of 4 cells. days, can express the pluripotency genes Oct4, Sox2 and Nanog. Furthermore, treatment of these aggregates with the DETAILED DESCRIPTION OF THE INVENTION Small molecule compound programin or a derivative thereof increased the expression of these three pluripotent genes by I. Introduction 25-200-fold compared to the unaggregated, untreated differ 0029. The present invention is based, in part, on the sur entiated cell. prising discovery that exposing cell aggregates (e.g., embry BRIEF DESCRIPTION OF THE DRAWINGS oid-like bodies) derived from somatic cells to a small mol ecule compound greatly improves the efficiency of induction 0019 FIG. 1 shows a schematic diagram of an exemplary of pluripotency in Such cells. Accordingly, the present inven method for inducing pluripotency from fibroblasts. tion provides compounds, compositions and methods for 0020 FIGS. 2A-2D show the effect of programin on dedifferentiating (e.g., reprogramming) lineage committed mouse fibroblasts cultured as a monolayer. The fibroblasts mammalian cells into pluripotent stem cells. More specifi were derived from the tail of Oct4-EGFP transgenic mice. cally, the present invention provides compounds of Formulas FIG. 2A shows CD34 fibroblasts. FIG. 2B Shows the I-IX (e.g., programin and derivatives thereof) and are useful absence of GFP' fibroblasts. FIGS. 2C and 2D show fibro of dedifferentiating a lineage committed cell or for inducing blasts after exposure to programin for 2 days. pluripotent stem cells from a lineage committed cell. In addi 0021 FIGS. 3A-3D show the effect of programin on tion, the methods provided herein include forming cell aggre mouse Oct4-EGFP fibroblasts cultured in aggregates or gates (e.g., embryoid-like bodies) by mechanical means. The embryoid-like bodies. FIG. 3A shows the aggregate at day 3 induced pluripotent stem cells generated from the methods of culturing on a non-adherent culture plate. FIG. 3B shows can be differentiated into lineage committed cells. an aggregate after 24-hour exposure to programin. GFP are 0030 The present invention involves the use of programin detectable by microscopy. FIG. 3C shows an aggregate after and its derivatives to generate pluripotent stem cells from 48-hour exposure to programin. FIG. 3D shows an aggregate differentiated cells. The use of programin alone cannot acti after 96-hour exposure to programin. vate expression of the pluripotency genes Oct4. SoX2, Nanog 0022 FIG.4 shows that CD34" mouse fibroblasts cultured and E-Cadherin in Somatic cells. The cells, such as human and in a monolayer do not express Oct4, Sox2 and Nanog, as mouse fibroblasts, must first be mechanically aggregated into determined by qPCR. Expression of these pluripotency mark embryoid-like cell bodies for about 4 days before they are ers was higher in the cell aggregates and highest in the aggre able to respond to the reprogramming activity of programinor gates that were treated with programin. a derivative thereof. The cell aggregation process alone 0023 FIG. 5 provides qPCR data showing that programin causes the fibroblasts to express basal levels of Oct4, Sox2 derivatives can enhance Oct4, Sox2 and Nanog expression in and Nanog. In addition, the aggregation enables the fibro cell aggregates of CD34" mouse fibroblasts. The small mol blasts to respond to programin or a derivative thereof. The ecules tested include 237 (Formula II), 400 (Formula III),594 exposure of the embryoid-like cell bodies to 2-5 uM pro (Formula IV). 648 (Formula VIII), 668 (Formula V), 957 gramin for 30 hours resulted in a 25-200 fold increase in Oct4, (Formula IX), 958 (Formula VI), 959 (Formula VII) and Sox2 and Nanog expression compared to an untreated control programin (Formula I). . 0024 FIG. 6 shows that aggregates of human umbilical cord perivascular mesenchymal progenitor (HUCPV) cells II. General Methodology express higher levels of Oct4, Sox2 and Nanog after treatment with 0-5uM programin compared to similar cells cultured in 0031 Practicing this invention utilizes routine techniques a monolayer. in the field of molecular biology. Basic texts disclosing the 0025 FIG.7 provides a higher resolution of the qPCR data general methods of use in this invention include Sambrook in FIG. 6. and Russell, Molecular Cloning, A Laboratory Manual (3rd 0026 FIGS. 8A-8E provides qPCR results showing the ed. 2001); Kriegler, Gene Transfer and Expression: A Labo level of Oct4 (FIG. 8A), Nanog (FIG. 8B), Sox2 (FIG. 8C), ratory Manual (1990); and Current Protocols in Molecular c-Myc (FIG. 8D) and KLF4 (FIG. 8E) in human skin fibro Biology (Ausubel et al., eds., 1994)). blast grown in a monolayer or grown as an aggregate (e.g., 0032 For nucleic acids, sizes are given in either kilobases embryoid-like body), and treated with 5uM programin for 4 (kb) or base pairs (bp). These are estimates derived from days. agarose or acrylamide gel electrophoresis, from sequenced 0027 FIGS. 9A-9D show programin-treated aggregates nucleic acids, or from published DNA sequences. For pro (e.g., embryoid-like bodies) differentiated into osteogenic teins, sizes are given in kilodaltons (kDa) or amino acid (bone) cells. FIG. 9A shows an aggregate prior to differen residue numbers. Protein sizes are estimated from gel elec tiation. FIG.9B shows the cells after treatment with 10 uM trophoresis, from sequenced proteins, from derived amino Wnt agonist. Osteogenic nodules were visible after 2 days acid sequences, or from published protein sequences. exposure to the Wnt agonist. FIG. 9C shows Alizen Rad S 0033 Oligonucleotides that are not commercially avail staining of the nodules. FIG. 9D shows that programin able can be chemically synthesized, e.g., according to the treated cells that were not mechanically formed into aggre solid phase phosphoramidite triester method first described gates underwent cell death. by Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 0028 FIGS. 10A-10D show programin-treated aggre (1981), using an automated synthesizer, as described in Van gates (e.g., embryoid-like bodies) differentiated into neural Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). cells. FIG. 10A shows an aggregate prior to differentiation. Purification of oligonucleotides is performed using any art US 2016/0177271 A1 Jun. 23, 2016 recognized strategy, e.g., native acrylamide gel electrophore teristics. For example, human pluripotent stem cells express sis oranion-exchange high performance liquid chromatogra at least Some, and in some embodiments, all of the markers phy (HPLC) as described in Pearson and Reanier, J. Chrom. from the following non-limiting list: SSEA-3, SSEA-4, TRA 255:137-149 (1983). 1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex 1, and Nanog. Cell morphologies associ III. Definitions ated with pluripotent stem cells are also pluripotent stem cell 0034. In this disclosure the term “or” is generally characteristics. employed in its sense including “and/or unless the content 0040. The term “non-pluripotent cells' refer to mamma clearly dictates otherwise. lian cells that are not pluripotent cells. Examples of such cells 0035. The term “Oct4” or “Oct-4” refers to a include differentiated cells as well as progenitor cells. factor belonging to the POU family. Oct4 is a marker of Examples of differentiated cells include, but are not limited undifferentiated cells (Okamoto et al., Cell 60:461-72, 1990). to, cells from a tissue selected from , skin, skel Octa is also reported to participate in the maintenance of etal muscle, fat tissue and peripheral blood. Exemplary cell pluripotency (Nichols et al., Cell 95:379-91, 1998). The types include, but are not limited to, fibroblasts, , human Oct4 polypeptide sequence are set forth in, e.g., Gen myoblasts, neurons, osteoblasts, osteoclasts, and lympho bank Accession Nos. NP 001167002, NP 001272915, cytes. NP 001272916, NP 002692, and NP 0976034. The human 0041. The term “lineage committed cell' refers to any cell Octa mRNA (coding) sequence are set forth in, e.g., Genbank that has or will differentiate into a particular cell type or Accession Nos. NM_001173531 NM_001285986, related cell types. Lineage committed cells include, for NM_001285987, NM_002701, and NM 203289. One example, osteoblasts, myoblasts, , neural cells, skilled in the art will appreciate that variants, isoforms, alter and adipocytes. native sequences of Oct4 are also useful in the present inven 0042. The term “dedifferentiate.” “dedifferentiation, or tion. “reprogramming as used herein, refers to the process by 0036. The term “Sox2 or “Sox-2 refers a member of the which lineage committed cells (e.g., myoblasts or osteo SRY-related HMG-box (SOX) family of transcription factors blasts) reverse their lineage commitment and can generate involved in regulation of embryonic development and the progeny of a new cell type, such as a , progenitor determination of cell fate. The human Sox2 polypeptide cell, multipotent stem cell, or pluripotent stem cell. For sequence is set forth in, e.g., Genbank Accession No. instance after Sufficient proliferation, a measurable propor NP 003097. The human Sox2 mRNA (coding) sequence is tion of progeny may have phenotypic characteristics of a new set forth in, e.g., Genbank Accession No. NM_003106. One cell type and this proportion is measurably more than before skilled in the art will appreciate that variants, isoforms, alter dedifferentiation or reprogramming. Under certain condi native sequences of Sox2 are also useful in the present inven tions, the proportion of progeny with characteristics of the tion. new cell type may be at least about 1%. 5%, 25% or more in 0037. The term “Nanog' refers to a transcription regulator order of increasing preference. Dedifferentiated cells can be involved in the proliferation and self-renewal of embryonic identified by loss of patterns of gene expression and cell stem cells and the . The human Nanog Surface protein expression associated with the lineage com polypeptide sequence is set forthin, e.g., Genbank Accession mitted cells. No. NP 0079141. The human Nanog mRNA (coding) 0043. The term “vessel” refers to a container, dish, plate, sequence is set forth in, e.g., Genbank Accession No. flask, bottle, cell culture tube, and the like that can be used to NM 024865. One skilled in the art will appreciate that vari culture, maintain or grow a cell. ants, isoforms, alternative sequences of Nanog are also useful 0044) The term “mechanically agitating refers to apply in the present invention. ing mechanical force to an object, Such as a group of cells or 0038. The term “pluripotent” or “pluripotency” refers to cell aggregates. cells with the ability to give rise to progeny cells that can undergo differentiation, under the appropriate conditions, 0045. The term “cellular aggregate' refers to a mass of at into cell types that collectively demonstrate characteristics least three cells that are in contact with each other and do not associated with cell lineages from all of the three germinal form a single layer of cells, e.g., a monolayer. layers (, , and ). Pluripotent 0046. The term “culturing, as used herein, refers to main stem cells can contribute to all embryonic derived tissues of a taining cells under conditions in which they can proliferate, prenatal, postnatal or adult animal. A standard art-accepted differentiate, and avoid senescence. Cells can be cultured in test, such as the ability to form a in 8-12 week old growth media containing appropriate growth factors. SCID mice, can be used to establish the pluripotency of a cell 0047. As used herein, “promote' or “increase.” or “pro population, however identification of various pluripotent moting or “increasing are used interchangeably herein. stem cell characteristics can also be used to detect pluripotent These terms refer to the increase in a measured parameter cells. (e.g., activity, expression, glycolysis, glycolytic metabolism, 0039. “Pluripotent stem cell characteristics' refer to char uptake, biosynthesis downstream of glycolysis) in a acteristics of a cell that distinguish pluripotent stem cells treated cell (tissue or Subject) in comparison to an untreated from other cells. The ability to give rise to progeny that can cell (tissue or Subject). A comparison can also be made of the undergo differentiation, under the appropriate conditions, same cell or tissue or subject between before and after treat into cell types that collectively demonstrate characteristics ment. The increase is sufficient to be detectable. In some associated with cell lineages from all of the three germinal embodiments, the increase in the treated cell is at least about layers (endoderm, mesoderm, and ectoderm) is a pluripotent 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, stem cell characteristic. Expression of certain combinations 2-fold, 3-fold, 4-fold or more in comparison to an untreated of molecular markers are also pluripotent stem cell charac cell. US 2016/0177271 A1 Jun. 23, 2016

0048. As used herein, “inhibit,” “prevent” or “reduce,” or readily undergo chemical changes under physiological con “inhibiting,” “preventing or “reducing are used inter ditions to provide the compounds of the present invention. changeably herein. These terms refer to the decrease in a Additionally, prodrugs can be converted to the compounds of measured parameter (e.g., activity, expression, mitochondrial the present invention by chemical or biochemical methods in respiration, mitochondrial oxidation, oxidative phosphoryla an ex vivo environment. For example, prodrugs can be slowly tion) in a treated cell (tissue or Subject) in comparison to an converted to the compounds of the present invention when untreated cell (tissue or subject). A comparison can also be placed in a transdermal patch reservoir with a suitable made of the same cell or tissue or subject between before and enzyme or chemical reagent. after treatment. The decrease is sufficient to be detectable. In Some embodiments, the decrease in the treated cell is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or IV. Programin and Derivatives Thereof completely inhibited in comparison to an untreated cell. In 0.052 The present invention provides compounds derived Some embodiments the measured parameter is undetectable from the compound known as reversine, programin, or 2-(4- (i.e., completely inhibited) in the treated cell in comparison to Morpholinoanilino)-6-cyclohexylamino-purine. In one the untreated cell. aspect, the compound has the Formula (I): 0049. The term “pharmaceutically acceptable salt” refers a salt of the active compound which is prepared with rela tively non-toxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by con r), NH tacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert N / Solvent. Examples of pharmaceutically acceptable base addi HN N N tion salts include Sodium, potassium, calcium, ammonium, - O CO organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contact 0053. In another aspect, the compound has the Formula ing the neutral form of Such compounds with a sufficient (II): amount of the desired acid, either neat or in a suitable inert Solvent. Examples of pharmaceutically acceptable acid addi OH tion salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogen N 2 carbonic, phosphoric, monohydrogenphosphoric, dihydro Sa Y.M genphosphoric, Sulfuric, monohydrogensulfuric, hydriodic, N H orphosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, Succinic, Suberic, 0054. In another aspect, the compound has the Formula fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsul (III): fonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric S H acids and the like (see, for example, Berge, S. M., et al. N "Pharmaceutical Salts', Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that HN Jr.N N allow the compounds to be converted into either base or acid addition salts. 0055. In yet another aspect, the compound has the For 0050. The neutral forms of the compounds may be regen mula (IV): erated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar Sol vents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention. try 0051. In addition to salt forms, the present invention pro vides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that US 2016/0177271 A1 Jun. 23, 2016

0056. In another aspect, the compound has the Formula 0060. In another aspect, the compound has the Formula (V): (IX):

NH2 N21 N-1- X

CN 0057. In another aspect, the compound has the Formula (VI): N21 1S NX

( \, 0061 The compounds can include all pharmaceutically N1 acceptable salts, isomers, Solvates, hydrates and prodrugs thereof. N1 N N 0062 One skilled in the art recognizes that 2-(4-morpholi noanilino)-6-cyclohexylamino-purine (programin or revers cy2 ine) and derivatives thereof may be prepared by either solid C N N phase synthesis or solution-phase synthesis. Detailed methods for synthesizing Such compounds are found in, e.g., Ding et al., J. Am. Chem. Soc., 124:1594 (2002); U.S. Pat. Nos. 7,176,312; 7,273,864; and 7,592, 177, the disclosures 0058. In some aspects, the compound has the Formula are herein incorporated by reference in their entirety for all (VII): purposes. 0063 Briefly, for solution phase synthesis of programin, cyclohexylamine and diisopropylethylamine may be added to a solution of 2-fluoro-6-chloropurine in n-butanol. The mix ture may be heated to 80° C. with vigorous stirring for 12 hours. The solvent can then be removed under reduced pres ( \, sure and the crude can be used directly in the next step N1 reaction without further purification. The crude 2-fluoro-6- cyclohexylamino-purine may be dissolved in ethanol, fol N1N N lowed by addition of 4-morpholinoaniline. The mixture may be heated to 75°C. in a sealed tube with vigorous stirring for cy2 24 hours. The solvent may then be removed under reduced C N N pressure and the crude material may be directly purified by flash chromatography to afford 2-(4-morpholinoanilino)-6- cyclohexylamino-purine as a pale white solid. It will be apparent to those skilled in the art that derivatives of 2-(4- 0059. In another aspect, the compound has the Formula morpholinoanilino)-6-cyclohexylamino-purine. Such as (VIII): compounds having the structure of Formulas II-IX, may be prepared via Solution-phase synthesis or Solid phase synthe S1S. NH2 V. Reprogramming to Induce Pluripotency in N21 Non-Pluripotent Cells 0064. A number of different methods and protocols have N F u-2N N been established for inducing non-pluripotent mammalian cells into induced pluripotent stem cells. iPSCs are similar to HO O ESCs in morphology, proliferation, and pluripotency, judged OH. by teratoma formation and contribution. Reprogram ming protocols include those involving the introduction of one or more reprogramming transcription factors selected from an Oct polypeptide (including but not limited to Oct 3/4), a Sox polypeptide (including but not limited to Sox2), a Klf polypeptide (including but not limited to Klf4) and/or a Myc polypeptide (including but not limited to c-Myc). US 2016/0177271 A1 Jun. 23, 2016

0065. Studies have shown that retroviral transduction of 0070 Differentiated cells are any cells forming the body mouse fibroblasts with four transcription factors that are of an organism, as opposed to germline cells (e.g., highly expressed in ESCs (Oct-3/4, Sox2. KLF4 and c-Myc) includes spermatozoa and ova). Every other cell type in the generate IPSCs. See, Takahashi, K. &Yamanaka, S. Cell 126, mammalian body (apart from the spermandova, gametocytes 663-676 (2006): Okita, K., Ichisaka, T. & Yamanaka, S. and undifferentiated stem cells) is a differentiated cell. Nature 448, 313-317 (2007); Wernig, M. et al. Nature 448, 0071. The cell can be, e.g., in culture or in a tissue, fluid, 318-324 (2007); Maherali, N. et al. Cell Stem Cell 1, 55-70 etc. and/or from or in an organism. In some embodiments, the (2007); Meissner, A., Wernig, M. & Jaenisch, R. Nature Bio differentiated cell is a primary cell line or is the progeny of a technol. 25, 1177-1181 (2007); Takahashi, K. et al. Cell 131, primary or secondary cell line. Cells that can be induced to 861-872 (2007); Yu, J. et al. Science 318, 1917-1920 (2007): pluripotency include, but are not limited to, fibroblast cells, Nakagawa, M. et al. Nature Biotechnol. 26, 101-106 (2007): blood cells, e.g., T cells, B cells, monocytes, myeloid pro Wernig, M., Meissner, A., Cassady, J. P. & Jaenisch, R. Cell genitor cells, etc., fibroblast cells, tumor cells, bone marrow Stem Cell. 2, 10-12 (2008). Studies have also demonstrated cells, stomach cells, liver cells, epithelial cells, nasal epithe reprogramming of human Somatic cells with transcription lial cells, mucosal epithelial cells, follicular cells, connective factors that are highly expressed in ESCs: Hockemeyer et al. tissue cells, muscle cells, bone cells, cells, gas Cell Stem Cell. 11:3(3):346-53 (2008); Lowry et al. Proc Natl trointestinal cells, splenic cells, cells, cells, tes AcadSci USA. 105 (8):2883-8 (2008); Parket al. Nature. 10; ticular cells, and nervous tissue cells. In some embodiments, 451(7175):14.1-6 (2008): Nakagawa et al. Nat Biotechnol. the human cell is a fibroblast, which may be conveniently January; 26(1): 101-6 (2008); Takahashi et al. Cell. 131(5): obtained from a subject by a punch biopsy. In other embodi 861-72 (2007); and Yu et al. Science. 318(5858): 1917-20 ments, the human cell is obtained from a sample, e.g., a hair (2007). follicle, a blood sample, a Swab sample (e.g., an oral Swab 0066. It has been shown that the efficiency of reprogram sample), and the like. When the reprogrammed cells (e.g. ming (e.g., the number of reprogrammed cells) can be induced pluripotent stem cells) are used for therapeutic treat enhanced by the addition of various Small molecules as ment of diseases, it is desirable to use differentiated cells (e.g. shown by, e.g., Shi, Y., et at (2008) Cell-StemCell 2:525-528, Somatic cells) isolated from patients. Huangfu, D., et at (2008) Nature Biotechnology 26(7):795 797, Marson, A., et at (2008) Cell-StemCell 3:132-135. It is 0072 Methods for isolation and culture of human and contemplated that the methods described herein can also be mammalian cells are well known in the art and have been used in combination with additional single small molecule (or described in, e.g., Humason, ANIMAL TISSUE TECH a combination of small molecules) that enhances the effi NIQUES, 4.sup.th ed., W. H. Freeman and Company (1979); ciency of production or a reprogrammed cell. In some Freshney et al., CULTURE OF ANIMAL CELLS (3rd ed. embodiments, some non-limiting examples of agents that 1994); and Ricciardelli et al., (1989) Cell Dev. Biol. enhance reprogramming efficiency include soluble Wnt, Wnt 25: 1016. conditioned media, BIX-01294 (a G9a histone methyltrans 0073 Suitable cell culture methods and conditions can be ferase), PD0325901 (a MEK inhibitor), DNA methyltrans determined by those of skill in the art using known method ferase inhibitors, (HDAC) inhibitors, val ology (see, e.g., Freshney et al., 1994, Supra). In general, the proic acid, 5'-azacytidine, dexamethasone, Suberoylanilide, cell culture environment includes consideration of Such fac hydroxamic acid (SAHA), and trichostatin (TSA), among tors as the substrate for cell growth, cell density and cell others. It is also contemplated herein that inhibitors of the contract, the gas phase, the medium, and temperature. Incu TGF-B signaling pathway (e.g. Repsox, E-616451 or bation of cells is generally performed under conditions SB431542, and anti-TGFB or RNAi agents) or known to be optimal for cell growth. Such conditions may inhibitors of SRC signaling, such as EI-275, or agonist of include, for example, a temperature of approximately 37°C. MEK/ERK cell signaling (e.g., prostaglandin2); inhibitors of and a humidified atmosphere containing approximately 5% Ca"/calmodulin signaling or EGF receptor tyrosine kinase CO. The duration of the incubation can vary widely, depend inhibitor; inhibitors of Na" channels or ATP-dependent ing on the desired results. In general, incubation is preferably potassium channel or agonists of MAPK signaling pathway continued until the cells express suitable Proliferation is con can be used either alone or in combination with another small veniently determined using: H thymidine incorporation or molecule (or combination of Small molecules) to enhance or BrdU labeling. Plastic dishes, flasks, or roller bottles may be increase the efficiency of producing reprogrammed cells from used to culture cells according to the methods of the present differentiated cells as disclosed herein. Additional agents invention. Suitable culture vessels include, for example, include PDK1 activators or compounds that promote glyco multi-well plates, petri dishes, tissue culture tubes, flasks, lytic metabolism, and/or Rho GTPase/ROCK inhibitors. roller bottles, and the like. 0067. It is believed that the small molecules described 0074 Defined cell media are available as packaged, pre herein can be used in combination with essentially any mixed powders or presterilized solutions. Examples of com method for generating iPSCs and thereby improve the effi monly used media include MEM-C. DME, RPMI 1640, ciency of the method. DMEM, Iscove’s complete media, or McCoy's Medium (see, 0068 A. Obtaining Non-Pluripotent Cells and Culturing e.g., GibcoBRL/Life Technologies Catalogue and Reference 0069. Non-pluripotent mammalian cells that can be used Guide: Sigma Catalogue). Typically, MEM-C, or DMEM are to generate iPSCs according to the methods described herein used in the methods of the invention. Defined cell culture can be differentiated cells from any suitable mammal (e.g., media are often supplemented with 5-20% serum, typically rodents such as, for example, mice, rats, guinea pigs, and heat inactivated serum, e.g., human, horse, calf, and fetal rabbits; non-rodent mammals such as, for example, dogs, bovine serum. Typically, 10% fetal bovine serum is used in cats, pigs, sheep, horses, cows, and goats; primates Such as, the methods of the invention. The culture medium is usually for example, chimpanzees and humans). buffered to maintain the cells at a pH preferably from about US 2016/0177271 A1 Jun. 23, 2016

7.2 to about 7.4. Other supplements to the media typically about 3 uM, about 4LM, about 5uM, about 6 uM, about 7 LM, include, e.g., antibiotics, amino acids, and Sugars, and growth about 8 uM, about 9 uM, or about 10 uM, of the small factors. molecule compound of Formula VI is used to treat the aggre 0075 B. Forming Cell Aggregates gates to induce pluripotency. In some embodiments, about 0 0076. To induce pluripotency, the somatic cells are cul uM to about 10uM, e.g., about OuM, about 1 uM, about 2 uM, tured in Suspension to form embryoid-like bodies. In some about 3 uM, about 4LM, about 5uM, about 6 uM, about 7 LM, embodiments, the Somatic cells are Suspended at a density of about 8 uM, about 9 uM, or about 10 uM, of the small about 1x10 cells/cm to about 1x10° cells/cm. In other molecule compound of Formula VII is used to treat the aggre embodiments, the cells are at a density of about 30,000 cells/ gates to induce pluripotency. In some embodiments, about 0 ml to about 500,000 cells/ml. The cells can be cultured in a uM to about 10uM, e.g., about OuM, about 1 uM, about 2 uM, non-adherent culture vessel. Such as a non-tissue culture about 3 uM, about 4LM, about 5uM, about 6 uM, about 7 LM, treated vessel or a low cell binding vessel. The cell can be about 8 uM, about 9 uM, or about 10 uM, of the small grown in any vessel that prevents the cells from forming a molecule compound of Formula VIII is used to treat the monolayer. aggregates to induce pluripotency. In some embodiments, 0077. To form aggregates or embryoid-like bodies, the about 0 uM to about 10 uM, e.g., about OuM, about 1 uM, Somatic or non-pluripotent cells are cultured in Suspension about 2 uM, about 3 LM, about 4 LM, about 5uM, about 6 uM, under conditions that allow the cells to develop into a loosely about 7 LM, about 8 LM, about 9 uM, or about 10 uM, of the compact mass of cells. In some instances, a Suspension of small molecule compound of Formula IX is used to treat the cells is mechanically manipulated. Such as mixed, stirred, aggregates to induce pluripotency. rocked, spun, titurated, and pipetted, such that the cells clump I0082 In some embodiments, the compound of Formula together into discrete three-dimensional aggregates. The cells II-IX is exposed to the aggregate at least 8 hours, e.g., about can be cultured in a vessel, e.g., roller bottle or stir bottle, and 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 periodically or continually agitated to promote the formation hours, 84 hours, 96 hours or more. The aggregate can be of aggregates. treated with any one of the compounds of Formula I-IX for at 0078. A mechanical force can be applied to the somatic least 1 day, e.g., 1 day, 2 days, 3 days, 4 days, 5 days 6, days, cells to preventorhinder the cells from adhering to the surface 7 days, 8 days, 9 days, 10 days or more. of the culture vessel. The magnitude of the mechanical force I0083. The aggregate can be treated with any one of the should not alter the integrity of the cells or induce cell death. compounds of Formula I-IX, or a combination of one or more Yet, the force should be sufficient to detach isolated cells and compounds of Formula I-IX. For instance, 2 compounds of aggregates from the Surface of a non-adherent culture vessel. Formula I-IX, such as those of Formulas I and II, Formulas I 007.9 The aggregates can beformed and cultured for about and III, Formulas I and IV. Formulas I and V. Formulas I and 2 to about 5 days, e.g., about 2 days, about 3 days, about 4 VI, Formulas I and VII, Formulas II and III, Formulas II and days, or about 5 days, after the start of the aggregation and IV. Formulas I and V. Formulas II and VI, Formulas II and VII, before small molecule treatment. Formulas II and VIII, Formulas II and IX, and the like can be 0080 C. Culturing Aggregates in Programin Compounds used. In some embodiments, at least 2, e.g., 2, 3, 4, 5, 6, 7, 8 0081. To induce pluripotency, the aggregates can be or 9, different compounds of Formula I-IX are useful for treated with (e.g., exposed to or cultured in) a derivative of inducing pluripotency in the aggregates. programin (compound of Formula I). In some embodiments, I0084 Reprogramming efficiency may be increased by about 0 uM to about 10 uM, e.g., about OuM, about 1 uM, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, about 2 uM, about 3 LM, about 4 LM, about 5uM, about 6 uM, 100%, 150%, 200%, 300%, 400%, 500% or more as com about 7 LM, about 8 uM, about 9 uM, or about 10 uM, of a pared with conventional methods. In some instances, the derivative of programin is used to treat the aggregates to reprogramming efficiency may be as high as 1%, 2%. 3%, induce pluripotent stem cells. In some embodiments, about 0 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40% or 50% uM to about 10uM, e.g., about OuM, about 1 uM, about 2 uM, (e.g., percent of induced pluripotent cells compared to total about 3 uM, about 4LM, about 5uM, about 6 uM, about 7 LM, number of starting Somatic cells). about 8 uM, about 9 uM, or about 10 uM, of the small I0085 Culturing of induced pluripotent stem cells gener molecule compound of Formula II is used to treat the aggre ated in this invention can use various media and techniques gates to induce pluripotency. In some embodiments, about 0 developed to culture primate pluripotent stem cells, more uM to about 10uM, e.g., about OuM, about 1 uM, about 2 uM, specially, embryonic stem cells, as described in U.S. Pat. No. about 3 uM, about 4LM, about 5uM, about 6 uM, about 7 LM, 7,442.548. It is appreciated that additional methods for the about 8 uM, about 9 uM, or about 10 uM, of the small culture and maintenance of human pluripotent stem cells, as molecule compound of Formula III is used to treat the aggre would be known to one of skill, may be used with the present gates to induce pluripotency. In some embodiments, about 0 invention. In certain embodiments, undefined conditions may uM to about 10uM, e.g., about OuM, about 1 uM, about 2 uM, be used; for example, pluripotent cells may be cultured on about 3 uM, about 4LM, about 5uM, about 6 uM, about 7 LM, fibroblastfeeder cells or a medium which has been exposed to about 8 uM, about 9 uM, or about 10 uM, of the small fibroblastfeeder cells in order to maintain the stem cells in an molecule compound of Formula IV is used to treat the aggre undifferentiated state. Alternately, pluripotent cells may be gates to induce pluripotency. In some embodiments, about 0 cultured and maintained in an essentially undifferentiated uM to about 10uM, e.g., about OuM, about 1 uM, about 2 uM, state using defined, feeder-independent culture system, Such about 3 uM, about 4LM, about 5uM, about 6 uM, about 7 LM, as a TeSR medium (Ludwig et al., 2006a, Ludwig et al., about 8 uM, about 9 uM, or about 10 uM, of the small 2006b). Feeder-independent culture systems and media may molecule compound of Formula V is used to treat the aggre be used to culture and maintain pluripotent cells. As described gates to induce pluripotency. In some embodiments, about 0 herein, various modifications may be made to these methods uM to about 10uM, e.g., about OuM, about 1 uM, about 2 uM, in order to reduce costs as desired. US 2016/0177271 A1 Jun. 23, 2016

I0086 For example, like human embryonic stem cells, iPS derm cells include Brachycury, GSC, LEF1, Mox1 and Tiel; cells can be maintained in 80% DMEM (Gibco #10829-018 and markers of ectoderm cells include Criptol, EN1, GFAP, or #11965-092), 20% defined fetal bovine serum (FBS) (or Islet 1, LIM1 and Nestin. Antibodies to markers of the three human AB serum), 1% non-essential amino acids, 1 mM germ layers are commercially available from, e.g., Abcam, L-glutamine, and 0.1 mM f-mercaptoethanol. Alternatively, EMD Millipore, Santa Cruz, Biotechnology, and the like. iPS cells can be maintained in serum-free medium, made with 0092. In some embodiments, the expression level (e.g., 80% Knock-Out DMEM (Gibco #10829-018), 20% serum amount) of Oct4, Nanog and Sox2 in the reprogrammed cell replacement (Gibco #10828-028), 1% non-essential amino is at least about at least about 2-fold higher, at least about acids, 1 mM L-glutamine, and 0.1 mM 0 mercaptoethanol. 4-fold higher, at least about 6-fold higher, at least about 8-fold Human bFGF may be added to a final concentration of about higher, at least about 10-fold higher, at least about 15-fold 4 ng/mL or bFGF may be used instead. higher, at least about 20-fold higher, at least about 40-fold I0087. D. Methods of Characterizing Pluripotent StemCell higher, at least about 80-fold higher, at least about 100-fold 0088 Chemically induced reprogrammed cells as dis higher, at least about 150-fold higher, at least about 200-fold closed herein are indistinguishable from higher, at least about 500-fold higher, or more than the or other pluripotent cells in morphology, proliferation, gene expression level (amount) of the same gene or gene productin expression, and teratoma formation. For example, chemically a differentiated cell from which the reprogrammed cell was induced human reprogrammed cells are expandable and derived. indistinguishable from human embryonic stem cells in mor 0093 E. Methods of Enriching and/or Isolating Repro phology and proliferation. Furthermore, these chemically grammed Cells induced reprogrammed cells can differentiate into cell types 0094. Another aspect of the present invention relates to the of the three germ layers in vitro and in . isolation of a population of reprogrammed cells from a het 0089 Analysis to assess the pluripotency characteristics erogeneous population of cells, such a mixed population of of the reprogrammed cells may be performed. The cells may cells comprising reprogrammed cells and differentiated cells be analyzed for different growth characteristics and embry from which the reprogrammed cells were derived. A popula onic stem cell like morphology. The pluripotent stem cells tion of reprogrammed cells produced by any of the method derived according to the methods described herein have char described herein can be enriched, expanded, isolated and/or acteristic antigens that can be identified or confirmed by purified by using any cell Surface marker present on the immunohistochemistry or flow cytometry, using antibodies reprogrammed cell which is not present on the differentiated for SSEA-1, SSEA-3 and SSEA-4 (Developmental Studies cell from which it was derived. Such cell Surface markers are Hybridoma Bank, National Institute of Child Health and also referred to as an affinity tag which is specific for repro Human Development, Bethesda Md.), and TRA-1-60 and grammed cells. Examples of affinity tags specific for repro TRA-1-81 (Andrews et al., 1987). Pluripotency of stem cells grammed cells are antibodies, ligands or other binding agents can also be confirmed by injecting approximately 0.5-10x10° that are specific to a marker molecule. Such as a polypeptide, cells into the rear leg muscles of 8-12 week old male SCID that is present on the cell Surface of a reprogrammed cell but mice. The appearance ofteratomas demonstrate that the stem which is not Substantially present on other cell types (e.g. on cells are differentiate to become at least one cell type of each differentiated cells). In some methods, an which of the three germ layers. binds to a cell Surface antigen on a reprogrammed cell (e.g. a 0090 Expression profiling of individual genes associated human reprogrammed cell) is used as an affinity tag for the with pluripotency may also be examined. Additionally, enrichment, isolation or purification of chemically induced expression of embryonic stem cell Surface markers may be reprogrammed cells produced by the methods described analyzed. Detection and analysis of a variety of genes or gene herein. In some embodiments, the reprogrammed cells can products known in the art to be associated with pluripotent also be isolated from the differentiated cells by fluorescence stem cells may include analysis of genes or gene products, activated cell sorting (FACS), affinity-based methods, and a such as, but not limited to, Oct4, Nanog, Sox2, Klf4, c-Myc, combination thereof. ABCG2, E-cadherin, B-tubulin III, C-Smooth muscle actin 0.095 The chemically induced reprogrammed cells can (C-SMA), fibroblast 4 (FGF4), Cripto, Dax 1. also be isolated by other techniques for cell isolation. Addi Zinc finger protein 296 (Zfp296), N-acetyltransferase-1 tionally, reprogrammed cells can also be enriched or isolated (Nat1), ES cell associated transcript 1 (ECAT1), ESG1/ by methods of serial subculture in growth conditions which DPPA5/ECAT2, ECAT3, ECAT6, ECAT7, ECAT8, ECAT9, promote the selective survival or selective expansion of the ECAT10, ECAT15-1, ECAT15-2, Fthl17, Sall4, undifferenti reprogrammed cells. Such methods are known by persons of ated embryonic cell (Utf1), Rex 1, p53, ordinary skill in the art. G3PDH, , including TERT, silent X chromosome 0096. In some embodiments, the cell culture or cell popu genes, Dnmt3a, Dnmt3b, TRIM28, F-box containing protein lation comprising the reprogrammed cells comprise at least 15 (Fbx15), Esrrb, TDGF1, GABRB3, Z?p42, FoxD3, GDF3, about 1% to 100%, e.g., at least about at least about 1%, at CYP25A1, developmental pluripotency-associated 2 least about 2%, at least about 3%, at least about 4%, at least (DPPA2), T-cell lymphoma breakpoint 1 (Tcl 1), DPPA3/ about 5%, at least about 6%, at least about 7%, at least about Stella, DPPA4, as well as other general markers for pluripo 8%, at least about 90%, at least about 9%, at least about 10%, tency, for example any gene or gene product used during at least about 11%, at least about 12%, at least about 13%, at induction to reprogram the cell. least about 14%, at least about 15%, at least about 16%, at 0091. The cells can also be characterized by the down least about 17%, at least about 18%, at least about 19%, at regulation of markers characteristic of the differentiated cells least about 20%, at least about 25%, at least about 30%, at from which the pluripotent cells are induced. Non-limiting least about 35%, at least about 40%, at least about 45%, at examples of markers of endoderm cells include Gata4, least about 50%, at least about 55%, at least about 60%, at FoXA2. PDX1, Nodal, Sox7 and Sox17; markers of meso least about 65%, at least about 70%, at least about 75%, at US 2016/0177271 A1 Jun. 23, 2016

least about 80%, at least about 85%, at least about 90%, at Example 1 least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at Protocol for Activating Pluripotent Gene Expression least about 97%, at least about 98%, at least about 99%, or in Mouse 100%, pluripotent stem cells. 0104. We have identified a small molecule via high 0097 F. Methods of Differentiating Reprogrammed Cells through-put screening of embryoid-like bodies fabricated 0098. Another method for assessing the pluripotency of from fibroblasts which carried the transgene Oct4 promoter the induced stem cells is to in vitro differentiate the cells into linked to an EGFP reporter (Oct4-EGFP).This synthetic het specific Somatic cell types. For example, certain growth fac erocyclic molecule, which we named “programin' (e.g., tors known to drive differentiation into particular cell types reversine), could induce mouse Somatic cells to strongly may be used. express Oct4, Sox2 and Nanog by 25-200 fold. In addition, the expression of E-cadherin (a cell adhesion gene that is 0099. In some embodiments, the stem cells generated crucial for maintaining pluripotency in embryonic cells) was according to the methods of the present invention are Subse induced and was increased by 5-fold compared to that of the quently induced to form, for example, hematopoietic (stem/ initial fibroblast. The reprogramming efficiency was approxi progenitor) cells, neural (stem/progenitor) cells (and option mately 20% and takes 7 days to accomplish. The expression ally, more differentiated cells, such as Subtype specific of Oct4, Sox2 and Nanog was not induced (activated) when neurons, , etc.), pancreatic cells (e.g., endo the fibroblasts were conventionally grown on normal adhe crine or pancreatic hormone-expressing sive culture dishes as a monolayer. cells), hepatocytes, cardiovascular (stem/progenitor) cells (e.g., cardiomyocytes, endothelial cells, Smooth muscle 0105. The process of forming these bodies itself activates cells), retinal cells, etc. A variety of methods are known for low basal levels of Oct4, Sox2 and Nanog expression (2-fold inducing differentiation of pluripotent stem cells into desired higher compared to growing, culture dish-adherent fibro cell types. A non-limiting examples of methods for inducing blasts). This mechanically-induced bioactivity was validated differentiation of stem cells into various cell fates are by qPCR analysis. Treating the cell aggregates with pro gramin further increased the expression of these three pluri described in, for example, U.S. Pat. Nos. 8,187,878; 7,781, potent genes, for example, by 20-200-fold compared to con 214; 7.757,762; 7,541,186: Kaiming Ye and Sha Jin, eds., trols. These results have also been replicated many times and Human Embryonic and Induce Pluripotent Stem Cells. Lin also using human fibroblasts. In Summary, programin or eage-specific Differentiation Protocols, New York: Humana derivatives thereofalone cannot active Oct4, Sox2 and Nanog Press, 2011; and Schlaeger et al., Current Protocols in Stem expression unless the Somatic cells are mechanically induced Cell Biology, New York: Wiley, 2014. to aggregate and prevented from adhering to culture dishes. 01.00 G. Pharmaceutical Formulations 0106 The programin-induced pluripotent (PIP) stem cells 0101. When used for pharmaceutical purposes, the small are capable of forming osteocytes when induced by osteo molecule compound of the present invention is generally genic inducing medium, as well as neural cells when treated formulated in a suitable buffer, which can be any pharmaceu with neural inducing Supplements. This novel approach for tically acceptable buffer, such as phosphate buffered saline or producing pluripotent stem cells is simple, fast, efficient and sodium phosphate/sodium sulfate, Tris buffer, glycine buffer, cost effective. As such, PIP stem cells provide a valuable and sterile water, and other buffers known to the ordinarily skilled Versatile source of cells for use in generating organs and artisan such as those described by Good et al. Biochemistry tissue transplants for use in regenerative medicine. Further 5:467 (1966). more, human PIP stem cells can be induced to differentiate into specific cell types such as liver, heart and nerve cells for 0102 The compositions can additionally include a stabi toxic and bioactivity testing of drugs by the pharmaceutical lizer, enhancer or other pharmaceutically acceptable carriers industry. or vehicles. A pharmaceutically acceptable salt or carrier can contain a physiologically acceptable compound that acts, for 0107 This example illustrates the use of a novel com example, to stabilize the Small molecule compound. A physi pound that enables reprogramming of mouse somatic cells ologically acceptable compound can include, for example, into induced pluripotent stem cells (iPSCs) that express pluri carbohydrates, such as glucose. Sucrose or dextrans, antioxi potent stem cell markers. dants, such as ascorbic acid or glutathione, chelating agents, 0.108 Materials required: Culture medium, supplemented low molecular weight proteins or other stabilizers or excipi with 10% FBS, 1% PS in DMEM (Invitrogen); Mouse ESC ents. Other physiologically acceptable compounds include medium, supplemented with 1xE27 (Invitrogen), 1,000 Wetting agents, emulsifying agents, dispersing agents or pre U/mL Mouse Leukemia Inhibitory Factor (LIF) in DMEM/ servatives, which are particularly useful for preventing the F12 culture medium (Invitrogen); Programin (frozen stock growth or action of microorganisms. Various preservatives dissolved in DMSO); Working concentration=1-5uM; Sterile are well known and include, for example, phenol and ascorbic 35 mm non-adhesive culture dish (SPL: 11035). acid. Examples of carriers, stabilizers or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Pub Procedures: lishing Company, Philadelphia, Pa., 17th ed. (1985). 0109 Day 1 of PIP stem cells production: VI. Examples 0110 1. Source mouse tail fibroblasts (passage 2-6 and in log phase of growth). Trypsinize the fibroblasts using stan 0103) The following examples are offered to illustrate, but dard protocols to obtain a suspension of the cells in culture not to limit the claimed invention. medium. US 2016/0177271 A1 Jun. 23, 2016 12

0111 2. Seed the fibroblasts or other somatic cells onto a and Hoechst dye for the presence of live cells (blue arrows) sterile 35 mm non-adhesive culture dish at density of 1x10 (FIGS. 3A-3D). Under the confocal microscope, Oct4-EGFP 1x10° cells/cm in 3 mL of culture medium. expression (green arrows) was undetectable in the live cells of 0112. 3. Incubate the cells at 37° C. and 5% CO, inside a non-treated embryoid-like bodies (FIG. 3A). Embryoid-like cell incubator. cell bodies treated with 5uM of Programin for 24 hours (FIG. 0113 4. Gently pipette the culture media to prevent the 3B), 48 hours (FIG. 3C), and 69 hours (FIG. 3D) expressed cells from adhering to culture dish Surface. Two sessions per Octá-EGFP. Programin was able to activate Oct4-EGFP day, 2 min for each session, must be performed. A standard 1 expression (green arrows) in the CD34 fibroblasts 24-69 mL pipette and plastic tip was used for pipetting the cultures. hours after treatment. The pipetting has to done very slowly (approximately 0.5-1 I0129. The qPCR results show that CD34" mouse fibro mL/second generates Sufficient force to detach cells from blasts when cultured as a monolayer do not express Oct4. non-adhesive culture dish Surface). Sox2 and Nanog (FIG. 4). In contrast, when the fibroblasts are 0114 Day 2 of PIP stem cell production. mechanically manipulated to form embryoid-like cell bodies, 0115 1. The cells would start to aggregate into embryoid they are induced to express pluripotent genes, such as Oct4. like bodies after day 1. These bodies will grow in numbers Sox2 and Nanog at low basal levels. Upon treating these and size during the period of culture. embryoid-like cell bodies with 5um Programin, Oct4, Sox2 0116 2. Gently pipette the culture media to prevent the and Nanog expression is significantly enhanced. embryoid-like cell bodies from attaching on culture dish sur I0130. The qPCR results show the efficacy of various Pro face. gramin derivatives to enhance Oct4, Sox2 and Nanog expres 0117 3. Add 1 mL of fresh culture medium. sion (FIG. 5). Derivatives 237, 400, 594,648, 668,957, 958 0118 Day 4 of PIP stem cell production. and 959 significantly increased Oct4, Sox2 and Nanog 0119) 1. Collect cell suspension and pellet cells by cen expression in embryoid-like cell bodies of CD34" mouse trifugation at 800 rpm for 5 min. Gentle centrifugation and fibroblasts. Levels of the pluripotent stem cell markers were pipetting do not de-aggregate the embryoid-like bodies. Wash higher in the embryoid-like cell bodies treated with pro cell once with mouse ESC medium and Programin (1-5uM) gramin or derivatives thereof, compared to untreated embry or programin derivatives. oid-like cell bodies and CD34" mouse fibroblasts cultured as 0120 2. Pellet the embryoid-like bodies again and re a monolayer. suspend them in 3 mL mouse ESC medium and Programinor derivatives thereof. Example 2 0121 3. Seed the embryoid-like cell bodies onto a sterile 35mm non-adhesive culture dish. Incubate the embryoid-like Protocol for Activating Pluripotent Gene Expression bodies at 37° C. and 5% CO, inside a cell incubator for 2-3 in Human Umbilical Cord Peri-Vascular days. Mesenchymal Progenitor Cells 0122 4. Perform daily pipetting twice in a day to prevent the embryoid-like cell bodies from sticking to the culture I0131 This example illustrates the use of a novel com dish. pound that promotes reprogramming of human progenitor 0123 5. Add 1 mL of fresh mouse ESC medium on day 5 cells, e.g., human umbilical cord peri-vascular mesenchymal (day 2 post-Programin treatment). progenitor cells into iPSCs that express pluripotent stem cell (0.124 Day 6-7 of PIP stem cell production. markers. 0125 1. Harvest the embryoid-like cell bodies for PI and 0.132. Materials required: HUCPV medium, consisting of Hoechst staining for confocal analysis. The presence of green 15% ESQ-FBS (Invitrogen) and 1% PS in MEM/F12 (Invit fluorescence and Hoechst staining in cells indicate the pres rogen), Essential 6 (Invitrogen) supplemented with 100 ence of live PIP cells expressing Oct4-EGFP. ng/mL basic fibroblast growth factor (bFGF) (Invitrogen), 0126 2. Harvest the embryoid-like cell bodies for qPCR Programin or derivatives thereof (frozen stock dissolved in analysis to validate expression of pluripotent genes: Oct4. DMSO); Working concentration=1-5uM; Sterile non-adhe Sox2 and Nanog expression. In addition, expression of other sive 24-well culture plate (SPL: 32024). key stem cell-associated genes. Procedures: Results: 0.133 1. Primary human umbilical cord peri-vascular 0127. A schematic diagram of an exemplary method of the (HUCPV) cells were maintained in HUCPV culture medium present invention is provided in FIG. 1. Tail fibroblasts and passaged by trypsinization. Only HUCPV cells below extracted from the tail of Oct4-EGFP transgenic mice and cell passage 6 were used. stained with CD34 (FIG. 2). The cells were sorted by flow cytometry for only those expressing the Surface marker 0.134 2. HUCPV cultures are trypsinize and dissociate CD34. FIG. 2B shows CD34" fibroblasts grown on adhesive into single cells. culture dishes for 3 days. CD34 fibroblasts grown on adhe I0135 3. The dissociated HUCPV cells are re-suspend as sive culture dishes and treated with 5uM programin for 3 days 3x10 cells in 600 uL of HUCPV medium and seeded into died and did not express Oct4 (FIGS. 2C and 2D). each well of a non-adhesive 24-well culture plate. 0128. CD34 fibroblasts harvested from the tail of an (0.136 4. The cells are incubated at 37° C. and 5% CO, Octá-EGFP transgenic mouse were mechanically pipetted inside a cell incubator. and maintained on non-adhesive culture plates so that the 0.137 5. On following day, until the end of experiment, the cells became aggregated to form embryoid-like bodies (FIG. cells were pipetted twice a day to prevent cells attaching onto 3A). The embryoid-like bodies in this study were stained with culture plates which caused the HUCPV cells to aggregate PI dye to determine the presence of dead cells (red arrows) into embryoid-like cell bodies. US 2016/0177271 A1 Jun. 23, 2016

0138 6. 200 uL of fresh HUCPV medium were added to gramin or derivatives thereof (frozen stock dissolved in the cultures, every 2 days. DMSO); Working concentration=5 uM, and sterile 35 mm 0139 7. After 4 days culture, the embryoid-like cell bodies non-adhesive culture dish (SPL: 11035). were centrifuged at 800 rpm for 5 minto remove the culture medium. Ensure that centrifugation and pipetting are gentile Procedure: which do not disaggregate the embryoid-like bodies. 0149 1. Primary human skin fibroblasts were cultured in 0140) 8. Wash embryoid-like cell bodies once with fibroblast medium and passaged by trypsinization. Passage 5 HUCPV medium supplemented with Essential 6 and 1-5uM fibroblasts were used for experimentation. Programin or derivatives thereof. Remove medium by cen 0150 2. After dissociating the cultures into single cells trifugation with trypsin, 2x10 cells were resuspended in 3 mL fibroblast 0141 9. Re-suspend the embryoid-like bodies in 600 uL of medium in non-adhesive 35 mm dish and maintained in 37° HUCPV medium supplemented with Essential 6 and 1-5uM C. incubator with 5% CO. Programin or derivatives thereof into each of the wells of a 0151. 3. Several hours later, aggregates were formed. Gen sterile non-adhesive 24-well culture plate. tly breakup excessively large aggregates by pipetting. 0142 10. Thirty hours after Programin treatment, the Pro 0152 4. The next day, compact aggregates were formed. gramin treated embryoid-like cell bodies were harvested for Again gentry breakup aggregates into Smaller size (100-120 qPCR analysis to validate expression of the pluripotent genes: um diameter) by pipetting. Octa, Sox2 and Nanog. Other key stem cells-associate genes 0153. 5. Three days after cell aggregate/embryoid-like cell were also analyzed. body formation, change the medium to human ESC medium supplemented with 5uM Programin or derivatives thereof. Results: 0154 6. Replenish medium with fresh Programin or 0143 Human umbilical cord perivascular mesenchymal derivatives thereof 2 days after. progenitor (HUCPV) cells were allowed to adhere to culture (O155 7. Harvest cells after 4 days of treatment with Pro dishes and then were treated with 0-5uM of Programin for 30 gramin or derivatives thereof. Extract total RNA using Trizol hours. qPCR analysis showed that Programin did not activate Reagent and purify using column. Octa, Sox2, KLF4, c-Myc and, Nanog expression in the cells 0156 8. Perform reverse-transcription and qPCR for key cultured as a monolayer. stem cells-associate genes. 0144 HUCPV cells that were aggregated into embryoid like bodies showed higher expression of Oct4, Sox2 and Results: Nanog compared to those cells cultured as a monolayer (FIG. 0157. After 4 days of compound treatment, stem cell-as 6). However, Klf4 and c-Myc expression were not activated. Sociated genes Such as Oct4, Nanog, Sox2, c-Myc and KLF4 (0145 The embryoid-like bodies formed from HUCPV are expressed at higher levels in the embryoid-like bodies cells were treated with 1-5 uM Programin for 30 hours. treated with programin than embryoid-like bodies treated Embryoid-like bodies exposed to Programin at all concen with vehicle, a monolayer of human skin fibroblasts treated trated tested showed significantly enhanced Oct4, Sox2, with programin, and a monolayer of human skin fibroblasts KLF4, c-Myc and, Nanog expression compared to the treated with vehicle (FIGS. 8A-E). Thus, Programinactivates untreated embryoid-like body controls (OuM) (FIGS. 6 and fibroblasts cultured in an aggregate, but not in a monolayer, to 7). 4 LM Programin induced the highest level of Oct4, Sox2, strongly express pluripotency-associated genes including: Klf4 and Nanog compared to the other concentrations. Octa, Sox2, Nanog. KLF4 and c-Myc. 0146 The data illustrates that HUCPV cells when physi cally aggregated into embryoid-like bodies respond to Pro Example 4 gramin by activating Oct4, Sox2, Klf4, c-Myc and, Nanog expression in the cells. The embryoid-like bodies formed Method for Testing the Developmental Potential of originally from HUCPV cells differentiate into pluripotent the Embryoid-Like Bodies Generated According to stem cells when grown on non-adhesive culture dishes and the Examples Above prevented from Sticking to the dish Surface. 0158. This example illustrates methods of differentiating the iPSCs produced according to the methods described in Example 3 Examples 1-3 to osteogenic cells or neural cells. Protocol for Activating Pluripotent Gene Expression Method for Osteogenic (Bone) Induction: in Human Skin Fibroblasts 0159 Programin-treated embryoid-like cell bodies 0147 This example illustrates a method of inducing the (1x10 cells/10 cm) were maintained as a suspension culture formation of iPSCs from human skin fibroblasts. The method in DMEM/F12, 1000 U/mL LIF and 1xE27 on non-adhesive includes forming the human fibroblasts into cell aggregates culture dishes for 4 days. The embryoid-like cell bodies were and then exposing the aggregates to the compound programin then transferred to culture dishes coated with 0.1% gelatin or a derivative thereof, e.g., programin 2-9. (FIG. 9A). 10 uM Wntagonist (Cathé81665, Calbiochem) 0148 Materials required: Fibroblast medium, supple was added to the culture medium (DMEM/F12, 1000 U/mL mented with 15% FBS, 1% non-essential amino acids, 1% PS LIF and 1xE27). Bone-like nodules were evident in the cul in Eagle's MEM medium (Invitrogen), human ESC medium, tures after 2-3 days (FIG. 9B). Alizen Reds staining of the supplemented with 20% knockout serum replacement (Invit nodules confirmed that the differentiated cells were osteo rogen), 2 mM L-glutamine, 0.1 mM non-essential amino cytes (FIG.9C). Programin-treated cells that were not aggre acids, 0.1 mM mercaptoethanol, 10 ng/mL fibroblast growth gated underwent cell death following exposure to the Wnt factor-basic (bFGF), 1%PS in DMEM/F12 (Invitrogen), Pro agonist (FIG.9D). US 2016/0177271 A1 Jun. 23, 2016

Method for Nerve Cell Induction: -continued (VII) (0160 Programin treated embryoid-like cell bodies (1x10' cells/10 cm) were maintained as a suspension culture in DMEM/F12, 1000 U/mL LIF and 1XB27 on non-adhesive culture dishes for 4 days. The embryoid-like cell bodies were N1 then transferred to culture dishes coated with 0.1% gelatin (FIG. 10A). No nerve-inducing small molecules were added N to the media because the B27 in the culture medium can N1 N induce neurogenesis (FIG.10B). Neurons were evident on the 2 0.1% gelatin coated culture plates after 3 days culture, and C us N Ny more obvious after 7 days (FIGS. 10C and D). NH (VIII) 0161 All patents, patent applications, and other publica 2 tions, including GenBank Accession Numbers, cited in this application are incorporated by reference in the entirety for all N21 purposes. N F u?N N 1. A method for producing a pluripotent stem cell from differentiated cells, the method comprising: HO O (a) culturing the differentiated cells in a vessel; OH, or (b) mechanically agitating the differentiated cells to form a Hó cellular aggregate; (IX) (c) exposing the cellular aggregate to an effective amount of a compound of Formula II-IX having any one of the following structures:

(II) OH

CN N 21 Sn O.M N21 N H (III) N X S C N N

(d) detecting the expression level of Oct4, Sox2 or Nanog " X in the cells of the aggregate; HN s N (e) determining any cell is a pluripotent stem cell if the (IV) expression level of Oct4, Sox2 or Nanogin the cell of the S aggregate is higher than the corresponding expression level in a differentiated cell; and (f) isolating the pluripotent stem cell. 2. The method of claim 1, wherein the differentiated cells " X of step (a) is obtained from a subject or a cell line. N N 3. The method of claim 2, wherein the subject is a mam (V) NH2 malian Subject. 4. The method of claim 2, wherein the cell line is a mam malian cell line. N21 5. The method of claim 1, wherein the differentiated cells are fibroblasts. l?s N 6. The method of claim 1, wherein the differentiated cells (VI) are progenitor cells. 7. The method of claim 1, wherein the differentiated cells ( \, of step (a) are at a density of about 1x10-1x10° cells/cm. N1 8. The method of claim 1, wherein the cellular aggregate does not adhere to an inner surface of the vessel. 9. The method of claim 8, wherein the inner surface of the vessel is coated with a non-adherent Substrate. y 10. The method of claim 1, wherein mechanically agitating C us N 2 N comprises titurating, stirring or rocking the differentiated cells. US 2016/0177271 A1 Jun. 23, 2016

11. The method of claim 1, wherein step (d) comprises an -continued amplification-based assay, a hybridization-based assay, an (VI) immunoassay or a flow cytometry assay. ( \, 12. The method of claim 1, wherein the higher expression N1 level of Oct4, Sox2 or Nanog is at least about 20-fold to about 200-fold higher compared to the differentiated cell. 13. The method of claim 1, further comprising, subsequent 1y to step (e), expanding the pluripotent stem cell. --- N 14. The method of claim 1, further comprising Subsequent to step (e), inducing the pluripotent stem cell to undergo differentiation. (VII) 15. (canceled) 16. A method for producing a cellular aggregate from dif ferentiated cells, wherein the cellular aggregate comprise a N1 cell that expresses at least one pluripotent stem cell marker, the method comprising: N1 N N (a) culturing the differentiated cells at a density of about 2 1x10 to about 1x10 cells/ml in a vessel; C ul-IXN N (b) mechanically agitating the differentiated cells to form the cellular aggregate; and NH (VIII) (c) culturing the cellular aggregate under conditions to 2 produce a cell that expresses at least one pluripotent stem cell marker. N21 17. The method of claim 16, further comprising (d) expos lsS X ing the cellular aggregate to an effective amount of a com F N N pound of Formula II-IX having any one of the following HO O Structures: OH, or

(II) HG OH

N 21 (IX) N Y.M N H (III) S H N ON " N X HN N N (IV) N21 S Sn H C ul?N N N

" N X N N (V) 18. The method of claim 16, wherein the at least one NH2 pluripotent stem cell marker is Oct4, Sox2 or Nanog. 19. The method of claim 16, further comprising subsequent N21 to step (c) expanding (c) the cell that expresses the at least one pluripotent stem cell marker. Sn l?N N 20. The method of claim 16, further comprising subsequent to step (c) isolating the cell that expresses the at least one pluripotent stem cell marker. US 2016/0177271 A1 Jun. 23, 2016 16

21. A compound of Formula II-IXhaving the any one of the -continued following structures: (VII)

(II) ( \, OH N1 N 21 N1 N N S. O. 2 N N C us N Ny (III) (VIII) S NH2 H N N21 " N X N HN N N F u?N N (IV) S HO O H N OH, or Hó " N X N N (IX) (V) NH Na

(VI) CN N21 N X C N N

22.-24. (canceled)