JP2016135102A - Combinational use of mechanical manipulation and programin to generate pluripotent stem cells from somatic cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods and compositions for inducing pluripotency in differentiated mammalian cells.SOLUTION: The present invention relates to a method for producing a pluripotent stem cell (iPS cell) from differentiated cells, the method comprising: (a) culturing the differentiated cells in a vessel; (b) mechanically agitating the differentiated cells to form a cellular aggregate; (c) exposing the cellular aggregate to an effective amount of a compound having any one of compositions of compounds derived from programin which is a derivative of pyrimidine or purine; (d) detecting the expression level of Oct4, Sox2 or Nanog in the cells of the aggregate; (e) determining that any cell is a pluripotent stem cell if the expression level of Oct4, Sox2 or Nanog in the cell of the aggregate is higher than the corresponding expression level in a differentiated cell; and (f) isolating the pluripotent stem cell.SELECTED DRAWING: Figure 1

Description

  The present invention utilizes mechanical aggregation of somatic cells to form embryoid somatic cell bodies, and programming for reprogramming somatic cells to produce embryoid-like cell bodies Or it relates to the treatment of these aggregates with derivatives thereof. Such reprogrammed cells are useful in the field of regenerative medicine.

  Stem cells have been widely published as a solution to alleviate the serious shortage of tissues and organs for transplantation. To date, many types of natural and artificially produced stem cells have been identified and created. However, all of them have drawbacks. Induced pluripotent stem cells (iPSC) (Takahashi and Yamanaka, Cell, 126 (4): 663-76 (2006)) produced by reprogramming somatic cells such as dermal fibroblasts are currently being used in regenerative medicine It is considered the most promising candidate. However, iPSC production is still a very slow (about 4 weeks) and inefficient (<0.01%) process that results in heterogeneous cell populations and requires special expertise in human pluripotent cell culture (Takahashi et al., Cell, 131, 861-72 (2007); Yu et al., Science, 318, 1917-20 (2007)). An alternative approach uses small chemical molecules that can directly activate the pluripotency genes Oct4, Sox2, and Nanog.

  Programin was first synthesized by Chen et al. (J. Am. Chem. Soc., 2004 126: 410-411). This was used to transdifferentiate C2C12 myogenic cells into bone cells and adipocytes. Later, using the proteome comparison technique, it was shown that the polycomb gene in C2C12 was probably involved in the transdifferentiation process (Shen et al., Proteomics, 2007, 7 (23): 4303-4316). Furthermore, it has been reported that transdifferentiation to skeletal muscle cells can be induced by treating fibroblasts with reversin (Luigi et al., 2007, Cell Death and Differentiation, 13 (12): 2042-2051; Chiara et al. al., 2009, Electrophoresis, 30 (12): 2193-2206). Mandal et al. (Biochip Journal, 2013, 7 (3): 278-287) examined the transcriptome profile of reversin-mediated transdifferentiation of NIH3T3 fibroblasts into osteoblasts. Studies have not shown that reversin activates the expression of pluripotent genes such as Oct4, Sox2, and Nanog.

  There is a need for a simple and efficient method based on the use of small molecule compounds to make iPSCs. The present invention fulfills this and other needs.

  In one aspect, the present invention provides a method for producing pluripotent stem cells from differentiated cells. The method includes: (a) culturing the differentiated cells in a container; (b) mechanically stirring the differentiated cells to form cell aggregates; and (c) an effective amount of the cell aggregates. (D) detecting an expression level of Oct4, Sox2 or Nanog in the cells of the aggregate, (e) exposure to a compound having any one of the structures represented by the following formulas II to IX: Determining that the cell is a pluripotent stem cell if the expression level of Oct4, Sox2, or Nanog in the cell of the aggregate is higher than the corresponding expression level in the differentiated cell; and (f) the multiplicity Isolating competent stem cells. In some embodiments, steps (b) and (c) are performed simultaneously. In another embodiment, steps (b) and (c) are performed sequentially. For example, step (c) may comprise about 24 to about 96 hours, or more after step (b), such as about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, It can be performed after about 96 hours or more.

The differentiated cells of step (a) can be obtained from the subject or cell line. In some embodiments, the subject is a mammalian subject. The cell line can be a mammalian cell line. In some embodiments, the differentiated cell is a fibroblast or a progenitor cell. The differentiated cells of step (a) can be at a density of about 1 × 10 ⁵ to 1 × 10 ⁶ cells / cm ² .

  Cell aggregates do not adhere to the inner surface of the container. In some embodiments, the inner surface of the container is coated with a non-adhesive material (eg, a substrate that prevents or prevents adhesion of cells to the site).

  In some embodiments, mechanical agitation is performed by rubbing differentiated cells, for example, by shaking or rotating the culture vessel in which the cells are maintained. Including stirring or rocking. For example, a pipette tip with a large pore size can be used to pipet the cells and apply mechanical force to the cells or cell aggregates to prevent or prevent their attachment to the culture vessel.

  In some embodiments, step (d) of the above method comprises an amplification based assay, a hybridization based assay, an immunoassay, or a flow cytometry assay. After step (e), the method may further comprise expanding pluripotent stem cells (proliferate). Further, the method may comprise inducing differentiation of pluripotent stem cells after step (e).

  In some cases, the higher expression level of Oct4, Sox2, or Nanog is at least about 20 to about 200 times higher than the differentiated cell.

  In another aspect of the invention, a pluripotent cell or cell line obtained by any of the above example embodiments of the method is provided.

  In yet another aspect, the present invention provides a method of making a cell aggregate comprising a cell that expresses at least one pluripotent stem cell marker from a differentiated cell. The method comprises: (a) culturing the differentiated cells in a container at a density of about 1 × 10 5 to about 1 × 10 6 cells / ml; (b) mechanically agitating the differentiated cells to form the cell aggregate And (c) culturing the cell aggregate under conditions that yield cells expressing the at least one pluripotent stem cell marker.

  In some embodiments, the method further comprises exposing to an effective amount of a compound having any one of the structures shown in Formulas II-IX below.

  The method may further comprise expanding cells expressing the at least one pluripotent stem cell marker after step (c). If desired, the method may comprise separating cells expressing the at least one pluripotent stem cell marker after step (c). The at least one pluripotent stem cell marker can be Oct4, Sox2, or Nanog.

  In another aspect, the present invention provides a compound having any one of the structures shown in Formulas II-IX below.

  In some cases, the compound is present in a mixture comprising differentiated cells. For example, a compound of formula II, III, IV, V, VI, VII, VIII, or IX and a differentiated cell may be mixed together to form a mixture. In some embodiments, the differentiated cells of the mixture are present in aggregates.

  In another aspect, the present specification provides a pharmaceutical composition for generating pluripotent stem cells from differentiated cells, comprising the aforementioned compound and a pharmaceutically acceptable salt.

  The inventors surprisingly found that pluripotency was observed when fibroblasts were mechanically formed into embryoid-like bodies and maintained on non-adherent culture dishes for 4 days. It has been discovered that the genes Oct4, Sox2, and Nanog can be expressed. Furthermore, when these aggregates were treated with the small molecule compound programin or derivatives thereof, the expression of these three pluripotency genes was increased 25-200 times compared to non-aggregated non-treated differentiated cells.

It is a schematic diagram of an example of a method for inducing pluripotency from fibroblasts. It is a figure which shows the influence of programin on the mouse | mouth fibroblast cultured in a monolayer. Fibroblasts were derived from the tail of Oct4-EGFP transgenic mice. FIG. 2 (A) shows CD34 ⁺ fibroblasts. FIG. 2 (B) shows the absence of GFP ⁺ fibroblasts. FIG. 2 (C) and FIG. 2 (D) show fibroblasts after exposure to programin for 2 days. It is a figure which shows the influence of programin on the mouse Oct4-EGFP fibroblast cultured by the aggregate or the embryoid body-like body. FIG. 3 (A) shows an aggregate on the third day of culture on a non-adhesive culture plate. FIG. 3 (B) shows the aggregate after 24 hours exposure to programin. GFP ⁺ can be detected with a microscope. FIG. 3 (C) shows the aggregate after 48 hours exposure to programin. FIG. 3 (D) shows aggregates after 96 hours exposure to programin. FIG. 4 shows that CD34 ⁺ mouse fibroblasts cultured in a monolayer do not express Oct4, Sox2, and Nanog as measured by qPCR. The expression of these pluripotency markers was higher with cell aggregates and was greatest with aggregates treated with programin. FIG. 4 shows qPCR data showing that programin derivatives can increase Oct4, Sox2 and Nanog expression in cell aggregates of CD34 ⁺ mouse fibroblasts. The small molecules tested included 237 (Formula II), 400 (Formula III), 594 (Formula IV), 648 (Formula VIII), 668 (Formula V), 957 (Formula IX), 958 (Formula VI), 959 (Formula VII) and programin (Formula I) are included. Diagram showing that human umbilical cord perivascular mesenchymal progenitor (HUCPV) cell aggregates express higher levels of Oct4, Sox2 and Nanog after treatment with 0-5 μM programin than similar cells cultured in monolayers. It is. It is a figure which shows the qPCR data of FIG. 6 with higher resolution. Oct4 (FIG. 8 (A)), Nanog (FIG. 8 (B) in human skin fibroblasts grown as monolayers or aggregates (eg, embryoid bodies) and treated with 5 μM programin for 4 days. )), Sox2 (FIG. 8C), c-Myc (FIG. 8D) and KLF4 (FIG. 8E) showing the results of qPCR. It is a figure which shows the programin processing aggregate (for example, embryoid body-like body) differentiated into the osteogenic (bone) cell. FIG. 9A shows an aggregate before differentiation. FIG. 9 (B) shows the cells after treatment with 10 μM Wnt agonist. Osteogenic nodules were visible after 2 days of exposure to Wnt agonists. FIG. 9 (C) shows Alizen Rad S staining of nodules. FIG. 9 (D) shows that cell death occurred in programin-treated cells that did not mechanically form aggregates. It is a figure which shows the programin process aggregate (for example, embryoid body-like body) differentiated into the nerve cell. FIG. 10A shows an aggregate before differentiation. FIG. 10 (B) shows cells treated by supplementation with B27. FIG. 10 (C) and FIG. 10 (D) show neurite growth from differentiated cells.

I. Introduction The present invention is surprising that, in part, by exposing cell aggregates derived from somatic cells (eg, embryoid bodies) to small molecule compounds, the pluripotency induction efficiency in such cells is greatly improved. Based on discovery. Accordingly, the present invention provides compounds, compositions, and methods for differentiating (eg, reprogramming) lineage committed mammalian cells into pluripotent stem cells. More specifically, the present invention provides compounds of formulas I-IX (eg, programin and derivatives thereof) that are useful for differentiation of differentiation lineage determining cells or induction of pluripotent stem cells from differentiation lineage determining cells. To do. Furthermore, the methods described herein include forming cell aggregates (eg, embryoid bodies) by mechanical means. Induced pluripotent stem cells produced by this method can differentiate into differentiation lineage-determining cells.

  The present invention includes the use of programin and its derivatives to make pluripotent stem cells from differentiated cells. The use of programin alone cannot activate the expression of pluripotency genes Oct4, Sox2, Nanog, and E-cadherin in somatic cells. Cells, such as human and mouse fibroblasts, must first mechanically aggregate into embryoid body-like cell bodies for about 4 days before they can respond to the reprogramming activity of programin or its derivatives. . The cell aggregation process alone causes fibroblasts to express basal levels of Oct4, Sox2, and Nanog. In addition, aggregation allows the fibroblasts to react with programin or its derivatives. Exposure of embryoid body-like cell bodies to 2-5 μM programin for 30 hours increased Oct4, Sox2, and Nanog expression by 25-200 fold compared to untreated control fibroblasts.

II. General Methods The practice of the present invention uses conventional techniques in the field of molecular biology. Basic texts disclosing general methods useful in the present invention include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990) And Current Protocols in Molecular Biology (Ausubel et al., Eds., 1994)).

  Nucleic acid sizes are indicated in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, sequenced nucleic acids, or public DNA sequences. Protein size is expressed in kilodaltons (kDa) or number of amino acid residues. Protein size is an estimate from gel electrophoresis, sequenced protein, derived amino acid sequence, or published protein sequence.

  Non-commercial oligonucleotides are described in, for example, Beaucage and Caruthers, Tetrahedron Lett. Using an automated synthesizer as described in Van Devanter et.al., Nucleic Acids Res. 12: 6159-6168 (1984). 22: 1859-1862 (1981) and can be synthesized chemically according to the solid phase phosphoramidite triester method first reported. Oligonucleotide purification can be accomplished by any art-known strategy such as native acrylamide gel electrophoresis or anion as described in Pearson and Reanier, J. Chrom. 255: 137-149 (1983). Performed using exchange high performance liquid chromatography (HPLC).

III. Definitions In this disclosure, the term “or” or “or” is generally used in its sense including “and / or” unless the content clearly dictates otherwise.

  The terms “Oct4” or “Oct-4” refer to transcription factors belonging to the POU family. Oct4 is a marker for undifferentiated cells (Okamoto et al., Cell 60: 461-72, 1990). Oct4 has also been reported to be involved in maintaining pluripotency (Nichols et al., Cell 95: 379-91, 1998). Human Oct4 polypeptide sequences are shown, for example, in Genbank accession numbers NP_001167002, NP_001272915, NP_001272916, NP_002692, and NP_0976034. Human Oct4 mRNA (coding) sequences are shown, for example, in Genbank accession numbers NM_001173531 NM_001285986, NM_001285987, NM_002701, and NM_203289. Those skilled in the art will appreciate that Oct4 variants, isoforms, and alternative sequences are also useful in the present invention.

  The terms “Sox2” or “Sox-2” refer to members of the SRY-related HMG box (SOX) family of transcription factors involved in embryonic development control and cell fate determination. The human Sox2 polypeptide sequence is shown, for example, in Genbank accession number NP_003097. The human Sox2 mRNA (coding) sequence is shown, for example, in Genbank accession number NM_003106. Those skilled in the art will appreciate that variants, isoforms and alternative sequences of Sox2 are also useful in the present invention.

  The term “Nanog” refers to a transcriptional regulator involved in the growth and self-renewal of embryonic stem cells and inner cell masses. The human Nanog polypeptide sequence is shown, for example, in Genbank accession number NP_0079141. The human Nanog mRNA (coding) sequence is shown, for example, in Genbank accession number NM_024485. Those skilled in the art will appreciate that Nanog variants, isoforms, and alternative sequences are also useful in the present invention.

  The term “pluripotent” or “pluripotency” collectively refers to characteristics associated with cell lines derived from all three germ layers (endoderm, mesoderm, and ectoderm) A cell that has the ability to generate progeny cells that can differentiate under appropriate conditions into a cell type. Pluripotent stem cells can contribute to all embryonic tissues of prenatal, postnatal, or adult animals. Although standard art accepted tests such as the ability to form teratomas in 8-12 week old SCID mice can be used to demonstrate cell population pluripotency, various pluripotency Pluripotent cells can also be detected by identifying the characteristics of stem cells.

  “Characteristics of pluripotent stem cells” refers to the characteristics of cells that distinguish pluripotent stem cells from other cells. The ability to generate progeny cells that can differentiate under appropriate conditions into cell types that collectively display characteristics associated with cell lines derived from all three germ layers (endoderm, mesoderm, and ectoderm) are pluripotent stem cells It is a characteristic. Expression of specific combinations of molecular markers is also a characteristic of pluripotent stem cells. For example, human pluripotent stem cells express at least some of the following non-limiting list of markers, in some embodiments, all: SSEA-3, SSEA-4, TRA-1-60, TRA- 1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rexl, and Nanog. Cell morphology associated with pluripotent stem cells is also a characteristic of pluripotent stem cells.

  The term “non-pluripotent cell” refers to a mammalian cell that is not a pluripotent cell. Examples of such cells include differentiated cells and progenitor cells. Examples of differentiated cells include, but are not limited to, cells derived from tissue selected from bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood. Examples of cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and lymphocytes.

  The term “lineage committed cell” refers to any cell that exhibits or differentiates into a particular cell type or related cell type. Examples of differentiated lineage determining cells include osteoblasts, myoblasts, chondrocytes, nerve cells, and adipocytes.

  As used herein, the terms “dedifferentiate”, “dedifferentiation”, or “reprogramming” mean that a differentiation line determining cell (eg, myoblast or osteoblast) reverses its lineage determination and progenitor Refers to a process that can give rise to progeny of new cell types such as somatic cells, progenitor cells, multipotent stem cells, or pluripotent stem cells. For example, after sufficient growth, a measurable proportion of progeny can exhibit the phenotypic characteristics of a new cell type, which is measurable above that before dedifferentiation or reprogramming. Under certain conditions, the proportion of progeny with new cell type characteristics can be at least about 1%, 5%, 25%, or more, in order of increasing preference. Dedifferentiated cells can be identified by the loss of the pattern of gene expression and cell surface protein expression associated with the lineage-determining cells.

  The term “vessel” refers to containers, dishes, plates, flasks, bottles, cell culture tubes, and the like that can be used for culturing, maintaining, or growing cells.

  The term “mechanical agitation” refers to applying mechanical force to a subject, such as a population of cells or cell aggregates.

  The term “cell aggregate” refers to a mass of at least three cells that are in contact with each other and do not form a single layer of cells, eg, a monolayer.

  As used herein, the term “culture” refers to maintaining a cell under certain conditions that allow the cell to proliferate, differentiate, and avoid senescence. The cells can be cultured in a growth medium containing appropriate growth factors.

  As used herein, “promote” or “increase / increase” or “promoting” or “increase / increase” are interchangeable herein. Used for. These terms refer to measured parameters (eg, activity, expression, glycolysis, glycolysis metabolism, glucose uptake, glycolysis in treated cells (tissue or subject) compared to untreated cells (tissue or subject). Refers to an increase in downstream biosynthesis. The comparison may be performed on the same cell, tissue, or subject before and after treatment. The rise is fully detectable. In some embodiments, the increase in treated cells is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1% compared to untreated cells. Double, double, triple, quadruple or more.

  As used herein, “inhibit”, “prevent”, or “reduce” or “inhibit”, “preventing”, or “reduce” (Reducing) "is used interchangeably herein. These terms refer to a decrease in measured parameters (eg, activity, expression, mitochondrial respiration, mitochondrial oxidation, oxidative phosphorylation) in treated cells (tissue or subject) compared to untreated cells (tissue or subject). The comparison may be performed on the same cell, tissue, or subject before and after treatment. The decrease is fully detectable. In some embodiments, the reduction in treated cells is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or complete compared to untreated cells. It is inhibition. In some embodiments, the parameter being measured is undetectable (ie, completely inhibited) in the treated cells as compared to untreated cells.

  The term “pharmaceutically acceptable salt” refers to a salt of an active compound prepared using a relatively non-toxic acid or base, depending on the particular substituents found on the compounds described herein. Point to. Where a compound of the present invention contains a relatively acidic functional group, the base is obtained by contacting the neutral form of such compound with a sufficient amount of the desired base neat or in a suitable inert solvent. Addition salts can be obtained. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. Where the compounds of the invention contain relatively basic functional groups, acid addition salts by contacting the neutral form of such compounds with a sufficient amount of the desired acid neat or in a suitable inert solvent. Can be obtained. Examples of pharmaceutically acceptable acid addition salts include hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrocarbonic acid, phosphoric acid, monohydrophosphoric acid, dihydrogenphosphoric acid ( derived from inorganic acids such as sulfuric acid, monohydrogen sulfate, hydroiodic acid, or phosphorous acid, and acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid And salts derived from relatively non-toxic organic acids such as fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid and methanesulfonic acid. Further, salts of amino acids such as alginate and organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge, SM, et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into base or acid addition salts.

  The neutral forms of the compounds may be regenerated 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 solvents, but for other purposes the salt is the Equivalent to the parent form.

  In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

IV. Programin and its derivatives The present invention provides compounds derived from compounds known as reversin, programin, or 2- (4-morpholinoanilino) -6-cyclohexylamino-purine. In some embodiments, the compound is represented by Formula (I).

  In another embodiment, the compound is represented by formula (II).

  In another embodiment, the compound is represented by formula (III).

  In yet another embodiment, the compound is represented by formula (IV).

  In another aspect, the compound is represented by formula (V).

  In another embodiment, the compound is represented by formula (VI).

  In some embodiments, the compound is represented by Formula (VII).

  In another embodiment, the compound is represented by formula (VIII).

  In another embodiment, the compound is represented by formula (IX).

  The compound can include all pharmaceutically acceptable salts, isomers, solvates, hydrates, and prodrugs thereof.

  One skilled in the art will appreciate that 2- (4-morpholinoanilino) -6-cyclohexylamino-purine (programin or reversin) and its derivatives can be prepared by either solid phase synthesis or liquid phase synthesis. Details of how to synthesize such compounds can be found, for example, in Ding et al., J. Am. Chem. Soc., 124: 1594 (2002), the entire disclosure of which is incorporated herein by reference for all purposes. U.S. Pat. Nos. 7,176,312; 7,273,864; and 7,592,177.

  Briefly, in the liquid phase synthesis of programin, cyclohexylamine and diisopropylethylamine can be added to a solution of 2-fluoro-6-chloropurine in n-butanol. The mixture can be heated to 80 ° C. with vigorous stirring for 12 hours. The solvent can then be removed under reduced pressure and the crude can be used directly in the next step reaction without further purification. Crude 2-fluoro-6-cyclohexylamino-purine can be dissolved in ethanol and then 4-morpholinoaniline can be added. The mixture can be heated to 75 ° C. for 24 hours with vigorous stirring in a sealed tube. The solvent can then be removed under reduced pressure and the crude material can be purified directly by flash chromatography to give 2- (4-morpholinoanilino) -6-cyclohexylamino-purine as a pale white solid. It will be apparent to one skilled in the art that derivatives of 2- (4-morpholinoanilino) -6-cyclohexylamino-purine, such as compounds having the structure of Formula II-IX, can be prepared by liquid phase synthesis or solid phase synthesis. .

V. Reprogramming to induce pluripotency in non-pluripotent cells Several different methods and protocols have been established to induce non-pluripotent mammalian cells into induced pluripotent stem cells. iPSCs are similar to ESCs in terms of morphology, proliferation, and pluripotency, and are judged by teratoma formation and chimera contribution. Reprogramming protocols include Oct polypeptide (but not limited to, for example, Oct3 / 4), Sox polypeptide (but not limited to, for example, Sox2), Klf polypeptide (but not limited to Includes the introduction of one or more reprogramming transcription factors selected from, for example, Klf4), and / or Myc polypeptides (including but not limited to c-Myc).

  Studies have shown that IPSCs are generated by retroviral transduction of mouse fibroblasts using four transcription factors (Oct-3 / 4, Sox2, KLF4, and c-Myc) that are highly expressed in ESCs. It is shown. Takahashi, K. & Yamanaka, S. Cell 126, 663-676 (2006); Okita, K., Ichisaka, T. & Yamanaka, S. Nature 448, 313-317 (2007); Wernig, M. et al. Nature 448, 318-324 (2007); Maherali, N. et al. Cell Stem Cell 1, 55-70 (2007); Meissner, A., Wernig, M. & Jaenisch, R. Nature Biotechnol. 25, 1177- 1181 (2007); Takahashi, K. et al. Cell 131, 861-872 (2007); Yu, J. et al. Science 318, 1917-1920 (2007); Nakagawa, M. et al. Nature Biotechnol. 26 , 101-106 (2007); Wernig, M., Meissner, A., Cassady, JP & Jaenisch, R. Cell Stem Cell. 2, 10-12 (2008). In addition, studies have shown reprogramming of human somatic cells by transcription factors that are highly expressed in ESCs: Hockemeyer et al. Cell Stem Cell. 11; 3 (3): 346-53 (2008); Lowry et al. Proc Natl Acad Sci US A. 105 (8): 2883-8 (2008); Park et al. Nature. 10; 451 (7175): 141-6 (2008); Nakagawa et al. Nat Biotechnol. Jan; 26 (1): 101-6 (2008); Takahashi et al. Cell. 131 (5): 861-72 (2007); and Yu et al. Science. 318 (5858): 1917-20 (2007) .

For example, Shi, Y., et al (2008) Cell-Stem Cell 2: 525-528, Huangfu, D., et al (2008) Nature Biotechnology 26 (7): 795-797, Marson, A., et al ( 2008) As shown by Cell-Stem Cell 3: 132-135, the addition of various small molecules can increase the efficiency of reprogramming (eg, the number of reprogrammed cells). It is contemplated that the methods described herein can be used in combination with additional single small molecules (or combinations of small molecules) that increase the efficiency of production of reprogrammed cells. In some embodiments, some non-limiting examples of agents that increase reprogramming efficiency include, among others, soluble Wnt, Wnt conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), and trichostatin (TSA). Further, as used herein, inhibitors of the TGF-β signaling pathway (eg, Repsox, E-616451, or SB431542, and anti-TGFβ antibody or RNAi agent), inhibitors of SRC signaling, eg, EI-275, or MEK / ERK cell signaling agonists (eg, prostaglandin 2); Ca ²⁺ / calmodulin signaling inhibitor or EGF receptor tyrosine kinase inhibitor; Na ²⁺ channel or ATP-dependent potassium channel inhibitor or MAPK signal Agonists of the transduction pathway alone or in combination with another small molecule (or combination of small molecules) enhance or increase the production efficiency of reprogrammed cells from differentiated cells as disclosed herein And can be used for Conceivable. Additional agents include PDK1 activators, compounds that promote glycolytic metabolism, and / or Rho GTPase / ROCK inhibitors.

  It is believed that the small molecules described herein can be used in combination with essentially any method for making iPSCs, thereby improving the efficiency of the method.

A. Non-pluripotent cell acquisition and culture Non-pluripotent mammalian cells that can be used to generate iPSCs according to the methods described herein can be any suitable mammal (eg, rodent, eg, mouse). , Rats, guinea pigs, and rabbits; differentiated cells derived from non-rodent mammals such as dogs, cats, pigs, sheep, horses, cows and goats; primates such as chimpanzees and humans) .

  A differentiated cell is any cell that forms the body of an organism, in contrast to germline cells (eg, gametes including sperm and eggs). All other cell types in the mammalian body are differentiated cells (apart from sperm and eggs, germ cells and undifferentiated stem cells).

  A cell can be, for example, in culture, tissue, fluid, etc. and / or derived from or in an organism. In some embodiments, the differentiated cell is a primary cell line or is a descendant of a primary or second generation cell line. Cells that can be induced pluripotently include, but are not limited to, fibroblasts, blood cells such as T cells, B cells, monocytes, bone marrow progenitor cells, fibroblasts, tumor cells, bone marrow Cells, gastric cells, liver cells, epithelial cells, nasal mucosal epithelial cells, mucosal epithelial cells, follicular cells, connective tissue cells, muscle cells, bone cells, chondrocytes, gastrointestinal cells, splenocytes, kidney cells, lung cells, testis cells , And neural tissue cells. In some embodiments, the human cell is a fibroblast, which can be conveniently obtained from a subject by punch biopsy. In another embodiment, the human cells are obtained from a sample, such as a hair follicle, a blood sample, a swab sample (eg, an oral swab sample) and the like. When reprogrammed cells (eg induced pluripotent stem cells) are used for therapeutic treatment of disease, it is desirable to use differentiated cells (eg somatic cells) isolated from the patient.

Methods for isolating and culturing human and mammalian cells are well known in the art, for example, Humason, ANIMAL TISSUE TECHNIQUES, 4 ^th ed., WH Freeman and Company (1979); Freshney et al., CULTURE OF ANIMAL CELLS (3rd ed. 1994); and Ricciardelli et al., (1989) In Vitro Cell Dev. Biol. 25: 1016.

Suitable cell culture methods and conditions can be determined by those skilled in the art using known methods (see, for example, Freshney et al., 1994, supra). In general, the cell culture environment includes consideration of factors such as substrate for cell growth, cell density, and cell contact, gas phase, media, and temperature. Cell incubation is usually performed under conditions known to be optimal for cell growth. Such conditions may include, for example, a humid atmosphere containing a temperature of about 37 ° C. and about 5% CO ₂ . The incubation period can vary widely depending on the desired result. In general, the incubation is preferably continued until the cells have a suitable expression. Proliferation is preferably measured by ³ H thymidine incorporation or BrdU labeling. Plastic dishes, flasks, or roller bottles can be used to culture cells according to the method of the present invention. Suitable culture vessels include, for example, multiwell plates, petri dishes, tissue culture tubes, flasks, roller bottles and the like.

  A defined cell culture medium is available as a packaged, pre-mixed powder or pre-sterilized solution. Examples of commonly used media include MEM-α DME, RPMI 1640, DMEM, Iscove complete media, or McCoy media (eg, Gibco BRL / Life Technologies catalog and Reference guide; see Sigma catalog). Typically, MEM-α or DMEM is used in the method of the present invention. Limited cell culture media is often supplemented with 5-20% serum, typically inactivated serum such as human, horse, calf, and fetal bovine serum. Typically, 10% fetal calf serum is used in the methods of the present invention. The culture medium is usually buffered so that the cells are preferably maintained at a pH of about 7.2 to about 7.4. Other supplements to the medium include, for example, antibiotics, amino acids, sugars, and growth factors.

B. Formation of cell aggregates To induce pluripotency, somatic cells are cultured in suspension to form embryoid bodies. In some embodiments, somatic cells are suspended at a density of about 1 × 10 ⁵ to about 1 × 10 ⁶ cells / cm ² . In another embodiment, the cells are from about 30,000 to about 500,000 cells / ml. The cells can be cultured in a non-adherent culture vessel such as a non-tissue culture treatment vessel or a low cell binding vessel. The cells can be grown in any vessel that prevents monolayer formation by the cells.

  To form aggregates or embryoid bodies, somatic or non-pluripotent cells are cultured in suspension under conditions that allow the cells to form a loosely packed cell mass. In some cases, the cell suspension is mechanically manipulated, eg, mixed, agitated, shaken, spun, rubbed, and pipetted so that the cells aggregate to form separate three-dimensional aggregates. . Cells can be cultured in containers, such as roller bottles or agitation bottles, and can be agitated periodically or continuously to promote aggregate formation.

  Mechanical forces can be applied to the somatic cells to prevent or prevent the cells from adhering to the culture vessel surface. The magnitude of the mechanical force should not change cell integrity or induce cell death. However, the force should be sufficient to detach the detached cells and aggregates from the surface of the non-adhesive culture vessel.

  Aggregates can be formed and cultured for about 2 to about 5 days, for example, about 2 days, about 3 days, about 4 days, or about 5 days after initiation of aggregation and prior to small molecule treatment.

C. Cultivation of aggregates in programin compounds To induce pluripotency, aggregates can be treated (eg, exposed or cultured therein) with a derivative of programin (compound of formula I). In some embodiments, from about 0 to about 10 μM, such as about 0 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM The derivatives are treated with aggregates to induce pluripotent stem cells. In some embodiments, a formula of about 0 to about 10 μM, such as about 0 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM. Aggregates are treated with a small molecule compound of II to induce pluripotency. In some embodiments, a formula of about 0 to about 10 μM, such as about 0 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM. Aggregates are treated with III small molecule compounds to induce pluripotency. In some embodiments, a formula of about 0 to about 10 μM, such as about 0 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM. Aggregates are treated with IV small molecule compounds to induce pluripotency. In some embodiments, a formula of about 0 to about 10 μM, such as about 0 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM. Aggregates are treated with small molecule compounds of V to induce pluripotency. In some embodiments, a formula of about 0 to about 10 μM, such as about 0 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM. Aggregates are treated with small molecule compounds of VI to induce pluripotency. In some embodiments, a formula of about 0 to about 10 μM, such as about 0 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM. Aggregates are treated with small molecule compounds of VII to induce pluripotency. In some embodiments, a formula of about 0 to about 10 μM, such as about 0 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM. Aggregates are treated with small molecule compounds of VIII to induce pluripotency. In some embodiments, a formula of about 0 to about 10 μM, such as about 0 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM. IX small molecule compounds are used to treat aggregates to induce pluripotency.

  In some embodiments, the compounds of Formulas II-IX are aggregated into the aggregate for at least 8 hours, such as about 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours. , Or more. Aggregates are at least 1 day, such as 1 day, 2 days, 3 days, 4 days, 5 days 6, 7 days, 8 days, 9 days, any one of compounds of formulas I-IX, 10 Can be processed for days or more.

  The aggregate may be treated with any one or a combination of one or more compounds of formulas I-IX. For example, two compounds of Formulas I-IX, such as Formulas I and II, Formulas I and III, Formulas I and IV, Formulas I and V, Formulas I and VI, Formulas I and VII, Formulas II and III, Formulas II and Compounds of formula IV and formulas I and V, formulas II and VI, formulas II and VII, formulas II and VIII, formulas II and IX, etc. can be used. In some embodiments, at least 2, for example 2, 3, 4, 5, 6, 7, 8, or 9 different compounds of Formulas I-IX are useful for inducing pluripotency in aggregates. is there.

  Reprogramming efficiency is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% compared to the conventional method It can be increased by 300%, 400%, 500%, or more. In some cases, the reprogramming efficiency is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40% or 50% (Eg, the percentage of induced pluripotent cells compared to the total number of starting somatic cells).

  In culturing induced pluripotent stem cells produced in the present invention, primate pluripotent stem cells, more specifically embryonic stem cells, are cultured as described in US Pat. No. 7,442,548. Various media and techniques developed in the past can be used. It will be appreciated that additional methods for culturing and maintaining human pluripotent stem cells can be used with the present invention, as is known to those skilled in the art. In certain embodiments, undefined conditions may be used, for example, pluripotent cells are exposed on or to fibroblast feeder cells to maintain stem cells in an undifferentiated state. It can be cultured on a medium. Alternatively, pluripotent cells are essentially produced using a feeder-independent culture system such as TeSR medium (Ludwig et al., 2006a; Ludwig et al., 2006b). Can be cultured and maintained in an undifferentiated state. Pluripotent cells can be cultured and maintained using culture systems and media that do not involve feeders. As described herein, various modifications can be made to these methods to reduce costs as desired.

  For example, like human embryonic stem cells, iPS cells are 80% DMEM (Gibco, # 10829-018 or # 11965-092), 20% limited fetal bovine serum (FBS) (or human AB serum), 1% non-essential It can be maintained in amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Alternatively, iPS cells may contain 80% Knock-Out DMEM (Gibco, # 10829-018), 20% serum replacement (Gibco, # 10828-028), 1% non-essential amino acids, 1 mM L-glutamine, and 0 Can be maintained in serum-free medium composed of 1 mM β-mercaptoethanol. Human bFGF may be added at a final concentration of about 4 ng / mL, or zebrafish bFGF may be used instead.

D. Methods for characterizing pluripotent stem cells Chemically induced reprogrammed cells as disclosed herein are embryonic stem cells or other multipotents in terms of morphology, proliferation, gene expression, and teratoma formation. Indistinguishable from competent cells. For example, chemically induced human reprogrammed cells are indistinguishable from human embryonic stem cells in terms of morphology and proliferation and can proliferate. Furthermore, these chemically induced reprogrammed cells can differentiate into three germ layer cell types in vitro and in teratomas.

Analysis can be performed to assess the pluripotency characteristics of reprogrammed cells. Cells can be analyzed for various growth characteristics and embryonic stem cell-like morphology. Pluripotent stem cells derived according to the methods described herein include SSEA-1, SSEA-3, and SSEA-4 (Developmental Studies Hybridoma Bank, National Institute of Child Health and Human Development, Bethesda Md.), And It has characteristic antigens that can be identified or confirmed by immunohistochemistry or flow cytometry using antibodies against TRA-1-60 and TRA-1-81 (Andrews et al., 1987). The pluripotency of stem cells can also be confirmed by injecting about 0.5-10 × 10 ⁶ cells into the muscles of the hind legs of 8-12 week old male SCID mice. The appearance of teratomas indicates that the stem cells differentiate into at least one cell type for each of the three germ layers.

  The expression profiles of individual genes associated with pluripotency can also be examined. In addition, the expression of embryonic stem cell surface markers can be analyzed. Detection and analysis of various genes or gene products known in the art to be associated with pluripotent stem cells include, but are not limited to, Oct4, Nanog, Sox2, Klf4, c-Myc, ABCG2, E-cadherin, β-tubulin III, α-smooth muscle actin (α-SMA), fibroblast growth factor 4 (FGF4), Cripto, Daxl, zinc finger protein 296 (Zfp296) , N-acetyltransferase-1 (Nat1), ES cell-related transcript 1 (ECAT1), ESG1 / DPPA5 / ECAT2, ECAT3, ECAT6, ECAT7, ECAT8, ECAT9, ECAT10, ECAT15-1, ECAT15-2, Fthll7, Sall4 , Differentiated germ cell transcription factor (Utf1), Rex1, p53, G3PDH, telomerase such as TERT, silent X chromosome gene, Dnmt3a, Dnmt3b, TRIM28, F box-containing protein 15 (Fbx15), Esrrb, TDGF1, GABRB3, Zfp42, FoxD3 , GDF3, CYP25A1, developmental pluripotency-associated 2 (DPPA2), T cell lymphoma breakpoint 1 (Tcl1), DPPA3 / Stella, DPPA4, and other pluripotent markers Analysis of any gene or gene product may be included.

  Cells can also be characterized by a decrease in markers characteristic of differentiated cells from which pluripotent cells are derived. Non-limiting examples of endoderm cell markers include Gata4, FoxA2, PDX1, Noda1, Sox7, and Sox17, and mesoderm cell markers include Brachycury, GSC, LEF1, Mox1, and Tie1 Examples of markers of ectoderm cells include Cripto1, EN1, GFAP, Islet 1, LIM1, and nestin. Antibodies against the three germ layer markers are commercially available from, for example, Abcam, EMD Millipore, Santa Cruz Biotechnology, and the like.

  In some embodiments, the expression level (eg, amount) of Oct4, Nanog, and Sox2 in the reprogrammed cell is at least greater than the expression level (amount) of the same gene or gene product in the differentiated cell from which the reprogrammed cell is derived. About at least about 2 times, at least about 4 times, at least about 6 times, at least about 8 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 40 times, at least about 80 times, at least about 100 times At least about 150 times, at least about 200 times, at least about 500 times, or more.

E. Methods for enriching and / or isolating reprogrammed cells Another aspect of the present invention is the reprogramming cell population from a heterogeneous cell population, such as a mixed cell population comprising reprogrammed cells and differentiated cells from which the reprogrammed cells are derived. About separation. A population of reprogrammed cells produced by any of the methods described herein can be used with any cell surface marker present on reprogrammed cells that is not present on differentiated cells from which the reprogrammed cells are derived. It can be concentrated, grown, isolated, and / or purified. Such cell surface markers are also referred to as affinity tags specific for reprogrammed cells. Examples of affinity tags specific to reprogrammed cells include markers such as polypeptides that are present on the cell surface of reprogrammed cells but are not substantially present on other cell types (eg, on differentiated cells). Examples include molecules specific antibodies, ligands, or other binding agents. In some methods, an antibody that binds to a cell surface antigen on a reprogrammed cell (eg, a human reprogrammed cell) is enriched for chemically induced reprogrammed cells produced by the methods described herein. Used as an affinity tag for isolation or purification. In some embodiments, reprogrammed cells can also be isolated from differentiated cells by fluorescence activated cell sorting (FACS), affinity-based methods, and combinations thereof.

  Chemically induced reprogrammed cells may be isolated by other techniques for cell isolation. Furthermore, reprogrammed cells can be enriched or isolated by continuous subculture methods in growth conditions that promote selective survival or selective proliferation of the reprogrammed cells. Such methods are known to those skilled in the art.

  In some embodiments, the cell culture or cell population comprising the reprogrammed cells is at least about 1-100%, such as at least about at least about 1%, at least about 2%, at least about 3%, at least about 4%. At least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 90%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13% At least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35% At least about 40%, at least about 45%, at least about 50%, at least about 55%, low At least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, At least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of pluripotent stem cells.

F. Differentiation Methods for Reprogrammed Cells Another way to assess the pluripotency of induced stem cells is to differentiate the cells into specific somatic cell types in vitro. For example, specific growth factors known to drive differentiation into specific cell types can be used.

  In some embodiments, stem cells generated according to the methods of the invention are then treated with, for example, hematopoietic (stem / progenitor) cells, neural (stem / progenitor) cells (and optionally further differentiated cells such as subtype specific Neurons, oligodendrocytes, etc.), pancreatic cells (eg, endocrine precursor cells or pancreatic hormone-expressing cells), hepatocytes, cardiovascular (stem / progenitor) cells (eg, cardiomyocytes, endothelial cells, smooth muscle cells), retina Induced to form cells and the like. Various methods are known for inducing differentiation of pluripotent stem cells into a desired cell type. Non-limiting examples of methods for inducing stem cell differentiation to various cell fates include, for example, US Pat. Nos. 8,187,878; 7,781,214; 7,757,762. No. 7,541,186; Kaiming Ye and Sha Jin, eds., Human Embryonic and Induce Pluripotent Stem Cells: Lineage-specific Differentiation Protocols, New York: Humana Press, 2011; and Schlaeger et al., Current Protocols in Stem Cell Biology, New York: Wiley, 2014.

G. Pharmaceutical Formulations When used in pharmaceutical applications, the small molecule compounds of the present invention are typically formulated in a suitable buffer, such as phosphate buffered saline or sodium phosphate / sodium sulfate, Tris buffer. Can be any pharmaceutically acceptable buffer, such as glycine buffer, sterile water, and other buffers known to those skilled in the art such as those described in Good et al. Biochemistry 5: 467 (1966).

  The composition may further comprise a stabilizer, enhancer, or other pharmaceutically acceptable carrier or vehicle. Pharmaceutically acceptable salts or carriers can include physiologically acceptable compounds that act, for example, to stabilize small molecule compounds. Physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients. obtain. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preserving agents that are particularly useful for preventing microbial growth or action. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).

VI. Examples The following examples are provided to illustrate the claimed invention and are not intended to be limiting.

Example 1: Protocol for activating pluripotent gene expression in mouse somatic cells We made from fibroblasts carrying the transgene Oct4 promoter (Oct4-EGFP) linked to an EGFP reporter Small molecules were identified by high-throughput screening of embryoid bodies. This synthetic heterocyclic molecule, which we named “programin” (eg, reversin), was able to induce 25-200-fold stronger expression of Oct4, Sox2, and Nanog in mouse somatic cells. . Furthermore, the expression of E-cadherin (a cell adhesion gene important for maintaining pluripotency of embryonic cells) was induced and increased by a factor of 5 compared to the first fibroblast. Reprogramming efficiency is about 20% and takes 7 days to achieve. When fibroblasts were grown as a monolayer by a conventional method on a normal adhesive culture dish, the expression of Oct4, Sox2 and Nanog was not induced (activated).

  These body formation processes themselves activate low basal levels of Oct4, Sox2, and Nanog expression (2 times higher than growing cultured dish-adherent fibroblasts). This mechanically induced biological activity was confirmed by qPCR analysis. Treatment of cell aggregates with programin further increased the expression of these three pluripotency genes by 20-200 fold compared to, for example, controls. These results were reproduced many more times and were also reproduced using human fibroblasts. In summary, programin or its derivatives alone cannot activate Oct4, Sox2, and Nanog expression unless they are mechanically induced so that somatic cells aggregate to prevent adherence to culture dishes.

  Programin-induced pluripotent (PIP) stem cells can form bone cells when induced by an osteogenesis-inducing medium and can form nerve cells when treated with a nerve-inducing supplement. This novel approach for generating pluripotent stem cells is simple, rapid, efficient and cost effective. Thus, PIP stem cells provide a valuable and versatile cell source for use in making organs and tissue grafts for use in regenerative medicine. Furthermore, human PIP stem cells can induce differentiation into specific cell types such as liver, heart, and nerve cells for drug toxicity and bioactivity studies by the pharmaceutical industry.

  This example illustrates the use of novel compounds that allow reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) that express pluripotent stem cell markers.

  Required Materials: Culture medium supplemented with DMEM (Invitrogen) containing 10% FBS, 1% PS; DMEM / F12 containing 1 × B27 (Invitrogen), 1,000 U / mL mouse leukemia inhibitory factor (LIF) Mouse ESC medium supplemented with culture medium (Invitrogen); programin (frozen stock dissolved in DMSO); working concentration = 1-5 μM; sterile 35 mm non-adhesive culture dish (SPL; 11035).

Procedure Day 1 of PIP stem cell production:

  1. Mouse tail fibroblasts (passage 2-6 and growth log phase) are procured. Fibroblasts are trypsinized using a standard protocol to obtain a cell suspension in culture medium.

2. Fibroblasts or other somatic cells are seeded onto sterile 35 mm non-adherent culture dishes at a density of 1 × 10 ⁵ to 1 × 10 ⁶ cells / cm ² in 3 mL culture medium.

3. Incubate the cells in a cell incubator at 37 ° C. and 5% CO ₂ .

  4). Gently pipet the culture medium to prevent cells from adhering to the culture dish surface. Must be done twice a day for 2 minutes each. Standard 1 mL pipettes and plastic tips were used for pipetting cultures. Pipetting needs to be done very slowly (approx. 0.5-1 mL / sec produces enough force to detach the cells from the non-adherent culture dish surface).

  Day 2 of PIP stem cell production.

  1. Cells begin to aggregate into embryoid bodies after the first day. During the culture period, the number and size of these bodies increase.

  2. Gently pipet the culture medium to prevent embryoid body-like cell bodies from attaching to the surface of the culture dish.

  3. Add 1 mL of fresh culture medium.

  Day 4 of PIP stem cell production.

  1. Collect cell suspension and pellet cells by centrifugation at 800 rpm for 5 minutes. Gentle centrifugation and pipetting does not disaggregate embryoid bodies. Cells are washed once with mouse ESC medium and programin (1-5 μM) or programin derivative.

  2. Embryoid bodies are pelleted again and they are resuspended in 3 mL mouse ESC medium and programin or derivatives thereof.

3. Embryoid somatic cell bodies are seeded in sterile 35 mm non-adherent culture dishes. Embryoid bodies are incubated at 37 ° C., 5% CO ₂ for 2-3 days in a cell incubator.

  4. Perform pipetting twice a day daily to prevent embryoid body-like cell bodies from sticking to the culture dish.

  5.5 Add 1 mL of fresh mouse ESC medium on day 5 (day 2 after programin treatment).

  Days 6-7 of PIP stem cell production.

  1. Embryoid somatic cell bodies are harvested for PI and Hoechst staining for confocal analysis. The presence of green fluorescence and Hoechst staining in the cells indicates the presence of live PIP cells expressing Oct4-EGFP.

  2. To verify the expression of pluripotency genes, ie, Oct4, Sox2, and Nanog, as well as the expression of other major stem cell-related genes, embryoid somatic cell bodies are collected for qPCR analysis.

Results A schematic diagram of an example method of the present invention is shown in FIG. Tail fibroblasts extracted from the tail of Oct4-EGFP transgenic mice and stained with CD34 (FIG. 2). Only cells expressing the surface marker CD34 were sorted by flow cytometry. FIG. 2 (B) shows CD34 ⁺ fibroblasts grown for 3 days on adherent culture dishes. CD34 ⁺ fibroblasts grown on adherent culture dishes and treated with 5 μM programin for 3 days died and did not express Oct4 (FIGS. 2C and 2D).

CD34 ⁺ fibroblasts recovered from the tail of Oct4-EGFP transgenic mice are mechanically pipetted and maintained on non-adherent culture plates so that the cells aggregate to form embryoid bodies. (FIG. 3A). The embryoid bodies in this experiment were stained with PI dye to determine the presence of dead cells (red arrow) and stained with Hoechst dye to determine the presence of live cells (blue arrow) (FIG. 3A ) To (D)). Under confocal microscopy, Oct4-EGFP expression (green arrow) was undetectable in living cells of untreated embryoid bodies (FIG. 3 (A)). Embryoid body-like cell bodies treated with 5 μM programin for 24 hours (FIG. 3 (B)), 48 hours (FIG. 3 (C)), and 69 hours (FIG. 3 (D)) expressed Oct4-EGFP. . Programin was able to activate Oct4-EGFP expression in CD34 ⁺ fibroblasts 24-69 hours after treatment (green arrow).

The qPCR results indicate that CD34 ⁺ mouse fibroblasts do not express Oct4, Sox2, and Nanog when cultured as a monolayer (FIG. 4). In contrast, when fibroblasts are mechanically engineered to form embryoid somatic cell bodies, they induce pluripotent genes such as Oct4, Sox2, and Nanog to be expressed at low basal levels. Is done. Treatment of these embryoid somatic cell bodies with 5 μM programin significantly increases Oct4, Sox2, and Nanog expression.

The qPCR results show the effectiveness of various programin derivatives that enhance Oct4, Sox2 and Nanog expression (FIG. 5). Derivatives 237, 400, 594, 648, 668, 957, 958, and 959 significantly increased Oct4, Sox2, and Nanog expression in embryoid-like cell bodies of CD34 ⁺ mouse fibroblasts. The level of pluripotent stem cell markers is compared to CD34 ⁺ mouse fibroblasts and untreated embryoid body-like cell bodies cultured as monolayers in embryoid body-like cell bodies treated with programin or its derivatives it was high.

Example 2: Protocol for activating pluripotency gene expression in human umbilical cord perivascular mesenchymal progenitor cells In this example, human progenitor cells to iPSCs expressing pluripotent stem cell markers, such as human umbilical cord blood vessels The use of novel compounds that promote reprogramming of surrounding mesenchymal progenitor cells is described.

  Necessary materials: programin or derivatives thereof (frozen stock dissolved in DMSO), Essential 6 (Invitrogen) supplemented with 100 ng / mL basic fibroblast growth factor (bFGF) (Invitrogen), 15% ESQ-FBS ( HUCPV medium consisting of Invitrogen) and MEM / F12 (Invitrogen) containing 1% PS; working concentration = 1-5 μM; sterile non-adherent 24-well culture plate (SPL; 32024).

Procedure 1. Primary human umbilical cord perivascular (HUCPV) cells were maintained in HUCPV culture medium and passaged by trypsinization. Only HUCPV cells below the sixth cell passage were used.

  2. HUCPV cultures are trypsinized and dissociated into single cells.

3. Dissociated HUCPV cells are resuspended at 3 × 10 ⁵ cells in 600 μL of HUCPV medium and seeded into each well of a non-adherent 24-well culture plate.

4). Cells are incubated in a cell incubator at 37 ° C. and 5% CO ₂ .

  5. The next day, until the end of the experiment, HUCPV cells were aggregated into embryoid body-like cell bodies by pipetting the cells twice a day so that the cells did not adhere on the culture plate.

  6. Every 2 days, 200 μL of fresh HUCPV medium was added to the culture.

  After culturing for 7.4 days, the embryoid body-like cell body was centrifuged at 800 rpm for 5 minutes to remove the culture medium. Centrifugation and pipetting should be gentle so that the embryoid bodies do not disaggregate.

  8). The embryoid body-like cell bodies are washed once with HUCPV medium supplemented with Essential 6 and 1-5 μM programin or derivatives thereof. Remove the medium by centrifugation.

  9. Resuspend embryoid bodies in 600 μL HUCPV medium supplemented with Essential 6 and 1-5 μM programin or derivatives thereof in each well of a sterile non-adherent 24-well culture plate.

  10. Thirty hours after programin treatment, programin-treated embryoid body-like cell bodies were collected for qPCR analysis to verify the expression of pluripotency genes Oct4, Sox2, and Nanog. Other major stem cell related genes were also analyzed.

Results Human umbilical cord perivascular mesenchymal progenitor (HUCPV) cells were adhered to a culture dish and then treated with 0-5 μM programin for 30 hours. qPCR analysis showed that programin did not activate Oct4, Sox2, KLF4, c-Myc, and Nanog expression in cells cultured as monolayers.

  HUCPV cells aggregated into embryoid bodies showed higher Oct4, Sox2 and Nanog expression compared to cells cultured as monolayers (FIG. 6). However, Klf4 and c-Myc expression was not activated.

  Embryoid bodies formed from HUCPV cells were treated with 1-5 μM programin for 30 hours. Embryoid bodies exposed to programin at all concentrations tested showed a significant increase in Oct4, Sox2, KLF4, c-Myc, and Nanog expression compared to untreated embryoid body controls (0 μM). (FIGS. 6 and 7). 4 μM programin induced the highest levels of Oct4, Sox2, Klf4, and Nanog compared to other concentrations.

  The data show that HUCPV cells respond to programin by activating Oct4, Sox2, Klf4, c-Myc, and Nanog expression in the cells when physically aggregated into embryoid bodies. Show. Embryoid bodies initially formed from HUCPV cells differentiate into pluripotent stem cells when grown on non-adhesive culture dishes and prevented from sticking to the dish surface.

Example 3: Protocol for activating pluripotent gene expression in human dermal fibroblasts This example describes a method for inducing the formation of iPSCs from human dermal fibroblasts. The method includes causing human fibroblasts to form cell aggregates and then exposing the aggregates to the compound programin or a derivative thereof, such as programins 2-9.

  Necessary materials: Fibroblast medium supplemented with Eagle MEM medium (Invitrogen) containing 15% FBS, 1% non-essential amino acid, 1% PS, 20% knockout serum replacement (Invitrogen), 2 mM L-glutamine, 0.1 mM non-essential amino acid, 0.1 mM mercaptoethanol, 10 ng / mL fibroblast growth factor-basic (bFGF), DMEM / F12 (Invitrogen) containing 1% PS, programin or a derivative thereof (dissolved in DMSO) Human ESC medium supplemented with (frozen stock); working concentration = 5 μM, and sterile 35 mm non-adherent culture dish (SPL; 11035).

Procedure 1. Primary human dermal fibroblasts were cultured in fibroblast medium and passaged by trypsinization. Passage 5 fibroblasts were used in the experiments.

2. After dissociating the culture into single cells using trypsin, 2 × 10 ⁶ cells were resuspended in 3 mL fibroblast medium in a non-adherent 35 mm dish and 5% CO ₂ in a 37 ° C. incubator. Maintained.

  3. After several hours, aggregates formed. Gently break overly large aggregates by pipetting.

  4). The next day compact aggregates were formed. Again, gently break the aggregate to smaller sizes (100-120 μm diameter) by pipetting.

  5. Three days after cell aggregate / embryoid body-like cell body formation, the medium is converted to human ESC medium supplemented with 5 μM programin or a derivative thereof.

  6.2 After two days, the medium is supplemented with fresh programin or a derivative thereof.

  7). Cells are harvested after 4 days of treatment with programin or its derivatives. Total RNA is extracted using Trizol reagent and purified using a column.

  8). Reverse transcription and qPCR are performed on major stem cell associated genes.

result:
After 4-day compound treatment, stem cell-related genes such as Oct4, Nanog, Sox2, c-Myc, and KLF4 are treated with programin in an embryoid body that has been treated with medium. Is expressed at higher levels than human fibroblast monolayers treated with, and human skin fibroblast monolayers treated with vehicle (FIGS. 8 (A)-(E)). Thus, programin activates fibroblasts cultured in aggregates to strongly express pluripotency-related genes including Oct4, Sox2, Nanog, KLF4, and c-Myc, but cultured in monolayers The activated fibroblasts are not activated.

Example 4: Method for testing the feasibility of embryoid bodies produced according to the above examples In this example, iPSCs produced according to the methods described in Examples 1 to 3 were used as osteogenic or neuronal cells. A method of differentiating into is described.

Bone formation (bone) induction method Programin-treated embryoid body-like cell bodies (1 × 10 ⁶ cells / 10 cm ² ) were placed in DMEM / F12, 1000 U / mL LIF, and 1 × B27 on non-adhesive culture dishes. Maintained as a suspension culture for 4 days. Thereafter, the embryoid body-like cell body was transferred to a culture dish coated with 0.1% gelatin (FIG. 9A). 10 μM Wnt agonist (catalog number 681665, Calbiochem) was added to the culture medium (DMEM / F12, 1000 U / mL LIF and 1 × B27). Bone-like nodules were observed in the cultures after 2-3 days (FIG. 9B). The differentiated cells were confirmed to be bone cells by Alizen Red staining of the nodules (FIG. 9C). The programin-treated cells that did not aggregate died after exposure to Wnt agonist (FIG. 9 (D)).

Method for Inducing Neurons Programin-treated embryoid body-like cell bodies (1 × 10 ⁶ cells / 10 cm ² ) were used as suspension cultures in DMEM / F12, 1000 U / mL LIF, and 1 × B27 on non-adhesive culture dishes. Maintained for 4 days. Thereafter, the embryoid body-like cell body was transferred to a culture dish coated with 0.1% gelatin (FIG. 10 (A)). Since B27 in the culture medium can induce neurogenesis, the nerve-inducing small molecule was not added to the medium (FIG. 10B). After 3 days of culture, neurons were seen on 0.1% gelatin-coated culture plates, which were more apparent after 7 days (FIGS. 10 (C) and (D)).

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Claims (24)

A method of producing pluripotent stem cells from differentiated cells,
(A) culturing the differentiated cells in a container;
(B) mechanically stirring the differentiated cells to form cell aggregates;
(C) exposing the cell aggregate to an effective amount of a compound having any one of the structures represented by the following formulas II to IX;
(D) detecting the expression level of Oct4, Sox2 or Nanog in the cells of the aggregate;
(E) determining that the cell is a pluripotent stem cell if the expression level of Oct4, Sox2, or Nanog in the cell of the aggregate is higher than the corresponding expression level in the differentiated cell; and (f) Isolating said pluripotent stem cells:

.   2. The method of claim 1, wherein the differentiated cells of step (a) are obtained from a subject or cell line.   The method of claim 2, wherein the subject is a mammalian subject.   The method of claim 2, wherein the cell line is a mammalian cell line.   The method of claim 1, wherein the differentiated cell is a fibroblast.   The method of claim 1, wherein the differentiated cell is a progenitor cell. The method of claim 1, wherein the differentiated cells of step (a) have a density of about 1 × 10 ⁵ to 1 × 10 ⁶ cells / cm ² .   The method of claim 1, wherein the cell aggregate does not adhere to the inner surface of the container.   The method of claim 8, wherein the inner surface of the container is coated with a non-adhesive substrate.   The method of claim 1, wherein mechanical agitation comprises rubbing, agitating, or shaking the differentiated cells.   2. The method of claim 1, wherein step (d) comprises an amplification based assay, a hybridization based assay, an immunoassay, or a flow cytometry assay.   2. The method of claim 1, wherein the higher expression level of Oct4, Sox2, or Nanog is at least about 20 to about 200 times higher than the differentiated cell.   The method of claim 1, further comprising expanding the pluripotent stem cells after step (e).   The method of claim 1, further comprising inducing the pluripotent stem cell to differentiate after step (e).   A pluripotent cell or cell line obtained by the method according to any one of claims 1 to 14. A method for producing a cell aggregate comprising cells expressing at least one pluripotent stem cell marker from differentiated cells, comprising:
(A) culturing the differentiated cells in a vessel at a density of about 1 × 10 ⁵ to about 1 × 10 ⁶ cells / ml;
(B) mechanically agitating the differentiated cells to form the cell aggregates; and (c) culturing the cell aggregates under conditions that produce cells expressing at least one pluripotent stem cell marker. Method. The method according to claim 16, further comprising the step of (d) exposing the cell aggregate to an effective amount of a compound having any one of the structures represented by the following formulas II to IX:

.   17. The method of claim 16, wherein the at least one pluripotent stem cell marker is Oct4, Sox2, or Nanog.   17. The method of claim 16, further comprising expanding cells expressing the at least one pluripotent stem cell marker after step (c).   17. The method of claim 16, further comprising isolating cells that express the at least one pluripotent stem cell marker after step (c). A compound having any one of the structures represented by the following formulas II to IX:

.   A pharmaceutical composition for producing pluripotent stem cells from differentiated cells, comprising the compound of claim 21 and a pharmaceutically acceptable salt. A mixture comprising differentiated cells and a compound having any one of the structures represented by the following formulas II to IX:

.   24. A mixture according to claim 23, wherein the differentiated cells are cell aggregates.