Methods of generating rock hyrax ipsc

Abstract

The disclosure is directed to methods of generating induced pluripotent Procavia capensis stem cells that combine transfection and chemical reprogramming. Specifically, the disclosure provides methods of generating induced pluripotent P. capensis stem cells that include generating primed P. capensis cells by chemically reprogramming primary P. capensis cells transfected with OCTA. 80X2. KI.F4. CMYC. and GLIS1 by culturing the cells, generating pre-induced P. capensis cells from the primed P. capensis cells by transfecting the primed P. capensis cells with OCT4. 80X2. KLF4, CMYC, and NANOG and culturing these cells, and generating the induced pluripotent P. capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P. capensis cells with OCT4, SOX2, KLF4. CMYC, NANOG, IJN28A. and SV40 and culturing the transfected cells. In other embodiments, the primary P. capensis cells are cultured to generate pre-induced P. capensis cells, which are then transfected with at least OCT4ISOX2IKLF4ICMYC. and SV40 T-antigen or an shRNA targeting TP53 and cultured again. The culturing uses a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and a TGF-β inhibitor. The disclosure is also directed to cell culture media useful in such methods and induced pluripotent P. capensis stem cells expressing at least OCT4, 80X2, KLF4, CMYC, NANOG, LIN28A, and SV40.

Description

METHODS OF GENERATING ROCK HYRAX IPSCCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 678,194 (filed on August 1, 2024) and U.S. Provisional Application No. 63 / 746,035 (filed on January 16. 2024), both of which is incorporated by reference in their entireties.FIELD OF THE INVENTION

[0002] This disclosure relates to methods for generating induced pluripotent rock hyrax (Procavia capensis) stem cells from primary rock hyrax cells (such as e.g., fibroblasts), including methods of generating the cells, media, and protocols used for the generation, as well as differentiation of these cells.BACKGROUND OF THE INVENTION

[0003] The rock hyrax (Procavia capensis) is a medium-sized mammal native to Africa and the Middle East. Rock hyraxes can reach a length of up to 50 cm and a weight of about 4-5 kg. Rock hyraxes, elephants, and mammoth are descended from a common ancestor, the tethytheria, that died out some 50 million years ago. As such, rock hyraxes are amongst the closest living genetic relatives of elephants.

[0004] Due to the size and associated ease of housing, rock hyrax provides an ideal model for studying genetic changes to elephant cells. In particular, rock hyrax stem cells can be used as model for elephant stem cells. However, given the limited number of naturally occurring stem cells, what is needed is a protocol to generate induced pluripotent stem cells from rock hyrax. Like elephants, it has been difficult to generate induced pluripotent rock hyrax stem cells.

[0005] This disclosure provides protocols that overcome the difficulties associated with generating induced pluripotent stem cells from primary rock hyrax cells.SUMMARY OF THE INVENTION

[0006] The disclosure provides methods of generating induced pluripotent Procavia capensis stem cells that express at least OCT4. SOX 2. KLF4, CMY C, NANOG. I.IN28A. and SV40, culture media for use in such methods, and induced pluripotent P. capensis stem cells that express at least OCT4, SOX2, KLF4, CMYC, NANOG, LIN28A, and SV40.

[0007] The methods of the disclosure combine transfection of pluripotency genes, such as OCT4. SOX2. KI 4 '4. CMYC, NANOG, LIN28A. and SV40, with the use of a chemicalreprogramming media containing an HD AC inhibitor (such as e.g., valproic acid (VPA)), a GSK-3 inhibitor (such as e.g, CHIR-99021), a monoamine oxidase inhibitor (such as e.g, tranylcypromine (2-PCPA) HC1), an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g., forskolin), a retinoid (such as e.g., CH55), a DOT1L inhibitor (such as e.g., EPZ004777), and a TGF-0 inhibitor (such as e.g., RepSox).

[0008] One embodiment of the disclosure is a method of generating induced pluripotent Procavia capensis stem cells including:(a) culturing primary P. capensis cells transfected with OCT4, SOX2, KLF4, CMYC, and GLIS1, and optionally an antibiotic resistance gene in a culture medium supplemented with an HD AC inhibitor (such as e.g., valproic acid (VPA)), a GSK-3 inhibitor (such as e.g., CHIR-99021), a monoamine oxidase inhibitor (such as e.g.. tranylcypromine (2-PCPA) HC1), an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g, forskolin), a retinoid (such as e.g., CH55), a DOT1L inhibitor (such as e.g., EPZ004777), and a TGF-0 inhibitor (such as e.g., RepSox) to generate primed P. capensis cells;(b) generating pre-induced P. capensis cells from the primed P. capensis cells by transfecting the primed P. capensis cells with OCT4, SOX2. KLF4, CMY C. and NANOG and culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor (such as e.g., valproic acid (VPA)), a GSK-3 inhibitor (such as e.g., CHIR-99021), a monoamine oxidase inhibitor (such as e.g., tranylcypromine (2-PCPA) HC1). an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g., forskolin). a retinoid (such as e.g., CH55), a DOT IL inhibitor (such as e.g., EPZ004777), and a TGF-f> inhibitor (such as e.g., RepSox); and(c) generating the induced pluripotent P. capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P. capensis cells with OCT4, SOX2, KLF4, CMYC, NANOG, LIN28A, and SV40 and culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor (such as e.g., valproic acid (VPA)), a GSK-3 inhibitor (such as e.g., CHIR-99021), a monoamine oxidase inhibitor (such as e.g., tranylcypromine (2-PCPA) HC1), an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g., forskolin), a retinoid (such as e.g., CH55). a DOT1L inhibitor (such as e.g., EPZ004777), and a TGF- inhibitor (such as e.g., RepSox).

[0009] In certain embodiments, the method also includes transfecting the primary' P. capensis cells with OCT4, SOX2, KLF4, CMYC, and GLIS1 and optionally an antibiotic resistance gene. The primary’ P. capensis cells, primed P. capensis cells, and pre-induced P. capensis cells may be transfected using a variety7of different vectors. In one embodiment,the primary P. capensis cells are transfected with a single stranded RNA replicon comprising OCT4. SOX2, KLF4. CMYC. GLISI. and optionally an antibiotic resistance gene. In another embodiment, the primed P. capensis cells are transfected with a plasmid comprising OCT4, SOX2, KLF4, CMYC, and NANOG. In an alternate embodiment, the preinduced P. capensis cells are transfected with a plasmid comprising OCT4, SOX2, KLF4, CMYC, NANOG, LIN28A, and SV40.

[0010] In certain embodiments, the method includes culturing primary P. capensis cells transfected with OCT4, SOX2, KLF4, CMYC, and GLISI, and an antibiotic resistance gene. In further embodiments, the method includes transfecting primary' P. capensis cells with OCT4, SOX2, KLF4, CMYC, and GLISI, and an antibiotic resistance gene.

[0011] Various media are suitable for use in the methods of the disclosure. In certain embodiments, the culture medium in (a), (b), and / or (c) is supplemented with from about 0.1 to about 1 mM of an HD AC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, from about 2 to about 10 pM of a DOT1L inhibitor, and from about 0.5 to about 4 pM of a TGF-P inhibitor.

[0012] In one embodiment, the generating pre-induced P. capensis cells from the primed P. capensis cells includes: (1) transfecting the primed P. capensis cells with OCT4, SOX2, KLF4. CMYC, and NANOG,' (2) culturing the transfected primed P. capensis cells in a culture medium supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and a ROCK inhibitor; and (3) culturing the transfected primed P. capensis cells in a culture medium devoid of a ROCK inhibitor supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and an agent to induce transcription of transfected genes. In other embodiments, the method includes culturing the transfected primed P. capensis cells in a culture medium supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and a ROCK inhibitor on a feeder layer.

[0013] In another embodiment, the generating the induced pluripotent Procavia capensis stem cells includes: (1) transfecting the pre-induced P. capensis cells with OCT4, SOX2, KLF4, CMYC, and NANOG: (2) culturing the transfected pre-induced P. capensiscells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and a ROCK inhibitor; and (3) culturing the transfected pre-induced P. capensis cells in a culture medium devoid of a ROCK inhibitor supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, a D0T1L inhibitor, a TGF-P inhibitor, and an agent to induce transcription of transfected genes. In certain embodiments, the method includes culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, a D0T1L inhibitor, a TGF- P inhibitor, and a ROCK inhibitor on a feeder layer.

[0014] In certain embodiments, the method also includes selecting for the cells after isolation using for example an antibiotic for which antibiotic resistance was introduced in state one. In some embodiments, the antibiotic resistance gene is puromycin-resistance, and the antibiotic is puromycin.

[0015] In one embodiment, the agent to induce transcription of transfected genes is doxycycline. A variety7of ROCK inhibitors may be used. In one embodiment, the ROCK inhibitor is Y-27632.

[0016] In one embodiment, the HD AC inhibitor is valproic acid. In another embodiment, the GSK-3 inhibitor is CHIR-99021. In an alternate embodiment, the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HO. In yet another embodiment, the activator of eukaryotic adenylyl cyclase is forskolin. In additional embodiments, the retinoid is Ch 55. In an embodiment, the DOT1L inhibitor is EPZ004777. In an embodiment, TGF-P inhibitor is RepSox. In some embodiments, any combination of these compounds may be used. Thus, in certain embodiments, the HD AC inhibitor can be valproic acid, the GSK-3 inhibitor can be CHIR-99021, the monoamine oxidase inhibitor can be tranylcypromine (2-PCPA) HC1, the activator of eukary otic adenylyl cyclase can be forskolin, the retinoid can be Ch 55, the DOT1L inhibitor can be EPZ004777, and / or the TGF-P inhibitor can be RepSox.

[0017] In certain embodiments, the culture medium in (a), (b), and / or (c) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox. In one embodiment, the culture medium in (a) is supplemented with valproic acid, CHIR-99021. tranylcypromine, forskolin, Ch 55, EPZ004777. and RepSox. In another embodiment, the culture medium in (b) is supplemented with valproicacid, CHIR-99021, tranylcypromine, forskolin, Ch 55. EPZ004777, and RepSox. In a further embodiment, the culture medium in (c) is supplemented with valproic acid, CHIR- 99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

[0018] A variety of primary P. capensis cells may be used in the methods. In one embodiment, the primary P. capensis cells are P. capensis fibroblasts. In another embodiment, the P. capensis fibroblasts are female P. capensis fibroblasts.

[0019] The method can include further steps. For example, in certain embodiments, the method further includes selecting for the induced pluripotent P. capensis stem cells.

[0020] A further aspect of the disclosure is a method of generating induced pluripotent Procavia capensis stem cells including: (a) culturing primary P. capensis cells in a culture medium supplemented with an HD AC inhibitor (such as e.g, valproic acid (VP A)), a GSK- 3 inhibitor (such as e.g, CHIR-99021), a monoamine oxidase inhibitor (such as e.g., tranylcypromine (2-PCPA) HC1), an activator of eukar otic adenylyl cyclase (a cAMP agonist, such as e.g., forskolin), a retinoid (such as e.g., CH55), a DOT1L inhibitor (such as e.g., EPZ004777), and a TGF-(3 inhibitor (such as e.g, RepSox) to generate pre-induced P. capensis cells; and (b) generating the induced pluripotent P. capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P. capensis cells with at least OCT4ISOX2IKLF4ICMYC, and SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes and culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor (such as e.g.. valproic acid (VPA)), a GSK-3 inhibitor (such as e.g, CHIR-99021), a monoamine oxidase inhibitor (such as e.g, tranylcypromine (2-PCPA) HC1), an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g., forskolin), a retinoid (such as e.g., CH55), a DOT1L inhibitor (such as e.g, EPZ004777), and a TGF-(3 inhibitor (such as e.g, RepSox).

[0021] In certain embodiments, these methods further include transfecting the preinduced P. capensis cells with an antibiotic resistance gene. In some embodiments, the methods include transfecting the pre-induced P. capensis cells with OCT4ISOX2IKLF4ICMYCILIN28A and SV40 T-antigen or an shRNA targeting TP53 retrogenes. In other embodiments, the methods include the pre-induced P. capensis cells with OCT4ISOX2IKLF4ICMYCILIN28A and (ii) SV40 T-antigen or an shRNA targeting TP53 retrogenes. In some embodiments, the OCT4ISOX2IKLF4ICMYC is from Loxodonta africana. In other embodiments, the OCT4ISOX2IKLF4ICMYCILIN28A is from Loxodonta africana. In further embodiments, the OCT4 / SOX2 / KLF4 CMYC / LIN28A / NANOG is from Loxodonta africana.

[0022] In certain embodiments of the methods, the cells are transfected with an SV40 T- antigen or an shRNA targeting TP53 retrogenes in Elephas maximus. In other embodiments, the pre-induced P. capensis cells are transfected with a single stranded RNA replicon containing OCT4ISOX2IKLF4ICMYC, and optionally an antibiotic resistance gene. In some embodiments, the primed P. capensis cells are transfected with a plasmid encoding OCT4ISOX2IKLF4ICMYC.

[0023] In other embodiments of the methods, the culture medium in (a) and / or (b) is supplemented with from about 0.1 to about 1 mM of an HDAC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 LIM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, from about 2 to about 10 pM of a DOT1L inhibitor, and from about 0.5 to about 4 pM of a TGF-[3 inhibitor.

[0024] In one embodiment, the HDAC inhibitor is valproic acid. In another embodiment, the GSK-3 inhibitor is CHIR-99021. In a further embodiment, the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1. In an alternate embodiment, the activator of eukaryotic adenylyl cyclase is forskolin. In a further embodiment, the retinoid is Ch 55. In yet another embodiment, the DOT1L inhibitor is EPZ004777. In an additional embodiment, the TGF-(3 inhibitor is RepSox.

[0025] In some embodiments of the methods, the culture medium in (a) and / or (b), is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin. Ch 55, EPZ004777, and RepSox. In an embodiment, the culture medium in (a) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox. In another embodiment, the culture medium in (b) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55. EPZ004777. and RepSox.

[0026] The methods may also include selecting for the induced pluripotent P. capensis stem cells. In certain embodiments, the selecting for induced pluripotent P. capensis cells includes treatment with doxycycline and antibiotic selection and wherein the transfected chemically reprogrammed P. capensis cells are resistant to the antibiotic used for selection. In certain embodiments, the antibiotic selection includes treatment with hygromycin or puromycin. In other embodiments, the antibiotic selection includes treatment with hygromycin, wherein the cells are treated every two days with hygromycin, and wherein the treatment lasts ten days. In certain embodiments, the antibiotic selection includes daily treatment with puromycin for five days.

[0027] In certain embodiments of the methods, the primary P. capensis cells are P. capensis fibroblasts. In some embodiments, the P. capensis fibroblasts are female P. capensis fibroblasts. The disclosure also includes induced pluripotent P. capensis stem cell produced by these methods as well as methods of differentiating the induced pluripotent P. capensis stem cell into endoderm, mesoderm, or ectoderm. In addition, the disclosure includes forming embryoid bodies from these

[0028] Another aspect of the disclosure is directed to induced pluripotent P. capensis stem cells. In one embodiment, the induced pluripotent P. capensis stem cell is produced by the methods described above. Another embodiment is an induced pluripotent P. capensis stem cell expressing at least OCT4, S X 2. K K4. CMYC. NANOG, LIN28A, and SV40. A further embodiment is an induced pluripotent P. capensis stem cell expressing at least OCT4. 80X2. KI.P'4. CMYC, NANOG, LIN28A, and SV40, whereby the stem cell is generated by transfection with at least OCT4, 80X2, KLF4, CMYC, NANOG, LIN28A, and SV40 and chemical reprogramming.

[0029] Y et another aspect of the disclosure is directed to a method of differentiating the induced pluripotent P. capensis stem cells described herein into endoderm, mesoderm, or ectoderm. In other embodiments, the P. capensis iPSCs are differentiated into primordial germ cells. A further embodiment of the disclosure is a method of forming an embry oid body from the induced pluripotent P. capensis stem cell of the disclosure.

[0030] Other features and advantages of the invention will be apparent from the detailed description and examples that follow.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The foregoing summary’, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figures demonstrate embodiments of the present invention. It should be understood, however, that the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0032] FIG. 1 shows a schematic diagram of a protocol for generating induced pluripotent rock hyrax (Procavia capensis) stem cells according to one embodiment of the disclosure.

[0033] FIG. 2 shows an image of a representative example of female rock hyrax fibroblasts (23Pc001-FFb). The image was taken on a Leica Flexicam with a 4x objective.

[0034] FIG. 3 shows an image of stem-like colony resulting from transfection 1 of female rock hyrax fibroblasts (23Pc001-FFb). The image was taken on a Leica Flexicam with a 4x objective.

[0035] FIG. 4 shows an image of stem-like colonies exhibiting heterogeneous morphologies, resulting from transfection stage 2 of female rock hyrax fibroblasts (23Pc001-FFb). The image was taken on a Leica Flexicam with a 4x objective.

[0036] FIG. 5 shows an image of a colony resulting from stage 3 (OCTA. SOX2, KLF4. CMYC, NANOG. and LIN28A as well as SV40); female rock hyrax fibroblasts (23Pc001- FFb). The image was taken on a Leica Flexicam with a lOx objective.

[0037] FIG. 6 shows an image of a representative colony of Procavia capensis iPSC line Single Factor after completing the transition out of the Chemical B Cocktail, followed by passaging in E8 Stem Flex media. The image was taken on a Leica Flexicam with a lOx objective.

[0038] FIG. 7 A is a chart showing core pluripotency gene expression (log fold change, with the wildtype as the reference) for P. capensis iPSCs (Pc iPSC) that have been subject to both Stages 2 and Stages 3.

[0039] FIG. 7B is a chart showing naive markers upregulated in Pc iPSCs.

[0040] FIG. 8 show s an image of representative chromosome staining for the P. capensis iPSC line coOSKMNL.

[0041] FIG. 9A-9J show characteristics of Procavia capensis (Rock hyrax) induced pluripotent stem cells produced by chemically inducing primary P. capensis cells to generate pre-iPSCs and then transfecting those cells and chemically reprogramming the generate the P. capensis iPSCs. FIG. 9A-9C show- phase contrast images of Procavia capensis fibroblasts (FIG. 9 A). P. capensis pre-iPSCs (pcPRCs) (FIG. 9B), and / t capensis iPSCs (pciPSCs) (FIG. 9C). FIG. 9D shows the results of a karyotype analysis of pciPSCs having fifty -four chromosomes. FIG. 9E shows immunofluorescent imaging for OCT4 and SOX2 in pciPSCs. FIG. 9F and FIG. 9G show' MA plot of RNA-seq data illustrating the transcriptional differences between P. capensis fibroblast cells (pcECs) and induced pluripotent stem cells (pciPSCs) for core pluripotency markers (FIG. 9F) as well as additional primed and naive pluripotency markers (FIG. 9G). All scale bars = 200pM. FIG. 9H show s bionano karyotyping of a clonal pciPSC line (Pla6T) demonstrating a normal karyotype. FIG. 91 shows pathways enriched for in pciPSCs are pathways enriched in embryonic cells. FIG. 9J shows the doubling time of Pla6T line compared to first generated human iPSCs

[0042] FIG. 9K shows RT-qPCR data for markers of primordial germ cells (PGCs) following differentiation of pciPSCs into putative PGG-like cells (PGCLCs).DETAILED DESCRIPTION

[0043] This disclosure is based on the discovery that it is possible to generate induced pluripotent Procavia capensis stem cells. As such, the disclosure is directed to methods of generating induced pluripotent Procavia capensis stem cells, culture media for use in such methods, and induced pluripotent P. capensis stem cells.

[0044] In one aspect, the disclosure is directed to methods of generating induced pluripotent Procavia capensis stem cells that express at least OCT4, 80X2, KLF4, CMYC, NANOG. I.IN28A. and SV40, culture media for use in such methods, and induced pluripotent P. capensis stem cells that express at least OCT4, SOX 2. KLF4, CMYC, NANOG, LIN28A, and SV40. The methods of the disclosure generate the induced pluripotent Procavia capensis stem cells that express at least OCT4. 0X2, KLF4, CMYC, NANOG, LIN28A, and SV40 using a combination of transfection and chemical reprogramming. The methods of this aspect of the disclosure involve three different stages: Stage 1 : Generation of primed P. capensis cells from primary P. capensis cells;Stage 2: Generation of pre-induced P. capensis cells from the primed P. capensis cells; and Stage 3 : Generation of pluripotent Procavia capensis stem cells from the pre-induced P. capensis cells.Each stage of the method combines use of transfection and a culture medium that chemically reprograms or at least partially chemically reprograms the cells. In Stage 1, the primary P. capensis cells are transfected with OCT4, SOX2, KLF4, CMYC, and GLIS1 and optionally an antibiotic resistance marker that is used to isolate the cells. In certain embodiments, the antibiotic resistance marker can be replaced with other markers that can be used to isolate the cells. In Stage 2, the primed P. capensis cells are transfected with OCT4, SOX2. KLF4, CMYC, and NANOG. In Stage 3, the pre-induced P. capensis cells are transfected with OCT4. SOX2, KLF4, CMYC, NANOG, LIN28A, and SV40.

[0045] In certain embodiments, the disclosure provides methods of generating induced pluripotent P. capensis stem cells that include:• generating primed P. capensis cells by culturing primary P. capensis cells transfected with OCT4. SOX2, KLF4, CMYC, and GLIS1 (and optionally an antibiotic resistance marker) in a culture medium supplemented with an HD AC inhibitor (such as e.g, valproic acid (VP A)), a GSK-3 inhibitor (such as e.g., CHIR-99021), a monoamineoxidase inhibitor (such as e.g., tranylcypromine (2-PCPA) HC1), an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g, forskolin). a retinoid (such as e.g., CH55), a DOT1L inhibitor (such as e.g., EPZ004777), and a TGF-[3 inhibitor (such as e.g., RepSox);• generating pre-induced P. capensis cells from the primed P. capensis cells by transfecting the primed P. capensis cells with OCT4, SOX2, KLF4, CMYC, wtA NANOG and culturing these cells in a culture medium supplemented with an HD AC inhibitor (such as e.g., valproic acid (VP A)), a GSK-3 inhibitor (such as e.g., CHIR-99021), a monoamine oxidase inhibitor (such as e.g., tranylcypromine (2-PCPA) HC1), an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g., forskolin), a retinoid (such as e.g.. CH55), a DOT1L inhibitor (such as e.g., EPZ004777), and a TGF- P inhibitor (such as e.g., RepSox); and• generating the induced pluripotent Procavia capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P. capensis cells with OCT4, SOX2, KLF4, CMYC, NANOG, L1N28A, and SV40 and culturing the transfected cells in a culture medium supplemented with an HD AC inhibitor (such as e.g., valproic acid (VP A)), a GSK-3 inhibitor (such as e.g., CHIR-99021), a monoamine oxidase inhibitor (such as e.g., tranylcypromine (2-PCPA) HC1), an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g, forskolin), a retinoid (such as e.g, CH55), a DOT1L inhibitor (such as e.g., EPZ004777), and a TGF- inhibitor (such as e.g., RepSox).In each stage of the protocol, the culture media chemically reprogram or at least partially chemically reprogram the cells. The antibiotic resistance marker is used for selection of the cells and as such is optional. In certain embodiments, the antibiotic resistance marker is included when generating primed P. capensis cells.

[0046] In another aspect, the disclosure is also directed to methods of generating induced pluripotent Procavia capensis stem cells that express at least at least OCT4, SOX2, KLF4, CMYC, (and optionally NANOG and / or LIN28A), and SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes, culture media for use in such methods, and induced pluripotent P. capensis stem cells that express at least OCT4, SOX2, KLF4, CMYC, and SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes. These methods of generating induced pluripotent Procavia capensis stem cells include: (a) culturing primary P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and a TGF-(3 inhibitor to generate pre-induced P. capensis cells; and (b) generating the induced pluripotent P. capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P. capensis cells with at least OCT4ISOX2IKLF4ICMYC, and SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes and culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOTIL inhibitor, and a TGF-P inhibitor.

[0047] The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.

[0048] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments, or applications of the present invention.I. Definitions

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods, and materials are now described.

[0050] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0051] It is noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Itis further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as an antecedent basis for use of such exclusive terminology as '‘solely,” “only” and the like in connection with the recitation of claim elements, or use of a '‘negative” limitation.

[0052] Each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0053] As used herein, the term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ± 20% or ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0054] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0055] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

[0056] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the appl i cabi 1 i ty of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0057] As used herein, the term “stem cell” refers to a cell that can self-renew and differentiate to at least one more differentiated or less developmentally capable phenotype. The term “stem cell” encompasses stem cell lines, induced stem cells, non-humanembryonic stem cells, pluripotent stem cells, multipotent stem cells, amniotic stem cells, placental stem cells, or adult stem cells.

[0058] An ‘'induced stem cell” is one derived from a non-pluripotent cell induced to a less differentiated or more developmentally capable phenotype by introduction of one or more reprogramming factors or genes. As the term is used herein, an induced stem cell need not be pluripotent, but has the capacity to differentiate, under appropriate conditions, to more than one more-highly-differentiated phenotype. It should be understood that the capacity was not present prior to the introduction of reprogramming factors. An induced stem cell will express at least one stem cell marker not expressed by the parent cell prior to introduction of reprogramming factors. In this context, a stem cell marker is exclusive of a factor introduced by reprogramming. An induced pluripotent stem cell. iPS cell, or iPSC, has the induced capacity to differentiate, under appropriate conditions, to a cell phenotype derived from each of the endoderm, mesoderm, and ectoderm germ layers.

[0059] As used herein, the term “somatic cell” refers to any cell other than a germ cell, a cell present in or obtained from a pre-implantation embryo, or a cell resulting from proliferation of such a cell in vitro. Stated another way. a somatic cell refers to any cells forming the body of an organism, excluding germ cells. Every cell type in the mammalian body-apart from the sperm and ova and the cells from which they are made (gametocytes) is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all substantially made up of somatic cells. In some embodiments the somatic cell is a “non-embryonic somatic cell,” by which is meant a somatic cell that is not present in or obtained from an embn o and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an “adult somatic cell,” by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro.

[0060] As used herein, the phrase “somatic rock hyrax cell” or “somatic P. capensis cell” refers to any cell from a rock hyrax (P. capensis) that is not a germ cell, a reproductive cell, or a stem / progenitor cell.

[0061] As used herein, the phrase “induced pluripotent rock hyrax stem cell” or ■‘induced pluripotent P. capensis stem cell” or '‘PC iPSC” or “pc iPSC” or “pciPSC” refers to any pluripotent stem cell that the been generated from a rock hyrax (P. capensis) somatic cell.

[0062] As used herein, the phrase “primary rock hyrax cell” or “primary P. capensis cell” refers to any rock hyrax (P. capensis) cell that is not pluripotent, including germ cellsand non-pluripotent cells. In certain embodiments, a primary P. capensis cell can be a somatic P. capensis cell.

[0063] As used herein, a “TP53 retrogene” is a copy of the TP53 gene that has been created through a process called retrotransposition, where a messenger RNA (mRNA) from the original TP53 gene is reverse-transcribed back into DNA and inserted into the genome. In certain embodiments, the TP53 retrogene may be truncated or contain other changes relative to wild type TP53.

[0064] As used herein, the terms “comprising,” “including,” “containing” and “characterized by” are exchangeable, inclusive, open-ended and do not exclude additional, unrecited elements or method steps. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements.

[0065] As used herein, the term “consisting of’ excludes any element, step, or ingredient not specified in the claim element.

[0066] Before certain embodiments are described in greater detail, it is to be understood that this invention is not limited to certain embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing certain embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0067] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0068] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0069] Before certain embodiments are described in greater detail, it is to be understood that this invention is not limited to certain embodiments described, as such may, of course, vary’. It is also to be understood that the terminology used herein is for the purpose ofdescribing certain embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0070] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments, or applications of the present invention.II. Culture media for seneratins PC iPSC

[0071] One aspect of the disclosure is directed to culture media that can be used in a protocol to generate iPSCs. In one embodiment, the culture media are used to generate PC iPSC from primary rock hyrax cells or somatic rock hyrax cells. In one embodiment, the culture media are used to generate PC iPSC from rock hyrax fibroblast. The culture media are uniquely formulated to facilitate chemical programming of P. capensis cells.

[0072] The culture media are basal culture media that have been supplemented with an HD AC inhibitor (such as e.g., valproic acid (VP A)), a GSK-3 inhibitor (such as e.g, CHIR- 99021). a monoamine oxidase inhibitor (such as e.g, tranylcypromine (2-PCPA) HC1). an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g, forskolin), a retinoid (such as e.g, CH55), a DOT1L inhibitor (such as e.g., EPZ004777), and a TGF-fi inhibitor (such as e.g., RepSox).

[0073] In some embodiments, the basal culture media are DMEM or Knockout-DMEM. The basal culture media may further be supplemented with one or more of bFGF. serum (e.g, FBS), KOSR, GlutaMAX™, NEAA, and 2-mercaptoethanol. In certain embodiments, GlutaMAX™ can be replaced with other solutions containing L-alanyl-L-glutamine dipeptide.

[0074] A variety of HD AC inhibitors may be used. In one embodiment, the HD AC inhibitor is valproic acid (2-propylpentanoic acid). Other suitable HD AC inhibitors include, but are not limited to, suberanilohydroxamic acid, trichostatin (A), cyclic tetrapeptides, benzamides, electrophilic ketones, sodium buty rate, and phenylbuty rate. In certain embodiments, the media contains from about 0. 1 to about 1 mM of the HD AC inhibitor.

[0075] In one embodiment, the GSK-3 inhibitor is CHIR-99021 (6-((2-((4-(2,4- Dichlorophenyl)-5-(4-methyl-lH-imidazol-2-yl)pyrimidin-2- yl)amino)ethyl)amino)nicotinonitrile). A variety of other GSK-3 inhibitors may be used in place of CHIR-99021. In certain embodiments, the media contain from about 10 to about 25 pM of the GSK-3 inhibitor.

[0076] A variety of monoamine oxidase inhibitor inhibitors may be used in the media. In one embodiment, monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1 (also known as tranylcypromine). In other embodiments, the monoamine oxidase inhibitor is selegiline. In yet another embodiment, the monoamine oxidase inhibitor is phenelzine. In certain embodiments, the cell culture medium is supplemented with from about 5 to about 25 pM of the monoamine oxidase inhibitor.

[0077] Similarly, a variety of activators of eukaryotic adenylyl cyclase may be used in the media. Suitable examples include, but are not limited to, forskolin ([(3R,4aR,5S,6S,6aS, 1 OS, 1 OaR, 10bS)-3-ethenyl-6, 10,10b-trihydroxy-3,4a,7,7, 10a- pentamethyl-l-oxo-5,6,6a,8,9,10-hexahydro-2H-benzo[f|chromen-5-yl] acetate) and 3- isobutyl-1 -methylxanthine. In one embodiment, the activator of eukaryotic adenylyl cyclase (cAMP agonist) in the culture media is forskolin. The culture media may be supplemented with from about 10 to about 30 pM of the activator of eukary otic adenylyl cyclase.

[0078] The culture media are also supplemented with a retinoid. In one embodiment, the retinoid is Ch 55 (4-[(lE)-3-[3.5-bis(l.l-Dimethylethyl)phenyl]-3-oxo-l- propenyl] benzoic acid). In other embodiments, the retinoid is all-trans-retinoic acid. Other examples of suitable retinoids include, but are not limited, to Re 80 (4-[l-hydroxy-3-oxo-3- (5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl- 2- naphthalenyl)-l-propenyl]benzoic acid). Am 580 (4-[(5.6.7.8-tetrahydro- 5.5.8.8-tetramethyl-2- naphthalenyl)carboxamido]benzoic acid) and Am 80 (4-[(5, 6,7, 8-tetrahydro-5, 5,8,8- tetramethyl-2-naphthalenyl)carbamoyl] benzoic acid. In certain embodiments, the culture media may be supplemented with from about 0.5 to about 3 pM of a retinoid.

[0079] In one embodiment, the DOT1L inhibitor is EPZ004777 (l-[3-[[(2R,3S.4R,5R)- 5-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxyoxolan-2-yl]methyl-propan-2- ylamino]propyl]-3-(4-tert-butylphenyl)urea). Other suitable DOT1L inhibitors include SGC0946 (l-(3-((((2R,3S,4R,5R)-5-(4-Amino-5-bromo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)- 3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert- butyl)phen l)urea). The culture media may be supplemented with from about 2 to about 10 pM of the DOT IL inhibitor.

[0080] One embodiment of a suitable TGF-fi inhibitor for use in the media is RepSox (2-(3-(6-Methylpyridin-2-yl)-lH-pyrazol-4-yl)-l,5-naphthyridine). Other exemplary TGF-|3 inhibitors include, but are not limited to, SB431542 (4-(4-(benzo[d][l,3]dioxol-5-yl)-5- (8yridine-2-yl)-lH-imidazol-2-yl)benzamide), LY2157299 (4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3-yl]quinoline-6-carboxamide), A83-01 3-(6- methylpyridin-2-yl)-N-phenyl-4-(quinoline-4-yl)-lH-pyrazole-l -carbothioamide), and tranilast. In some embodiments, the cell culture medium is supplemented with from about 0.5 to about 4 pM of the TGF-0 inhibitor.

[0081] In certain embodiments, the basal culture medium is supplemented with from about 0. 1 to about 1 mM of an HD AC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 0.5 to about 4 pM of a TGF-P inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, or from about 2 to about 10 pM of a DOT IL inhibitor. In other embodiments, the basal culture medium is supplemented with from about 0. 1 to about 1 mM of an HD AC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 0.5 to about 4 pM of a TGF-P inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, and from about 2 to about 10 pM of a DOT IL inhibitor.

[0082] In other embodiments, the basal culture medium is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin. Ch 55, EPZ004777, and RepSox. In certain embodiments, the culture medium is supplemented with one or more of 0. 1 to about 1 mM of valproic acid, from about 10 to about 25 pM of CHIR-99021, from about 0.5 to about 4 pM of RepSox. from about 5 to about 25 pM of tranylcypromine (2-PCPA) HC1, from about 10 to about 30 pM of forskolin, from about 0.5 to about 3 pM of Ch 55 or from about 2 to about 10 pM of EPZ004777. In other embodiments, the culture medium is supplemented with 0.1 to about 1 mM of valproic acid, from about 10 to about 25 pM of CHIR-99021. from about 0.5 to about 4 pM of RepSox, from about 5 to about 25 pM of tranylcypromine (2-PCPA) HCL from about 10 to about 30 pM of forskolin, from about 0.5 to about 3 pM of Ch 55, and from about 2 to about 10 pM of EPZ004777.III. Methods of seneratins PC iPSC - “Triple Transfection”

[0083] One aspect of the disclosure is directed to methods of generating the induced pluripotent Procavia capensis stem cells that express at least OCT4, SOX 2. KI.I- 4. CMYC , NANOG. LIN28A, and SV40 using a combination of transfection and chemical reprogramming.

[0084] The methods of this aspect of the disclosure rely on a stage-wise approach of generating the PC iPSC that involves: priming P. capensis primary cells (Stage 1);generating pre-induced P. capensis cells from the primed P. capensis cells (Stage 2); and generating the induced pluripotent Procavia capensis stem cells (Stage 3).

[0085] A variety of primary P. capensis cells may be used in the methods of the disclosure. In some embodiments, the primary P. capensis cells are somatic P. capensis cells. In some embodiments, the somatic P. capensis cells are endothelial cells, epithelial cells, or fibroblasts such as fetal fibroblasts, adult fibroblasts, and skin fibroblasts. In one embodiment, the primary P. capensis cells are fibroblasts, in particular fibroblast from a female P. capensis.

[0086] In certain embodiments, the methods of this aspect of the disclosure involve:• Stage 7: culturing primary P. capensis cells transfected with OCT4. SOX2. KLF4, CMYC. and GLIS1. and optionally an antibiotic resistance gene in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and a TGF-(3 inhibitor to generate primed P. capensis cells;• Stage 2: generating pre-induced P. capensis cells from the primed P. capensis cells by transfecting the primed P. capensis cells with OCT4. SOX2, KI.F4. CMY C, and NANOG and culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOTIL inhibitor, a TGF- inhibitor; and• Stage 3: generating the induced pluripotent P. capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P. capensis cells with OCT4, SOX2, KI.1' 4. CMYC, NANOG, LIN28A, and SV40 and culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and a TGF-P inhibitor.Thus, the methods of this aspect of the disclosure uniquely rely on a three-stage protocol that uses a culture medium supplemented with the same key chemical factors at each stage. The culture media chemically reprogram or at least partially chemically reprogram the cells.Culture Media for Stages 1-3

[0087] The methods of this aspect of the disclosure involve use of the same culture medium at Stages 1-3. The culture medium is supplemented with an HD AC inhibitor (such as e.g., valproic acid (VP A)), a GSK-3 inhibitor (such as e.g., CHIR-99021), a monoamineoxidase inhibitor (such as e.g., tranylcypromine (2-PCPA) HC1), an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g, forskolin), a retinoid (such as e.g., CH55), a D0T1L inhibitor (such as e.g., EPZ004777), and a TGF-P inhibitor (such as e.g., RepSox). Depending on the stage, in certain embodiments the culture medium is further supplemented with a ROCK inhibitor such as e.g. , Y-27362. In other embodiments, depending on the stage, the culture medium is further devoid of a ROCK inhibitor such as e.g., Y-27362 and supplement with an antibiotic, such as e.g.. puromycin. to which the cells have been induced to be resistant. The culture media are formulated to chemically reprogram the cells at Stages 1-3 of the methods.

[0088] In one embodiment, the culture medium is supplemented with from about 0.1 to about 1 mM, alternatively about 0.5 mM, alternatively at least 0.5 mM of an HD AC inhibitor. In another embodiment, the culture medium is supplemented with from about 10 to about 25 pM, alternatively about 15 pM. alternatively at least 15 pM of a GSK-3 inhibitor. In yet another embodiment, the culture medium is supplemented with from about 0.5 to about 4 pM, alternatively about 2 pM. alternatively at least 2 pM of a TGF- inhibitor. In further embodiments, from about 5 to about 25 pM, alternatively about 10 pM, alternatively at least 10 pM of a monoamine oxidase inhibitor. In further embodiments, the culture medium is supplemented with from about 10 to about 30 pM, alternatively about 20 pM, alternatively at least 20 pM of an activator of eukaryotic adenylyl cyclase. In yet further embodiments, the culture medium is supplemented with from about 0.5 to about 3 pM, alternatively about 1 pM, alternatively at least 1 pM of a retinoid. In additional embodiments, the culture medium is supplemented with from about 2 to about 10 pM, alternatively about 5 pM. alternatively at least 5 pM of a DOT1L inhibitor.

[0089] In another embodiment, the culture medium is supplemented with from about 0. 1 to about 1 mM of an HD AC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 0.5 to about 4 pM of a TGF-P inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, and from about 2 to about 10 pM of a DOT IL inhibitor.

[0090] In one embodiment, the HD AC inhibitor is valproic acid. In another embodiment, the GSK-3 inhibitor is CHIR-99021. In an alternate embodiment, the TGF- inhibitor is RepSox. In a further embodiment, the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1. In another embodiment, the activator of eukaryoticadenylyl cyclase is forskolin. In a further embodiment, the retinoid is Ch 55. In an alternate embodiment, the DOT1L inhibitor is EPZ004777.

[0091] Any of the HD AC inhibitors, the GSK-3 inhibitors, the TGF-0 inhibitors, the monoamine oxidase inhibitors, the activators of eukaryotic adenylyl cyclase, the retinoids, and the DOT IL inhibitors described in II. above can be used.

[0092] In one embodiment, the culture medium is also supplemented with one or more of GlutaMAX™ (L-alanyl-L-glutamine dipeptide), NEAA, 2-mercaptoethanol. KOSR. and FBS. In some embodiments, the culture medium is DMEM or Knockout DMEM. In further embodiments, the culture medium is the culture medium shown in Table 1-2 below.

[0093] In another embodiment, the culture medium contains about 10 ng / mL of bFGF, about 0.5 mM of VPA. about 15 pM of CHIR-99021, about 10 pM of Tranylcypromine, about 20 pM of Forskolin, about 1 pM of Ch55, about 5 pM of EPZ004777, about 2 pM of RepSox, and optionally about 10 pM ofY-27632. Alternatively, the culture medium contains at least 10 ng / mL of bFGF, at least 0.5 mM of VPA, at least 15 pM of CHIR- 99021, at least 10 pM of Tranylcypromine, at least 20 pM of Forskolin. at least 1 pM of Ch55. at least 5 pM of EPZ004777, at least 2 pM of RepSox, and optionally at least 10 pM ofY-27632.

[0094] In certain embodiments, the culture media are used in combination with a feeder layer such as a mouse embryonic fibroblast (MEF) feeder layer or a DR4 feeder layer.Stage 1: Generation of primed P. capensis primary cells

[0095] The first stage of the methods is generation of primed P. capensis primary' cells from primary P. capensis cells. In certain embodiments, this stage involves transfection of P. capensis primary’ cells (such as e.g.. P. capensis fibroblasts) with OCT4. KI.F4. SOX2. GI.IS1. CMYC, and optionally antibiotic resistance (e.g, puromycin-resistance).

[0096] In one embodiment, Stage 1 of the methods involve culturing primary’ P. capensis cells transfected with OCT4. SOX 2. KL F4, CMYC, and GLIS1 , and optionally an antibiotic resistance gene in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT IL inhibitor, and a TGF-(3 inhibitor to generate primed P. capensis cells.

[0097] In another embodiment, Stage 1 also involves transfecting the primary P. capensis cells with OCT4. SOX2, KLF4. CMYC. and GLIS1 and optionally an antibiotic resistance gene (such as e.g., puromycin for later selection of the cells). In certainembodiments, the antibiotic resistance gene is omitted. In some embodiments, the primary P. capensis cells are transfected with a single stranded RNA replicon comprising OCT4. SOX 2. KLF4, CMYC, GLIS1, and optionally an antibiotic resistance gene.

[0098] In one embodiment, Stage 1 of the methods involve culturing primary P. capensis cells transfected with OCT4. SOX2. KLF4, CMYC, and GLIS1 , and an antibiotic resistance gene in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and a TGF- inhibitor to generate primed P. capensis cells. This embodiment may also involve transfecting the primary P. capensis cells with OCT4, SOX 2. KLI'4. CMYC, and GLIS1 and an antibiotic resistance gene (such as e.g, puromycin for later selection of the cells). In some embodiments, the primary P. capensis cells are transfected with a single stranded RNA replicon comprising OCT4. SOX 2. KLF4, CMYC, GLIS1, and an antibiotic resistance gene.

[0099] In some embodiments, the culture medium in Stage 1 is supplemented with from about 0. 1 to about 1 mM of an HD AC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, from about 2 to about 10 pM of a DOT1L inhibitor, and from about 0.5 to about 4 pM of a TGF-(3 inhibitor.

[0100] In the culture medium used in Stage 1, the HD AC inhibitor can be valproic acid, the GSK-3 inhibitor can be CHIR-99021 , the monoamine oxidase inhibitor can be tranylcypromine (2-PCPA) HC1, activator of eukary otic adenylyl cyclase can be forskolin, the retinoid can be Ch 55, the DOT1L inhibitor can be EPZ004777, and / or the TGF-(3 inhibitor can be RepSox.

[0101] In one embodiment, the culture medium in Stage 1 is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

[0102] In certain embodiments, stage 1 of the methods involves transfecting the primary7P. capensis cells and culturing the transfected cells in non-chemical reprogramming medium prior to chemical reprogramming. Stage 1 may also include selecting cells exhibiting iPSC characteristics such as e.g., compact, high nucleus-to-cytoplasm ratio, and clear borders. The culturing and chemical reprogramming can be carried out under adherent conditions such as plates pre-coated with Matrigel™ or a similar substrate.

[0103] In certain embodiments, the Stage 1 cells are generated using the protocol in Example 4 below.Stage 2: Generation of PC pre-iPSC from primed P. capensis primary cells

[0104] The second stage of the methods is generation of pre-induced P. capensis cells from primed P. capensis primary cells. In certain embodiments, this stage involves transfection of the pre-induced P. capensis cells generated in Stage 1 with OCT4. SOX2, KI.F4. CMYC, and NANOG followed by chemical reprogramming of the cells. In some embodiments, this stage involves transfection of the pre-induced P. capensis cells generated in Stage 1 with Loxodonta africana OCT4. SOX 2. KLF4, CMYC, and NANOG followed by chemical reprogramming of the cells.

[0105] In some embodiments. Stage 2 of the methods involves generating pre-induced P. capensis cells from the primed P. capensis cells (generated in Stage 1) by transfecting the primed P. capensis cells with OCT4. SOX 2. KLI-4. CMYC, and NANOG and culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and a TGF-(3 inhibitor.

[0106] In certain embodiments, Stage 2 (generating pre-induced P. capensis cells from the primed P. capensis cells) includes:(1) transfecting the primed P. capensis cells obtained in Stage 1 with OCT4. SOX 2. KLF4, CMYC, and NANOG,'(2) culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-0 inhibitor, and a ROCK inhibitor; and(3) culturing the transfected primed P. capensis cells in a culture medium devoid of a ROCK inhibitor supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-J3 inhibitor, and an agent to induce transcription of transfected genes.In certain embodiments, the methods include culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-[3 inhibitor, and a ROCK inhibitor on a feeder layer after (3). Avariety of different agents can be used to induce transcription of transfected genes. In certain embodiments, the agent to induce transcription of transfected genes is doxycycline.

[0107] In other embodiments. Stage 2 (generating pre-induced P. capensis cells from the primed P. capensis cells) includes:(1 ) transfecting the primed P. capensis cells obtained in Stage 1 with OCT4, SOX2. KI.F4. CMYC, and NANOG(2) culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-(3 inhibitor, and a ROCK inhibitor; and(3) culturing the transfected primed P. capensis cells in a culture medium devoid of a ROCK inhibitor supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and doxycycline.In certain embodiments, the methods include culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and a ROCK inhibitor on a feeder layer after (3).

[0108] In some embodiments, the antibiotic resistance gene is puromycin-resi stance, and the antibiotic is puromycin. In other embodiments, the ROCK inhibitor is Y-27632.

[0109] In some embodiments, in Stage 2, the primed P. capensis cells are transfected with a plasmid comprising OCT4, SOX2, KLF4. CMYC. and NA NOG.

[0110] In some embodiments, the culture medium in Stage 2 is supplemented with from about 0. 1 to about 1 mM of an HD AC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, from about 2 to about 10 pM of a DOT1L inhibitor, and from about 0.5 to about 4 pM of a TGF-P inhibitor.[OHl] In the culture medium used in Stage 2, the HD AC inhibitor can be valproic acid, the GSK-3 inhibitor can be CHIR-99021, the monoamine oxidase inhibitor can be tranylcypromine (2-PCPA) HCL activator of eukaryotic adenylyl cyclase can be forskolm.the retinoid can be Ch 55, the D0T1L inhibitor can be EPZ004777, and / or the TGF- inhibitor can be RepSox.

[0112] In one embodiment, the culture medium in Stage 2 is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

[0113] In certain embodiments, stage 2 of the methods involves contacting the cells with a ROCK inhibitor (Y-27632) after transfection. After contacting with the ROCK inhibitor, the cells are washed, cultured in the medium supplemented with a ROCK inhibitor, followed by culturing in the medium devoid of a ROCK inhibitor supplemented with 2 pg / mL doxycycline (or other agents that may be used to induce transcription of transfected genes). The culturing includes culturing on a feeder layer, such as e.g., an MEF feeder layer.

[0114] In certain embodiments, the Stage 2 cells are generated using the protocol in Example 5 below.Stage 3 Generation of PC iPSCs from PC pre-iPSCs

[0115] The third stage of the methods is generation of induced pluripotent P. capensis stem cells from pre-induced P. capensis cells. In certain embodiments, this stage involves transfection of the pre-induced P. capensis cells generated in Stage 2 with OCT4. OX 2. KL] '4. CMYC, NANOG. IJN28A. and SV40 followed by culturing the cells in a culture medium that is capable of chemical reprogramming.

[0116] Stage 3 of the methods relies on the introduction of a cocktail of individual transcription factors into Procavia capensis “pre-iPSCs” to leverage the heterogeneous distribution of these transgenes to further enhance pluripotency by shifting the cell state to a more pluripotent state unique to the Procavia capensis, then allowing endogenous pluripotency factors to take over and maintain cell state.

[0117] In some embodiments, Stage 3 of the methods involves generating the induced pluripotent Procavia capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P. capensis cells with OCT4, SOX2, KLF4. CMYC, NANOG, LIN28A, and SV40 and culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and a TGF-P inhibitor.

[0118] In one embodiment, the generating of the induced pluripotent Pr ocavia capensis stem cells in stage 3 includes:(1) transfecting the pre-induced P. capensis cells (i.e., Stage 2 cells) with OCT4. SOX2.KLF4, C.MYC. and NA NOG(2) culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT IL inhibitor, a TGF-P inhibitor, and a ROCK inhibitor (e.g, Y-27632); and(3) culturing the transfected pre-induced P. capensis cells in a culture medium devoid of a ROCK inhibitor supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT IL inhibitor, a TGF-P inhibitor, and an agent to induce transcription of transfected genes.In certain embodiments, the methods include culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a D0T1L inhibitor, a TGF-P inhibitor, and a ROCK inhibitor on a feeder layer. A variety of different agents can be used to induce transcription of transfected genes. In certain embodiments, the agent to induce transcription of transfected genes is doxycycline.

[0119] In another embodiment, the generating of the induced pluripotent Procavia capensis stem cells in stage 3 includes:(1) transfecting the pre-induced P. capensis cells with OCT4. SOX 2. KI.I-4. CMYC, and NANOG,-(2) culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOTIL inhibitor, a TGF-P inhibitor, and a ROCK inhibitor (e.g., Y-27632); and(3) culturing the transfected pre-induced P. capensis cells in a culture medium devoid of a ROCK inhibitor supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and doxycycline.In certain embodiments, the methods include culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-[3 inhibitor, and a ROCK inhibitor on a feeder layer.

[0120] In some embodiments, the antibiotic resistance gene is puromycin-resistance and wherein the antibiotic is puromycin. In other embodiments, the ROCK inhibitor is Y- 27632.

[0121] In some embodiments, the culture medium in Stage 3 is supplemented with from about 0. 1 to about 1 mM of an HD AC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, from about 2 to about 10 pM of a DOT1L inhibitor, and from about 0.5 to about 4 pM of a TGF-(3 inhibitor.

[0122] In the culture medium used in Stage 3, the HD AC inhibitor can be valproic acid, the GSK-3 inhibitor can be CHIR-99021, the monoamine oxidase inhibitor can be tranylcypromine (2-PCPA) HC1, activator of eukary otic adenylyl cy clase can be forskolin, the retinoid can be Ch 55, the DOT1L inhibitor can be EPZ004777, and / or the TGF-(3 inhibitor can be RepSox.

[0123] In one embodiment, the culture medium in Stage 3 is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

[0124] In additional embodiments, the pre-induced P. capensis cells are transfected with a plasmid comprising OCT4. SOX2. KI.1- 4. CMYC. NANOG. IJN28A. and SV40.

[0125] In some embodiments, the culturing during Stage 3 involves culturing on a feeder layer, such as e.g., an MEF feeder layer. In certain embodiments, stage 3 of the methods involves contacting the cells with a ROCK inhibitor (Y-27632) after transfection. After contacting with the ROCK inhibitor, the cells are washed, cultured in the medium supplemented with a ROCK inhibitor, followed by culturing in the medium devoid of a ROCK inhibitor supplemented with 2 pg / rnL doxycycline (or other agents that may be used to induce transcription of transfected genes). The culturing includes culturing on a feeder layer, such as e.g., an MEF feeder layer.

[0126] In certain embodiments, the Stage 3 cells are generated using the protocol in Example 6 below .

[0127] In certain embodiments, the methods of the disclosure include maintenance and / or cry opreservation of the P. capensis iPSCs after Stage 3.

[0128] In one embodiment, the methods include maintaining the P. capensis iPSCs in a culture medium supplemented with NaB, FGF, and a TGF-0 inhibitor such as RepSox afterStage 3. The culture medium can be E-8-Flex with E8 Flex Supplement. In one embodiment, the medium in Table 1-3 is used. The cells can be maintained on a MEF feeder layer and passaged using trypsinization. Trypsinization is used and the culture medium is further supplemented with a ROCK inhibitor (e.g., Y-27362).

[0129] In another embodiment, the methods include culturing of the primary7P. capensis cells before differentiation (z.e., before Stage 1). The culturing may involve thawing cryopreserved primary P. capensis cells (such as e.g.. fibroblasts in particular female fibroblasts). The culturing also involves culturing the cells on a feeder layer (e.g. , an MEF feeder layer).

[0130] The methods also include selection, isolation, and / or purification of pc iPSCs or Stage 2 cells. In certain embodiments, the selection, isolation, and / or purification of cells uses the protocol of Example 7. In other embodiments, the cells may be isolated and / or purified using Fluorescence-Activated Cell Sorting (FACS). A suitable FACS protocol is described in Example 11 below.

[0131] In certain embodiments, Stage 2 and / or Stage 3 cells are isolated. In one embodiment, the isolation includes allowing the cells to reach approximately 80% confluence. The cells are then treated with 0.5 pg / mL puromycin in reprogramming media for 24 hours to eliminate non-transfected cells. The puromycin concentration is increased to 5 pg / mL and the treatment is continued for an additional 48 hours. Media is changed daily to prevent toxicity and support cell recovery.

[0132] In other embodiments, the methods include transitioning the P. capensis iPSCs from the chemical reprogramming media. In some embodiments, the transition involves the protocol in Example 8.

[0133] The methods can also include flash freezing of Stage 1, Stage 2, and / or Stage 3 cells. In certain embodiments, the flash freezing involves use of liquid nitrogen. In one embodiment, the flash freezing follows the protocol of Example 9.FIG. 1 Protocol for Generation of PC iPSCfrom PC pre-iPSC

[0134] A schematic representation of one embodiment of a method of the disclosure is shown in FIG. 1. The method shown in FIG. 1 involves isolating P. capensis primary cells (fibroblasts in this example) (pc Fibre).

[0135] In Stage 1 of the method, the cells are then primed by transfecting the . capensis primary cells with OCT4. SOX 2. KI.F4. CMYC. and GLIS1, and an antibiotic resistance gene (e.g., puromycin) (using for example ReproRNA priming) therebygenerating pc PRC1 cells. The pc PRC1 cells are chemically reprogrammed by culturing the cells in a culture medium supplemented with an HD AC inhibitor (such as e.g, valproic acid (VPA)), a GSK-3 inhibitor (such as e.g, CHIR-99021), a monoamine oxidase inhibitor (such as e.g., tranylcypromine (2-PCPA) HC1), an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g., forskolin), a retinoid (such as e.g., CH55), a DOTIL inhibitor (such as e.g, EPZ004777), and a TGF- inhibitor (such as e.g., RepSox) to generate pc PRC2 cells (also known as primed P. capensis cells).

[0136] The primed P. capensis cells are further reprogrammed in stage 2 of the method to generate pre-induced P. capensis cells by transfecting the primed P. capensis cells with OCT4, SOX2, KLF4, CMY C, and NANOG and culturing the cells in a culture medium supplemented with an HD AC inhibitor (such as e.g.. valproic acid (VPA)), a GSK-3 inhibitor (such as e.g., CHIR-99021), a monoamine oxidase inhibitor (such as e.g., tranylcypromine (2-PCPA) HC1), an activator of eukar otic adenylyl cyclase (a cAMP agonist, such as e.g., forskolin), a retinoid (such as e.g., CH55), a DOT1L inhibitor (such as e.g., PZ Q llT), and a TGF-(3 inhibitor (such as e.g, RepSox). In certain embodiments, the transfecting is achieved using loxOSKM reprogramming.

[0137] In stage 3 of the protocol, the induced pluripotent Procavia capensis stem cells are generated from the pre-induced P. capensis cells. Stage 3 involves transfecting the preinduced P. capensis cells with OCT4, SOX2. KLF4, CMY C, NANOG, LIN28A, and SV40 and culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor (such as e.g., valproic acid (VPA)), a GSK-3 inhibitor (such as e.g., CHIR-99021), a monoamine oxidase inhibitor (such as e.g., tranylcypromine (2-PCPA) HC1), an activator of eukaryotic adenylyl cyclase (a cAMP agonist, such as e.g, forskolin), a retinoid (such as e.g, CH55), a DOT1L inhibitor (such as e.g., EPZ004777), and a TGF-P inhibitor (such as e.g., RepSox). The Table below summarizes the starting cell, ending cells, and genes transfected at each stage of the method.IV. Methods of generating PC iPSC - “Single Transfection”

[0138] Another aspect of the disclosure is directed to methods of generating induced pluripotent Procavia capensis stem cells which do not rely on the triple transfection protocol. In this aspect of the disclosure, the primary P. capensis cells are cultured in a chemical induction medium to generate pre-induced (primed) Procavia capensis cells and the induced pluripotent P. capensis stem cells are generated by transfecting the pre-induced P. capensis cells with at least OCT4ISOX2IKLF4ICMYC and SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes and culturing in a chemical induction medium. The same culture media as used in the method described above in the section entitled ’‘Culture Media for Stages 1-3” can be used.

[0139] A variety of primary' P. capensis cells may be used in the methods. In some embodiments, the primary P. capensis cells are somatic P. capensis cells. In some embodiments, the somatic P. capensis cells are endothelial cells, epithelial cells, or fibroblasts such as fetal fibroblasts, adult fibroblasts, and skin fibroblasts. In one embodiment, the primary P. capensis cells are fibroblasts, in particular fibroblasts from a female P. capensis.

[0140] In one embodiment, the induced pluripotent P. capensis stem cells are generated by transfecting the pre-induced P. capensis cells with (i) OCT4ISOX2IKLF4ICMYC and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes. In another embodiment, the induced pluripotent / ’, capensis stem cells are generated by transfecting the pre-induced P. capensis cells with (i) OCT4ISOX2IKLF4ICMYCILIN28A and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes. In yet another embodiment, the induced pluripotent P. capensis stem cells are generated by transfecting the pre-induced P. capensis cells with (i) OCT4 / SOX2 / KLF4 / CMYC / LIN28A NANOG and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes.

[0141] In a further embodiment, the induced pluripotent P. capensis stem cells are generated by transfecting the pre-induced P. capensis cells with (i) Loxodonta africana OCT4ISOX2IKLF4ICMYC and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes. In another embodiment, the induced pluripotent P. capensis stem cells are generated by transfecting the pre-induced P. capensis cells with (i) Loxodonta africana OCT4ISOX2IKLF4ICMYCILIN28A and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes. In yet another embodiment, (i) Loxodonta africana OCT4 / SOX2 / KLF4XMYC LIN28A / NANOG and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes.

[0142] In an alternate embodiment, the induced pluripotent P. capensis stem cells are generated by transfecting the pre-induced P. capensis cells with (i) OCT4ISOX2IKLF4ICMYC and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes in Elephas maximus. In another embodiment, the induced pluripotent P. capensis stem cells are generated by transfecting the pre-induced P. capensis cells with (i) OCT4ISOX2IKLF4ICMYCILIN28A and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes in Elephas maximus. In yet another embodiment, (i) OCT4 / SOX2 / KLF4 / CMYC I.IN28A NANOG and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes in Elephas maximus.

[0143] In an alternate embodiment, the induced pluripotent P. capensis stem cells are generated by transfecting the pre-induced P. capensis cells with (i) Loxodonta africana OCT4ISOX2IKLF4ICMYC and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes in Elephas maximus. In another embodiment, the induced pluripotent P. capensis stem cells are generated by transfecting the pre-induced P. capensis cells with (i) L. africana OCT4ISOX2IKLF4ICMYCILIN28A and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes in Elephas maximus. In yet another embodiment, (i) L. africana OCT4 / SOX2 / KLF4 / CMYCLIN28A / NANOG and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes in Elephas maximus.

[0144] The methods for generating induced pluripotent Procavia capensis stem cells of this aspect generally include: (a) culturing primary P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOTIL inhibitor, and a TGF-0 inhibitor to generate pre-induced P. capensis cells; and (b) generating the induced pluripotent P. capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P. capensis cells with at least OCT4ISOX2IKLF4ICMYC, and SV40 T-antigenor an shRNA targeting TP53 and / or TP53 retrogenes and culturing the transfected preinduced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT IL inhibitor, and a TGF-P inhibitor.

[0145] In one embodiment, the methods include culturing the primary P. capensis cells in a basal culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-P inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor. A variety of basal culture media may be suitable. In one embodiment, the culture medium is Knock-out DMEM or DMEM. The basal culture media may further be supplemented with one or more of bFGF, serum (e.g., FBS). KOSR, GlutaMAX™. NEAA, and 2-mercaptoethanol. The culture medium may also further be supplemented with serum. In other embodiments, the culture medium is serum- free.

[0146] In certain embodiments, the chemically reprogramming includes passaging the cells for at least 2-3 passages. The passaging may involve media exchange.

[0147] In certain embodiments, commercially available transfection systems, such as the Neon transfection system (Invitrogen MPK100265), are used. In one embodiment, the method includes transfecting the pre-induced P. capensis cells with an antibiotic resistance gene.

[0148] In another embodiment, the methods include transfecting the pre-induced P. capensis cells with OCT4ISOX2IKLF4ICMYCILIN28A and SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes. In a further embodiment, the method includes transfecting the pre-induced P. capensis cells with OCT4ISOX2IKLF4ICMYCILIN28A / NANOG and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes. In another embodiment, the OCT4ISOX2IKLF4ICMYC is from L. africana. In a further embodiment, the OCT4ISOX2IKLF4ICMYCILIN28A is from L. africana. In an alternate embodiment, the OCT4 / SOX2 / KLF4 / CMYC / LIN28A / NANOG is from L. africana. In certain embodiments, the cells are transfected with an SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes in Elephas maximus.

[0149] The pre-induced P. capensis cells can be transfected using techniques known in the art. In one embodiment, pre-induced P. capensis cells are transfected with a single stranded RNA replicon comprising OCT4ISOX2IKLF4ICMYC, and optionally an antibiotic resistance gene. In another embodiment, the pre-induced P. capensis cells are transfected with a plasmid comprising OCT4ISOX2IKLF4ICMYC.

[0150] In certain embodiments, the culture medium in (a) and / or (b) is supplemented with from about 0. 1 to about 1 mM of an HD AC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, from about 2 to about 10 pM of a DOT IL inhibitor, and from about 0.5 to about 4 pM of a TGF-[3 inhibitor. In one embodiment, the HD AC inhibitor is valproic acid. In another embodiment, the GSK-3 inhibitor is CHIR-99021. In alternate embodiment, the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1. In yet another embodiment, the activator of eukary otic adenylyl cyclase is forskolin. In a further embodiment, the retinoid is Ch 55. In an additional embodiment, the DOT IL inhibitor is EPZ004777. In yet another embodiment, the TGF-0 inhibitor is RepSox.

[0151] In further embodiments, the culture medium in (a) and / or (b) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox. In one embodiment, the culture medium in (a) is supplemented with valproic acid, CHIR-99021. tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox. In another embodiment, the culture medium in (b) is supplemented with valproic acid. CHIR-99021, tranylcypromine, forskolin. Ch 55, EPZ004777, and RepSox.

[0152] The transfecting may include changing the culture medium every7two days. In some embodiments, where desired a selection marker, such as antibiotic resistance (to e.g., hygromycin or puromycin) is also transfected.

[0153] The transfection may be carried in the presence of absence of a feeder layer. In certain embodiments, the transfecting is carried out on cells on a matrix coating without feeder cells. In one embodiment, the matrix coating is laminin. In another embodiment, the coating is geltrex.

[0154] In certain embodiments, the methods include selecting for the induced pluripotent P. capensis stem cells. In some embodiments, the selecting includes culturing the induced pluripotent P. capensis stem cells with doxycycline and the antibiotic used for selection (such as e.g., hygromycin or puromycin) under feeder-free conditions, followed by culturing the induced pluripotent P. capensis species stem cells on a feeder layer along with doxycycline. In other embodiments, the selecting includes culturing the induced pluripotent P. capensis species stem cells with doxycycline and the antibiotic used for selection (such as e.g., hygromycin or puromycin) under feeder-free conditions, followed by culturing the induced pluripotent P. capensis species stem cells on a feeder layer along with doxycycline, followed by culturing the induced pluripotent P. capensis species cells on a feeder layerresistant to the antibiotic used for the selection and the antibiotic used for selection (such as e.g.. hygromycin or puromycin).

[0155] In another embodiment, the culture medium contains about 10 ng / mL of bFGF, about 0.5 mM of VP A, about 15 pM of CHIR-99021, about 10 pM of Tranylcypromine, about 20 pM of Forskolin, about 1 pM of Ch55, about 5 pM of EPZ004777, about 2 pM of RepSox, and optionally about 10 pM ofY-27632. Alternatively, the culture medium contains at least 10 ng / mL of bFGF, at least 0.5 mM of VPA, at least 15 pM of CHIR- 99021, at least 10 pM of Tranylcypromine, at least 20 pM of Forskolin, at least 1 pM of Ch55, at least 5 pM of EPZ004777, at least 2 pM of RepSox, and optionally at least 10 pM ofY-27632.

[0156] In certain embodiments, the culture media are used in combination with a feeder layer such as a mouse embryonic fibroblast (MEF) feeder layer or a DR4 feeder layer.

[0157] In some embodiments, the culturing during step (b) involves culturing on a feeder layer, such as e.g., an MEF feeder layer. In certain embodiments, step (b) of the methods involves contacting the cells with a ROCK inhibitor (Y-27632) after transfection. After contacting with the ROCK inhibitor, the cells are washed, cultured in the medium supplemented with a ROCK inhibitor, followed by culturing in the medium devoid of a ROCK inhibitor supplemented with 2 pg / mL doxycycline (or other agents that may be used to induce transcription of transfected genes). The culturing includes culturing on a feeder layer, such as e.g., an MEF feeder layer.

[0158] In certain embodiments, the method utilizes the protocol described in Example 12. In other embodiments, the method utilizes the protocol described in Example 12 but with a different basal medium. In certain embodiments of these methods of generating induced pluripotent P. capensis stem cells, the methods disclosed in PCT / US2024 / 056242 can be used except that primary P. capensis cells are used in place of primary Afrotheria species cells (such as e.g. somatic Afrotheria species cells). The disclosure of PCT / US2024 / 056242 as it pertains to differentiation protocols and media is hereby- incorporated by reference.

[0159] The methods also include selection, isolation, and / or purification of pc iPSCs. In certain embodiments, the selection, isolation, and / or purification of cells uses the protocol of Example 7. In other embodiments, the cells may be isolated and / or purified using Fluorescence-Activated Cell Sorting (FACS). A suitable FACS protocol is described in Example 11 below.

[0160] In certain embodiments, the pc iPSCs are isolated. In one embodiment, the isolation includes allowing the cells to reach approximately 80% confluence. The cells are then treated with 0.5 pg / mL puromycin in reprogramming media for 24 hours to eliminate non-transfected cells. The puromycin concentration is increased to 5 pg / mL and the treatment is continued for an additional 48 hours. Media is changed daily to prevent toxicity and support cell recovery’.

[0161] In other embodiments, the methods include transitioning the P. capensis iPSCs from the chemical reprogramming media. In some embodiments, the transition involves the protocol in Example 8.

[0162] The methods can also include flash freezing of P. capensis iPSCs. In certain embodiments, the flash freezing involves use of liquid nitrogen. In one embodiment, the flash freezing follows the protocol of Example 9.

[0163] Any of the primary P. capensis cells described above can be used with the methods. In certain embodiments, the primary P. capensis cells are P. capensis fibroblasts. In other embodiments, the P. capensis fibroblasts are female P. capensis fibroblasts.V. PC iPSCs

[0164] The disclosure also provides for induced pluripotent P. capensis stem cells (PC iPSC) that are generated from primary P. capensis cells, such as e.g., P. capensis fibroblasts. In certain embodiments, the induced pluripotent / ’, capensis stem cells are generated using any of the methods described herein.

[0165] One embodiment of the disclosure is an induced pluripotent P. capensis stem cell expressing OCTA. 80X2. KTT4. CMYC, NANOG. TIN28A. and SV40, wherein the stem cell is generated by transfecting and chemically reprogramming a primary P. capensis cell (such as e.g., a fibroblast (including a female fibroblast)). The cells may also express GLIS1.

[0166] Another embodiment of the disclosure is an induced pluripotent P. capensis stem cell expressing OCT . 80X2. KTT4. CMYC. NANOG. TIN28A. and SV40, wherein the stem cell is generated by chemically reprogramming a primary P. capensis cell and transfecting the reprogrammed P. capensis cell to express OCT4, SOX2, KLF4, CMYC, NANOG, LIN28A, and SV40. Yet another embodiment is an induced pluripotent P. capensis stem cell expressing OCT4, SOX2, KLF4, CMYC, NANOG, LIN28A, GLIS1, and SV40, wherein the stem cell is generated by chemically reprogramming a primary P. capensis cell andtransfecting the reprogrammed P. capensis cell to express OCT4, SOX2, KLF4, CMYC, NANOG. I.IN28A. GLIS1, and SV40.

[0167] In one embodiment, the induced pluripotent P. capensis stem cells of the disclosure express OCT4, SOX 2. KLF4. CMYC, NANOG, LIN28A, and SV40. In another embodiment, the induced pluripotent P. capensis stem cells of the disclosure express OCT4, SOX2, KLF4. CMYC, NANOG, LIN28A, GLIS1, and SV40.VI. Uses ofPCiPSCs

[0168] The disclosure also includes using the P. capensis iPSCs. In particular, the P. capensis iPSCs can be differentiated into each germ-layer (endoderm, mesoderm, and ectoderm). In other embodiments, embryoid bodies are formed from the cells. In further embodiments, the iPSCs are used to generate embryos. The cells may also be further edited using, for example, multiplex editing. In further embodiments, the P. capensis iPSCs can be used to generate chimeras, embryos, germ cell progenitors, and gametes. In other embodiments, the P. capensis iPSCs are differentiated into primordial germ cells.

[0169] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples, therefore, specifically point out the preferred embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.EXAMPLES

[0170] While the invention has been described and illustrated herein by references to various specific materials, procedures, and examples, it is understood that the invention is not restricted to the particular combinations of material and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary, only, with the true scope and spirit of the invention being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.Example 1: Culture Media

[0171] The following culture media were used in Examples 2 to 8.Growth Media & MEF Media: DMEM

[0172] This media formulation supports the plating of inactivated mouse embryonic fibroblasts (MEFs, serving as a feeder layer) as well as the propagation of primary rock hyrax (P. capensis) cells into higher passages (passage >20). This formulation is made up of:GlutaMAX™ contains 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl.Chemical De-Differentiation / Reprogramming and Maintenance Media

[0173] This media formulation supports the initial stages of reprogramming primary hyrax cells into pre-iPSCs through the transfection process and the maintenance / expansion of resultant stem-like and naive hyrax iPSCs.Growth / Maintenance iPSC Media

[0174] This media formulation supports reprogrammed hyrax cells iPSCs through high passage (n > 20).Example 2: Primary Culture of Procavia capensis Fibroblasts

[0175] Objective: The objective of this example was to establish a primary culture of Procavia capensis fibroblasts transitioning from a cryopreserved state to active growth in preparation for cellular reprogramming.

[0176] Materials: The following materials were used for testing in this example:• Cryovials containing female Procavia capensis fibroblasts (Identifier: 23Pc001-FFb)• Water / bead bath set to 37°C• Growth media: DMEM supplemented with 10% FBS, 1% GlutaMAX™• T75 tissue-culture treated flasks• 0.25% Trypsin-EDTA• Centrifuge• CO2 incubator set at 37°C, 5% CO2• Pipettes (pl 000) and pipette tips• Inactivated DR4 Mouse Embryonic Fibroblasts (MEFs)• Gelatin-coated plates• ROCK inhibitor Y-27632 (10 pM)Procedure:

[0177] Thawing Cells: The cryovial containing fibroblasts was warmed in a 37°C water bath until a small pellet of ice remained. Immediately after thawing, the vial was transferred to a biosafety cabinet to maintain sterility .

[0178] Preventing Osmotic Shock: Gradually pre-warmed growth media was added to the cryovial to dilute the cryoprotectant slowly and reduce osmotic shock. The cryovial was gently mixed by pipetting and then centrifuged at 300 x g for 5 minutes to pellet the cells. The supernatant was carefully aspirated to remove residual cryoprotectant.

[0179] Cell Plating: The cell pellet was resuspended in fresh growth media. The cells were then homogenized by gently pipetting up and down using a p 1000 pipette. The cells were seeded in a T75 flask and placed in a CO2 incubator. After seeding, the cells were allowed to adhere and recover for 48 hours without disturbance.

[0180] Media Change and Maintenance: While monitoring cell confluence, the media was replaced very’ 48-72 hours. The culture was maintained until cells reached approximately 70% confluence.

[0181] Preparing for Reprogramming: Once at 70% confluence, the cells were trypsinized with 0.25% Trypsin-EDTA for approximately 5 minutes. Try psin was neutralized with growth media containing 10 pM ROCK inhibitor Y-27632 to enhance cell survival. The cells were then centrifuged at 300 x g for 5 minutes and the supernatant was aspirated.

[0182] An image of a representative example of female rock hyrax fibroblasts after primary culturing is shown in FIG. 2.Example 3: Preparation of MEF Feeder Layer

[0183] Objective: The objective of this example was to prepare a stable and uniform feeder layer using DR4 Mouse Embryonic Fibroblasts (MEFs) for supporting the growth and maintenance of reprogrammed cells.

[0184] Materials: The following materials were used for testing in this example:• DR4 Mouse Embryonic Fibroblasts (MEFs) cryovials• Tissue culture-treated six-well plates or 10 cm dishes• 0.1% Gelatin solution (STEMCELL; 07903)• Growth media appropriate for MEFs• Water / bead bath set to 37°C• Centrifuge• CO2 incubator set at 37°C, 5% CO2• Pipettes (pl 000) and pipette tipsProcedure:

[0185] Coating Plates: Enough of 0. 1% gelatin solution was added to cover the surface of six-well plates or 10 cm dishes. The plates or dishes were then incubated at room temperature for 60 minutes to allow a thin layer of gelatin to form. The gelatin solution was aspirated, and the plates were rinsed once with sterile phosphate-buffered saline (PBS) to remove excess gelatin.

[0186] Thawing and Preparing MEFs: A cryovial of DR4 MEFs was removed from liquid nitrogen storage and immediately placed in a 37°C water bath. The cryovial was thawed until a small pellet of ice remained to minimize cell damage. The cryovial was then transferred to a biosafety cabinet and pre-warmed growth media was slowly added to dilute the cryoprotectant gently. The cell suspension was centrifuged at 300 x g for 5 minutes to pellet the cells. The supernatant was carefully aspirated to remove residual cryoprotectant.

[0187] Resuspending and Plating MEFs: The MEF pellet was resuspended in fresh growth media by gently pipetting up and down using a pl 000 pipette to ensure homogenous cell distribution. The cell suspension was plated onto the gelatin-coated plates, distributing cells evenly. The plating density was optimized based on the expected confluency required for supporting reprogrammed cells.

[0188] Incubating MEFs: The plated cells were placed in a 37°C, 5% CO2 incubator. The cells were allowed to adhere and form a monolayer without disturbance for 24 hours.Example 4: Transfection Protocol Stage 1: Priming Fibroblasts for Reprogramming with ReproRNA™-OKSGM and an Early-Stage Reprogramming Formulation

[0189] Objective: The objective of this example was to use ReproRNA™-OKSGM as a priming step prior to transcription factor-based reprogramming in Procavia capensis.

[0190] Materials: The following materials were used for testing in this example:• Procavict cctpensis fibroblasts• ReproRNA™-OKSGM (STEMCELL Technologies) (containing OCT4, KI. I A. SOX2. GLIS1, CMYC, and a puromycin-resistance gene)• Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen)• Opti-MEM I Reduced Serum Medium (Gibco)• iPSC culture medium (e.g. , mTeSR™l)• 6-well culture plates• CO2 incubator set at 37°C, 5% CO2• Pipettes and sterile tips• Centrifuge and tubesProcedure:

[0191] Preparation of Cell Culture: Procavia cctpensis fibroblasts were seeded at a density of 50,000 cells per well in a 6-well plate using a culture medium appropriate for fibroblasts. The cells were allowed to adhere and reach 70-80% confluence before transfection.

[0192] Transfection with ReproRNA™-OKSGM: ReproRNA™-OKSGM and Lipofectamine™ RNAiMAX were thawed at room temperature. The transfection mixture was prepared by diluting 10 pL of ReproRNA™-OKSGM in 250 pL of Opti-MEM I per well. In a separate tube, 5 pL of Lipofectamine™ RNAiMAX was diluted in 250 pL of Opti-MEM I. Both solutions were incubated at room temperature for 5 minutes, combined, and then mixed gently. The combined mixture was incubated for an additional 20 minutes at room temperature to form the transfection complexes. The culture medium from the fibroblasts was aspirated and the transfection mixture was gently added. The plate was rocked gently to distribute the mixture evenly. The cells were returned to the CO2 incubator and incubated for 6 hours; then the transfection mixture was replaced with fresh fibroblast medium.

[0193] Post-Transfection Care: 24 hours post-transfection, the medium was changed to iPSC culture medium (e.g., mTeSR™!) to support reprogramming. The medium was replaced daily to provide optimal growth conditions and remove any transfection reagent residues. After the transfection reagents were removed, subsequent media replacements consisted of chemical reprogramming media (see Table 1-2 above for the formulation). The cells were monitored daily under a microscope for signs of reprogramming. Typicalmorphological changes, such as colony formation and changes in cell size and shape, appeared within 7-14 days.

[0194] Colony Selection and Expansion: At day fourteen post-transfection, the culture was assessed for colonies exhibiting iPSC characteristics (compact, high nucleus-to- cytoplasm ratio, and clear borders). Promising colonies were manually picked using a sterile pipette tip or by mechanical dissection under sterile conditions. Individual colonies were transferred to new wells of a 24-well plate pre-coated with Matrigel™ or a similar substrate using chemical reprogramming medium for further expansion and characterization.

[0195] After conducting stage 1 of the transfection as outlined in this example, images of the resulting stem-like colonies were taken. Specifically, an image of stem-like colony resulting from transfection of female rock hyrax fibroblasts is shown in FIG. 3. For ease of reference, these cells were named “primed” cells.Example 5: Transfection Protocol Stage 2: Creating “Pre-iPSCs” with Afrotheria Transcription Factors

[0196] Objective: The objective of this example was to introduce pluripotency- associated transcription factors into reprogrammed Proccivia capensis iPSCs using a nucleofection method to enhance reprogramming efficiency.

[0197] Materials:• Stem-like colonies of Procavia capensis post-priming (primed cells) (see Example 4 above)• Lonza 4D-Nucleofector™ System• P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza) (containing 1 x 0.675 mL P3 Primary Cell Nucleofector® Solution; 1 x 0.15 mL Supplement 1; 50 pg pmaxGFP™ Vector (1 pg / pl in 10 mM Tris pH 8.0); 2 x 16-well Nucleocuvette® Strips (20 pl))• Pre- warmed reprogramming media supplemented with 10 pM ROCK inhibitor (Y - 27632)• Plasmid DNA mix containing Loxodonta africana Oct4 / Sox2 / Klf4 / cMyc, and NANOG• DR4 Mouse Embry onic Fibroblasts (MEFs), inactivated• Six-well plates• CO2 incubator set at 37°C, 5% CO2Procedure:

[0198] Cell Preparation: The cell count was standardized to 1 x 106cells per transfection. The cell suspension was centrifuged at 300 x g for 5 minutes to pellet the cells, and the supernatant was aspirated.

[0199] Nucleofection Setup: Each cell pellet was resuspended in 82 pL of P3 Primary Cell Solution. 18 pL of Supplement 1 (as provided in the P3 nucleofector kit) was added to each cell suspension. To each mixture, 5 pg of plasmid DNA was added, ensuring an equal ratio of the plasmids containing Loxodonta africana Oct4 / Sox2 / Klf4 / cMyc, and NANOG. The solution was then homogenized by gentle pipetting to ensure an even distribution of DNA throughout the cell suspension.

[0200] Transfection: The cell-DNA mixture was transferred into the cuvette provided with the nucleofector kit. The DS- 150 program on the Lonza 4D-Nucleofector was used to electroporate the cells.

[0201] Recovery and Seeding: Immediately after nucleofection, 500 pL of pre-warmed reprogramming media containing 10 pM ROCK inhibitor was added directly into the cuvette to aid cell recovery. The entire contents of the cuvette were gently transferred to a well of a six-well plate pre-seeded with inactivated DR4 MEFs. The volume in each well was adjusted to 2 mL with additional reprogramming media containing 10 pM ROCK inhibitor.

[0202] Post-Transfection Care: The transfected cells were incubated at 37°C in a 5% CO2 humidified incubator. After 24 hours, the media was replaced with fresh reprogramming media devoid of a ROCK inhibitor but supplemented with 2 pg / mL doxycycline to induce the transcription of the introduced genes.

[0203] After conducting stage 2 of the transfection as outlined in this example, images of the resulting stem-like colonies were taken. Specifically, an image of stem-like colonies exhibiting heterogeneous morphologies, resulting from transfection stage 2 of female rock hyrax fibroblasts is shown in FIG. 4. For ease of reference, these cells were named “pre- iPSCsTExample 6: Transfection Protocol Stage 3: “Boosting” of Endogenous Expression via a Cocktail of Afrotheria Transcription Factors

[0204] Objective: By introducing a cocktail of individual transcription factors into Proccivia capensis "pre-iPSCs" heterogeneous distribution of these transgenes was able to be leveraged to further enhance pluripotency by shifting the cell state to a more pluripotentstate that is unique to the target species, then allowing endogenous pluripotency factors to take over and maintain cell state.

[0205] Materials: The following materials were used for testing in this example:• ‘Pre-iPSC” colonies of P. capensis from Stage 2 (see Example 5 above)• Lonza 4D-Nucleofector™ Sy stem• P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza) (containing 1 x 0.675 mL P3 Primary Cell Nucleofector® Solution; 1 x 0.15 mL Supplement 1; 50 pg pmaxGFP™ Vector (1 pg / pl in 10 mM Tris pH 8.0); 2 x 16-well Nucleocuvette® Strips (20 pl))• Pre- warmed reprogramming media supplemented with 10 pM ROCK inhibitor (Y- 27632)• Plasmid DNA mix containing OCT4, 80X2. KLF4, CMYC, NANOG, LIN28A, and SV40• DR4 Mouse Embryonic Fibroblasts (MEFs). inactivated• Six-well plate• CO2 incubator set at 37°C, 5% CO2• Doxycycline (2 pg / mL Sigma D5207)Procedure:

[0206] Cell Preparation: The cell count was standardized to 1 x 106cells per transfection. The cells were centrifuged to pellet them, and the supernatant was carefully- removed.

[0207] Nucleofection Setup: Each cell pellet was resuspended in 82 pL of P3 Primary Cell Solution. 18 pL of Supplement 1 as provided in the P3 nucleofector kit was added. 5 pg of total plasmid DNA was added to the cell suspension, ensuring an equal ratio of OCT4. 80X2. KLF4. CMYC, NANOG, and LIN28A as well as SV40. The mixture was homogenized by gentle pipetting to ensure thorough mixing of DNA with the cells.

[0208] Transfection: The cell-DNA mixture was transferred into a cuvette provided with the nucleofector kit. The cells were electroporated using the DS-150 program on the Lonza 4D-Nucleofector.

[0209] Recovery and Seeding: Immediately post-nucleofection, 500 pL of pre- warmed reprogramming media containing 10 pM ROCK inhibitor was added directly into the cuvette to aid cell recovery. The entire contents of the cuvette were carefully transferred to a new well of a six-well plate pre-seeded with inactivated DR4 MEFs. The total volume ineach well was adjusted to 2 mL with additional reprogramming media containing 10 pM ROCK inhibitor.

[0210] Post-Transfection Care: The transfected cells were incubated at 37°C in a 5% CO2 humidified incubator. After 24 hours, the media w as replaced w ith fresh reprogramming media devoid of ROCK inhibitor but supplemented with 2 pg / mL doxycycline to induce the transcription of the introduced genes.

[0211] An image of a colony resulting from stage 3 (OCT4. SOX 2. KI.1' 4. CMYC, NANOG LIN28A and SV40) transfected female rock hyrax fibroblasts (23Pc001-FFb) is shown in FIG. 5.Example 7: Cell Selection and Purification / Isolation Protocol for Reprogrammed Procavia capensis cells

[0212] Objective: The objective of this example was to select for reprogrammed colonies based on transcription of exogenous factors, leading into isolation of stem-like colonies based on morphological indicators of pluripotency, then followed by expansion to establish stable lines of iPSCs.

[0213] Materials: The following materials were used for testing in this example:• Colonies of Stage 1 ("‘Primed’'), Stage 2 (“Pre-iPSCs”), or Stage 3 (“iPSCs”) P. capensis• Reprogramming media supplemented with puromycin (if in Stage 2 or 3) and ROCK inhibitor• DR4 Mouse Embryonic Fibroblasts (MEFs). inactivated• P20 pipette and sterile tips• TrypLE™ Select (Gibco)• Six-well and 24-well culture plates• Centrifuge• CO2 incubator set at 37°C, 5% CO2Procedure:

[0214] Antibiotic Selection (Stage 2 and 3 only): After transfection, the cells were allowed to recover until they reached approximately 80% confluence. The cells were treated with 0.5 pg / mL puromycin in reprogramming media for 24 hours to eliminate nontransfected cells. The puromycin concentration was increased to 5 pg / mL and the treatment was continued for an additional 48 hours. Media was changed daily to prevent toxicity and support cell recovery.

[0215] Initial Recovery: Following antibiotic treatment, the media was replaced with fresh reprogramming media without puromycin. and the cells were allowed to recover for 7- 10 days. Cell health and morphology was monitored closely during this recovery phase.

[0216] FACS (Stage 2 and 3 only): After cells recovered from puromycin selection, perform FACS (described in detail below) was performed. After FACS, each set of ~1 x 106recovered cells were plated into six wells of a six-well plate coated with gelatin and seeded with MEFs as previously described.

[0217] Colony Picking: The culture was inspected under a microscope and identify colonies exhibiting canonical pluripotent stem cell morphology (<?.g., compact, high nucleus-to-cytoplasm ratio, prominent borders) were identified. Using a sterile P20 pipette tip, surrounding non-pluripotent cells were gently dislodged and removed to isolate the target colony. The old media was carefully aspirated and replaced with fresh reprogramming media supplemented with ROCK inhibitor to support colony survival.

[0218] Transfer and Expansion: The isolated colonies were transferred to a 24-well plate pre-seeded with inactivated DR4 MEFs. This helps to provide a supportive microenvironment for the iPSCs. Colonies were allowed to adhere and expand for up to 7 days, along with changing media every other day and monitoring growth and morphology.

[0219] Further Isolation and Expansion: Once colonies were well-established, each selected colony was trypsinized using TrypLE™ Select for approximately 5 minutes. After centrifugation at 300 x g for 5 minutes, the supernatant was carefully aspirated, and the cells were resuspended in fresh reprogramming media. The cells were then plated into a six-well plate pre-seeded with DR4 MEFs, allowing for further expansion. The seeding density w as adjusted based on observed growth rates, ty pically expanding colonies 1 to 10 or 1 to 20.

[0220] Monitoring and Maintenance: The expanded colonies were continued to be monitored for pluripotent morphology and growth characteristics. Periodic passaging and media changes w ere conducted to maintain optimal cell health and proliferation.Example 8: Transition to Naive Media

[0221] Objective: The objective of this example was to remove chemicals and inducing agents associated with all previous stages of reprogramming of Procavia cctpensis iPSCs and continue the transition to stable pluripotency.

[0222] Materials: The following materials were used for testing in this example:B cocktail mediaE8 Stem Flex media (Thermo Fisher Scientific A2858501)PBS• CO2 incubator• Micropipettes and tips• TrypLE™ Select (Thermo Fisher Scientific 12563011)• Centrifuge• 15 mL conical tubesProcedure:

[0223] Initial Setup: P. capensis iPSCs were maintained in B cocktail media at an appropriate confluency (-70-80%) for transitioning. If necessary, the P. capensis iPSCs were cultured to achieve these conditions.

[0224] Day 1 - Initial Transition: A 75:25 mixture of B cocktail media to E8 Stem Flex media was prepared. The spent media from the iPSC cultures was aspirated. The cells were gently washed with PBS to remove any residual media. The prepared 75:25 mixture of B cocktail media to E8 Stem Flex media was added to the culture. Then, the cells were incubated at 37°C in a 5% CO2 incubator.

[0225] Day 2: A 50:50 mixture of B cocktail media to E8 Stem Flex media was prepared. The spent media was aspirated. The cells were washed with PBS. The prepared 50:50 mixture of B cocktail media to E8 Stem Flex media was added and the cells were incubated at 37°C in a 5% CO2 incubator.

[0226] Day 3: A 25:75 mixture of B cocktail media to E8 Stem Flex media was prepared. The spent media was aspirated. The cells were washed with PBS. The prepared 25:75 mixture of B cocktail media to E8 Stem Flex media was added. Subsequently, the cells were incubated at 37°C in a 5% CO2 incubator.

[0227] Day 4: A 10:90 mixture of B cocktail media to E8 Stem Flex media was prepared. The spent media was aspirated. The cells were washed with PBS. The prepared 10:90 mixture of B cocktail media to E8 Stem Flex media was added. Then, the cells were incubated at 37°C in a 5% CO2 incubator.

[0228] Day 5: A 5:95 mixture of B cocktail media to E8 Stem Flex media was prepared. The spent media was aspirated. The cells were washed with PBS. The prepared 5:95 mixture of B cocktail media to E8 Stem Flex media was added. Subsequently, the cells were incubated at 37°C in a 5% CO2 incubator.

[0229] Day 6: 100% E8 Stem Flex media was used. The spent media was aspirated. The cells were washed with PBS. E8 Stem Flex media was added to the culture and the cells were incubated at 37°C in a 5% CO2 incubator.

[0230] Day 7+: The cells were continued to be maintained in E8 Stem Flex media, with media changes every 48 hours and monitoring of the cells for any signs of differentiation or stress.

[0231] After culturing the cells were inspected. An image of a representative colony of Proccivia capensis iPSC line Single Factor after completing the transition out of the Chemical B Cocktail, followed by passaging in E8 Stem Flex media (as outlined in this Example) is shown in FIG. 6.Example 9: Fluorescence-Activated Cell Sorting (FACS) Protocol for Reprogrammed Cells

[0232] Objective: The objective of this example is to isolate reprogrammed iPSC populations expressing specific fluorescence markers (mCherry and BFP), ensuring high purity and optimal viability’ for subsequent analyses and experiments.

[0233] Materials: The following materials were used for testing in this example:• Flow cytometer with lasers suitable for mCherry (587 nm excitation)• Reprogrammed iPSC suspension expressing mCherry• PBS (Phosphate-Buffered Saline), sterile• FACS buffer: PBS with 2% Fetal Bovine Serum (FBS), sterile• 70 pm cell strainer• Collection tubes pre-coated with FACS buffer• Centrifuge• TrypLE™ Express Enzyme (IX), phenol red (Gibco)• Propidium iodide (PI) or 7-AAD for viability stainingProcedure:

[0234] Cell Preparation: Healthy iPSCs at -80% confluence should be dissociated using TrypLE™ Express Enzyme under sterile conditions. The cells were incubated at 37°C for 3-5 minutes until cell detachment was observed. TrypLE™ is neutralized with an equal volume of FACS buffer. The cells were then pipetted gently to disperse clumps andcreate a single-cell suspension. The cell suspension is filtered through a 70 pm cell strainer into a new tube to remove aggregates and ensure a uniform flow during sorting.

[0235] Instrument Calibration and Setup: Set detector for mCherry with a 610 nm emission filter. Forward and side scatter parameters are established to exclude debris and dead cells.

[0236] Sorting Gates and Parameters: Using fluorescence-minus-one (FMO) controls, gates were set for mCherry+ cells. Adjust Photomultiplier tube (PMT) voltages are adjusted to achieve clear discrimination: typically, PMT voltages might be set around 585 V for mCherry. Sort gates are configured based on fluorescence intensity and side scatter properties to enrich viable fluorescent cells.

[0237] Sorting Process: Cells are sorted at a moderate flow rate (about 300-500 cells per second) to maintain cell integrity and sorting accuracy. Sorted cells are collected into tubes containing chilled FACS buffer to immediately cool cells and preserve viability-.

[0238] Post-Sorting Handling: Immediately after sorting, collected cells are centrifuged at 300 x g for 5 minutes at 4°C. The supernatant is carefully aspirated, and the cells were resuspended in pre-warmed iPSC culture medium. Cells were then plated on a feeder layer or matrix-coated plates to promote recox en and growth.

[0239] Post-Sorting Analysis: A sample of the sorted cells is reanalyzed to confirm the purity, which should exceed 90% for effective downstream applications. Post-sort viabilityusing a viability assay (e.g, trypan blue) is assessed, aiming for a recovery of more than 95% viable cells.Example 10: RNA Sequencing and Analysis of Reprogrammed Procavia capensis iPSCs

[0240] Objective: The objective of this example was to conduct high-throughput RNA sequencing of reprogrammed Procavia capensis iPSCs to identify gene expression patterns and potential regulatory- mechanisms involved in pluripotency and differentiation.

[0241] Process Overview: The RNA samples from Procavia capensis iPSCs were prepared and sent to Novogene for next-generation sequencing. Post-sequencing, the raw data was analyzed using the FormBio platform.

[0242] Sample Preparation and Sequencing: Total RNA was extracted from the iPSCs using a high-integrity method to ensure a minimum RNA integrity number (RIN) of 8.0. The quality and concentration of RNA were assessed using a spectrophotometer inhouse; Novogene also performed quality- checks.Data Analysis Using FormBio:

[0243] Platform Configuration: Analysis of the sequencing data was configured on the FormBio platform with the following specific parameters:

[0244] Reference Genome: P. capensis (ProcapUConnvl) to align sequence reads.

[0245] Data Handling: Raw data files located in the directory CRI / ARCHIVED / were utilized.

[0246] Read Alignment Preferences: Marking of duplicates was skipped (markdups: skip) to maintain all read information for downstream analysis.

[0247] RNA Integrity and Handling: Strandedness was set to unstranded (stranded: U), and reads were trimmed (trim: true) using Trim Galore (trimalgo: trimgalore) to improve the quality of mapping.

[0248] Analysis Specifics: The workflow employed was maseq vl .1.2, optimized for RNA-Seq data analysis.Results and Reporting:

[0249] Data Interpretation: Initial analysis focused on gene expression profiling and identification of differentially expressed genes, with an emphasis on those associated with pluripotency and differentiation pathways.

[0250] Further Analysis: Data was not analyzed for alternative splicing (altsplice: false) or specific cancer markers (cancer: false) as the focus was on standard gene expression analysis in the context of stem cell biology.

[0251] Data Repository: All processed and raw data files are stored securely.

[0252] The results of this testing are shown in FIG. 7A and FIG. 7B. FIG. 7A shows core pluripotency gene expression (log fold change, with the wildtype as the reference) for P. capensis iPSCs (Pc iPSC) that have been subject to both Stage 2 and Stage 3. Figure 7B shows naive markers upregulated in Pc iPSCs.Example 11: Chromosome Counting Protocol for Procavia capensis iPSCs

[0253] Objective: The objective of this example was to perform chromosome counting on Procavia capensis lines to assess chromosomal integrity and stability.

[0254] Materials: The following materials were used for this Example.• Colchicine stock solution• Media (appropriate for iPSCs)• 70% methanol• 0.075 M KC1 solutionFixative solution (methanol: acetic acid, 3: 1)• Giemsa stain• PBS• 0.25% Trypsin-EDTA• Syringe filter• Centrifuge• CO2 incubator• Micropipettes and tips• Glass slides• Paper towels• -20°C freezer• Bright field microscopeProcedure:

[0255] Preparation: For the colchicine treatment. 5 pL of colchicine stock solution was added to 5 mL of media and the cells were incubated with colchicine for 3 hours. The solutions and slides were also prepared as follows. 70% methanol, 0.075 M KC1 solution, fixative (methanol: acetic acid, 3:1), and Giemsa stain was prepared. Glass slides were placed in cold 70% methanol during the incubation period. After incubation, the slides were removed from methanol, excess methanol was drained by touching the end to a paper towel, and the slides were stored at -20°C until use.

[0256] Cell Harvesting: For hypotonic treatment, the 0.075 M KC1 solution was syringed filed and warmed up to 37°C. The cells were collected with 0.25% Trypsin-EDTA and centrifuged at 900 rpm for 8 minutes. The supernatant was discarded, and the cells were resuspended in the remaining 200-500 pL of liquid by gentle pipetting. 1 mL of 0.075 M KC1 solution was added, mixed gently, then an additional 7 mL of 0.075 M KC1 solution w as added. The solution was mixed by slow inversion of the tube and then incubated at 37°C for 8 minutes.

[0257] For the initial fixation, 1 mL of fixative solution was added to the cell suspension and mixed gently by slow inversion of the tube. The mixture sat at room temperature for 10 minutes and was then centrifuged at 900 rpm for 8 minutes. The supernatant w as discarded, and the cells were resuspended in the remaining 200-500 pL of liquid by flicking the tube.

[0258] 1 mL of fixative solution was added, mixed gently, and an additional 5 mL of fixative solution was added. The solution was mixed by slow inversion of the tube. The resultant mixture sat at room temperature for 10 minutes and was then centrifuged at 900 rpm for 8 minutes.

[0259] For repeated fixation, the fixation steps described above were repeated twice more. After the final centrifugation, the supernatant was discarded, and the cells were resuspended in the remaining 0.3 mL of fixative solution by flicking the tube.

[0260] Slide Preparation: For slide preparation, the slides were removed from the - 20°C freezer. From a height of approximately 6 inches, 20 pL of the cell suspension was dropped onto the slide held at a 45° angle to spread the cells evenly. The slides were allowed to air dry completely and then stained. For the staining, the slides were stained with Giemsa stain for 5-10 minutes, rinsed with distilled water and allowed to air dry.

[0261] The results of this testing are show n in Table 11-1 and FIG. 8. FIG. 8 shows a representative chromosome staining for Procavia capensis iPSC line coOSKMNL.Example 12: Alternate protocol for Rock hyrax iPSCs generation

[0262] Procavia capensis (rock hyrax) fibroblasts were reprogrammed with a chemical reprogramming method that differs from the protocol described in Examples 1 -8 above. Specifically, in this protocol, primary’ P. capensis cells are cultured in a chemical induction medium to generate primed Procavia capensis cells and the induced pluripotent P. capensis stem cells are generated by transfecting the primed P. capensis cells with at leastOCT4ISOX2IKLF4ICMYC and SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes.

[0263] Following the protocol and timeline as described below rock hyrax fibroblasts were reprogrammed into colonies with characteristic stem cell morphology (see FIG. 9A- 9C). As additional validation of key stem cell features, karyotype after reprogramming was also assessed (FIG. 9D). The cells were stained for stem cell markers OCT4 and SOX2 (FIG. 9E), and the expression of core and secondary pluripotency genes (FIG. 9F and FIG. 9G). A GO pathway analysis was also performed. Interestingly, these pciPSCs showed similar expression patterns of secondary7pluripotency genes and were also characterized by NANOG expression. As with transgene-only reprogramming methods, a drop in the expression of KFL4 was observed, but the total expression remains high. In general fibroblasts do not express KI.1'4. but the tested Procavict capensis (Rock hyrax) fibroblasts were not extensively characterized. While Rock hyrax does not have TP53 retrogene expansion as elephants, they do indeed have the same expansion of LIF retrogenes.

[0264] The genomic integrity7and stability of the induced pluripotent Procavia capensis (rock hyrax) stem cells was also evaluated. The tested cell line resulted in a karyotypically normal cell line, and all screened clones were also karyoty pically normal (FIG. 9H). The cells were passaged through passage 25 with no observable tendency towards senescence.

[0265] Procavia capensis (rock hyrax) induced pluripotent stem cells (pciPSCs) were differentiated into putative primordial germ-cell like cells (PGCLCs) using the protocol shown below. When pciPSCs were placed under two PGCLC differentiation protocols (one developed for cows, the other for rhinoceros) for 5 to 10 days, the expression of canonical PGCLC markers TFAP2C, PRDM1, PRDM14, and NANOS3 was observed via RT-qPCR (FIG. 9K).Rock hyrax differentiation protocol

[0266] Procavia capensis (rock hyrax) fibroblasts were used as the starting cell line for reprogramming. These cells are maintained in 30% FBS / 1% antibiotic / antimycotic / !% Nonessential AA / EGM-2 media (Lonza) with Laminin521 coating (5 pg / ml; Gibco A29248). Once the cells became approximately 70% confluent, a chemical cocktail medium was used for partial reprogramming to Procavia capensis pre-iPSCs (pcPRCs) - KO DMEM (Gibco 10829-018) + 10% KOSR (Invitrogen) + 55 pM 2-mercaptoethanol (55 mM (1000X); Gibco 21985023) + 50 ng / ml bFGF (20 pg / ml; heat stable; Life technologies PHG0369) + 0.5 mM VPA (EtOH; Selleckchem S3944) + 5 pM CHIR-99021 (DMSO; Selleckchem S1263) + 2 pM RepSox (DMSO; Selleckchem S7223) + 10 pMTranylcypromine (2-PCPA) HC1 (DMSO; Selleckchem S4246) + 20 pM Forskolin (DMSO; Selleckchem S2449) + 1 pM Ch 55 (DMSO: Tocris 2020) + 5 pM EPZ004777 (DMSO;Selleckchem S7353). The medium was changed every two days until small pcPRC colonies were observed. Once colonies reached sufficient size, they were hand-picked and mechanically passaged 2-3 times. Once 2-3 million pcPRCs were available, they were nucleofected with plasmids encoding genome integrating (via Piggy Bac), inducible, polycistronic transgene expression cassettes. These cassettes contained one of Loxodonta africana OCT4ISOX2IKLF4ICMYC, or L. africana OCT4ISOX2IKLF4ICMYCILIN28A or L. africana OCT4 / SOX2 / KLF42CMYC / LIN28A / NANOG and SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes in Elephas maximus. Subsequently, the cells were recovered, selected with mammalian selection markers hygromycin and puromycin, and induced for 1 month using the same chemical cocktail that was used for partial reprogramming until full induced pluripotent stem cells (pciPSCs) morphology, growth, and molecular signature was observed. Afterwards, the cells w ere transferred onto mouse embryonic feeder cells (MEFs) for long-term maintenance and propagation (ThermoFisher A34966).PGCLC cell differentiation protocol

[0267] PGCLC differentiation was performed in tw o previously described protocols.Briefly, pciPSCs were transitioned to emE8 medium on fibronectin-coated plates (Sigma Fl 141). On day 8. the maintenance medium was replaced with PGCLC induction medium following either a rhinoceros-based protocol (Hayashi et al., Robust induction of primordial germ cells of w hite rhinoceros on the brink of extinction. Science Advances, 8(49): eabp9683, 2022) or a bovine protocol (Shirasawa et al. Efficient derivation of embryonic stem cells and primordial germ cell-like cells in cattle. Journal of Reproduction and Development, 70(2): 82-95, 2024). Both induction media shared a base of Glasgow Minimum Essential Medium (GMEM) (Gibco 11710035) supplemented with 15% (vol / vol) KnockOut Serum Replacement (KOSR) (Gibco 10828028), non-essential amino acids (NEAA) (Gibco 11140050), 0.1 mM 2-mercaptoethanol (Gibco 21985023), leukemia inhibitory factor (LIF) (StemCell 78055.2), stem cell factor (SCF) (StemCell 100-1712), epidermal growth factor (EGF) (StemCell 78006), 10 M Y-27632 (StemCell 72302), 3 M CHIR99021 (Selleckchem SI 263), and 2.5 uM IWR1 (StemCell 72564). The rhinoceros protocol medium was further supplemented with 10 uM forskolin (StemCell 100-0249) and 10 pM bpV (HOpic) (Selleckchem S8651) and utilized RPMI 1640 (Gibco61870036) plus 10% fetal bovine serum (FBS) (Gibco A5670701) in place of GMEM-KSR as the basal medium, whereas the bovine protocol medium included 2 mM GlutaMAX (Gibco 35050061), an additional NEAA supplement, 20 ng / mL basic fibroblast growth factor (bFGF) (StemCell 78003), and 1 / .zM SB590885 (StemCell 100-1656). Cultures were maintained under their respective induction conditions with media changes every 2-3 days. On day 15, all cells were transitioned back to the standard GMEM-based medium (without induction factors). Samples were collected at days 5 and 10 of differentiation for molecular analysis.EMBODIMENTS[026S] The invention also provides the following non-limiting embodiments.

[0269] Embodiment l is a method of generating induced pluripotent Procavia capensis stem cells comprising: (a) culturing primary P. capensis cells transfected with OCT4. SOX 2. KLF4, CMYC, and GLISl , and optionally an antibiotic resistance gene in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOTIL inhibitor, and a TGF-0 inhibitor to generate primed P. capensis cells; (b) generating pre-induced P. capensis cells from the primed P. capensis cells by transfecting the primed P. capensis cells with OCT 4. SO 2. KLF4, CMYC, and NANOG and culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and a TGF-[3 inhibitor; and (c) generating the induced pluripotent P. capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P. capensis cells with OCT4. SOX2, KLF4, CMYC, NANOG, TIN 28 A. and SV40 and culturing the transfected preinduced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT IL inhibitor, and a TGF-[3 inhibitor.

[0270] Embodiment 2 is the method of embodiment 1 further comprising transfecting the primary7P. capensis cells with OCT4, SOX2, KLF4, CMYC, and GLISl and optionally an antibiotic resistance gene.

[0271] Embodiment 3 is the method of embodiment 2, wherein the primary P. capensis cells are transfected with a single stranded RNA replicon comprising OCT4. SOX2, KLT4. CMYC, GLISl, and optionally an antibiotic resistance gene.

[0272] Embodiment 4 is the method of any one of embodiments 1 to 3, wherein the primed P. capensis cells are transfected with a plasmid comprising OCT4. SOX2. KLF4, CMYC, and NANOG.

[0273] Embodiment 5 is the method of any one of embodiments 1 to 4, wherein the preinduced P. capensis cells are transfected with a plasmid comprising OCT4. SOX2, KLF4, CMYC, NANOG, LIN28A, and SV40.

[0274] Embodiment 6 is the method of any one of embodiments 1 to 5. wherein the culture medium in (a), (b), and / or (c) is supplemented with from about 0. 1 to about 1 mM of an HD AC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, from about 2 to about 10 pM of a DOT1L inhibitor, and from about 0.5 to about 4 pM of a TGF- P inhibitor.

[0275] Embodiment 7 is the method of any one of embodiments 1 to 5, wherein generating pre-induced P. capensis cells from the primed P. capensis cells comprises: (1) transfecting the primed P. capensis cells with OCT4, SOX2. KLF4, CMYC. and NANOG: (2) culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF- inhibitor, and a ROCK inhibitor; and (3) culturing the transfected primed P. capensis cells in a culture medium devoid of a ROCK inhibitor supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and an agent to induce transcription of transfected genes.

[0276] Embodiment 8 is the method of embodiment 7, wherein the method comprises culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and a ROCK inhibitor on a feeder layer.

[0277] Embodiment 9 is the method of any one of embodiment 1 to 8, wherein the generating the induced pluripotent Procavia capensis stem cells comprises: (1) transfecting the pre-induced P. capensis cells with OCT4, SOX2, KLF4, CMY C, and NANOG: (2) culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator ofeukary otic adenylyl cyclase, a retinoid, a D0T1L inhibitor, a TGF-P inhibitor, and a ROCK inhibitor; and (3) culturing the transfected pre-induced P. capensis cells in a culture medium devoid of a ROCK inhibitor supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, a D0T1L inhibitor, a TGF-P inhibitor, and an agent to induce transcription of transfected genes.

[0278] Embodiment 10 is the method of embodiment 9. wherein the method comprises culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and a ROCK inhibitor on a feeder layer.

[0279] Embodiment 11 is the method of any one of embodiments 7 to 10, wherein the agent to induce transcription of transfected genes is doxycycline.

[0280] Embodiment 12 is the method of any one of embodiments 7 to 10, wherein the ROCK inhibitor is Y-27632.

[0281] Embodiment 13 is the method of any one of embodiments 1 to 12. wherein the HD AC inhibitor is valproic acid.

[0282] Embodiment 14 is the method of any one of embodiments 1 to 13, wherein the GSK-3 inhibitor is CHIR-99021.

[0283] Embodiment 15 is the method of any one of embodiments 1 to 14. wherein the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1.

[0284] Embodiment 16 is the method of any one of embodiments 1 to 15, wherein the activator of eukaryotic adenylyl cyclase is forskolin.

[0285] Embodiment 17 is the method of any one of embodiments 1 to 16. wherein the retinoid is Ch 55.

[0286] Embodiment 18 is the method of any7one of embodiments 1 to 17, wherein the DOT1L inhibitor is EPZ004777.

[0287] Embodiment 19 is the method of any one of embodiments 1 to 18. wherein the TGF-P inhibitor is RepSox.

[0288] Embodiment 20 is the method of any' one of embodiments 1 to 12, wherein the culture medium in (a), (b), and / or (c) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

[0289] Embodiment 21 is the method of any one of embodiments 1 to 12, wherein the culture medium in (a) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

[0290] Embodiment 22 is the method of any one of embodiments 1 to 12, wherein the culture medium in (b) is supplemented with valproic acid, CHIR-99021, tranylcy promine, forskolin. Ch 55, EPZ004777, and RepSox.

[0291] Embodiment 23 is the method of any one of embodiments 1 to 12. wherein the culture medium in (c) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

[0292] Embodiment 24 is the method of any one of embodiments 1 to 23. wherein the primary P. capensis cells are P. capensis fibroblasts.

[0293] Embodiment 25 is the method of embodiment 24, wherein the P. capensis fibroblasts are female P. capensis fibroblasts.

[0294] Embodiment 26 is the method of any one of embodiments 1 to 25, wherein step (a) comprises culturing primary P. capensis cells transfected with OCT4. SOX2. KI. 4. CMYC, and GLIS1. and an antibiotic resistance gene in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and a TGF-P inhibitor to generate primed P. capensis cells.

[0295] Embodiment 27 is the method of any one of embodiments 1 to 26. further comprising selecting for the induced pluripotent P. capensis stem cells.

[0296] Embodiment 28 is the method of embodiment 27, wherein the selecting comprises culturing the cells with an antibiotic for which the antibiotic resistance gene was transfected in (a).

[0297] Embodiment 29 is the method of embodiment 28, wherein the antibiotic resistance gene is puromycin-resistance and wherein the antibiotic is puromycin.

[0298] Embodiment 30 is an induced pluripotent P. capensis stem cell produced by the method of any one of embodiments 1 to 29.

[0299] Embodiment 31 is an induced pluripotent P. capensis stem cell expressing at least OCT4, 80X2. KLF4, CMYC. NANOG, LIN28A, and SV40.

[0300] Embodiment 32 is an induced pluripotent P. capensis stem cell expressing at least OCT4. SOX2. KI.F4. CMYC, NANOG. I.IN28A. and SV40, wherein the stem cell is generated by transfection with at least OCT4, SOX2. KLF4, CMYC. NANOG. LIN28A, and SV40 and chemical reprogramming.

[0301] Embodiment 33 is a method of differentiating the induced pluripotent P. capensis stem cell of any one of embodiments 30 to 32 into endoderm, mesoderm, or ectoderm.

[0302] Embodiment 34 is a method of forming an embryoid body from the induced pluripotent P. capensis stem cell of any one of embodiments 30 to 32.

[0303] Embodiment 35 is a method of generating induced pluripotent Procavia capensis stem cells comprising: (a) culturing primary P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOTIL inhibitor, and a TGF-0 inhibitor to generate pre-induced P. capensis cells; and (b) generating the induced pluripotent P. capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P. capensis cells with at least OCT4ISOX2IKLF4ICMYC. and SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes and culturing the transfected preinduced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and a TGF-(3 inhibitor.

[0304] Embodiment 36 is the method of embodiment 35 further comprising transfecting the pre-induced P. capensis cells with an antibiotic resistance gene.

[0305] Embodiment 37 is the method of embodiments 35 or 36 comprising transfecting the pre-induced P. capensis cells with OCT4ISOX2IKLF4ICMYCILIN28A and SV40 T- antigen or an shRNA targeting TP53 and / or TP53 retrogenes.

[0306] Embodiment 38 is the method of embodiments 35 or 36 comprising transfecting the pre-induced P. capensis cells with OCT4ISOX2IKLF4ICMYCILIN28AXANOG and (ii) SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes.

[0307] Embodiment 39 is the method of any one of embodiments 35 to 39, wherein the OCT4ISOX2IKLF4ICMYC is from Loxodonta africana.

[0308] Embodiment 40 is the method of embodiment 37, wherein the OCT4ISOX2IKLF4ICMY CILIN28A is from Loxodonta africana.

[0309] Embodiment 41 is the method of embodiment 38, wherein the OCT4 / SOX2 / KLF4 CMYC / LIN28A / NANOG is from Loxodonta africana.

[0310] Embodiment 42 is the method of any one of embodiments 35 to 41, wherein the cells are transfected with an SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes in Elephas maximus.

[0311] Embodiment 43 is the method of any one of embodiments 35 to 42, wherein the pre-induced P. capensis cells are transfected with a single stranded RNA replicon comprising OCT4ISOX2IKLF4ICMYC, and optionally an antibiotic resistance gene.

[0312] Embodiment 44 is the method of any one of embodiments 35 to 42, w herein the pre-induced P. capensis cells are transfected with a plasmid comprising OCT4ISOX2IKLF4ICMYC.

[0313] Embodiment 45 is the method of any one of embodiments 35 to 44, wherein the culture medium in (a) and / or (b) is supplemented with from about 0. 1 to about 1 mM of an HD AC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, from about 2 to about 10 pM of a DOT1L inhibitor, and from about 0.5 to about 4 pM of a TGF-0 inhibitor.

[0314] Embodiment 46 is the method of any one of embodiments 35 to 45, wherein the HD AC inhibitor is valproic acid.

[0315] Embodiment 47 is the method of any one of embodiments 35 to 46, wherein the GSK-3 inhibitor is CHIR-99021.

[0316] Embodiment 48 is the method of any one of embodiments 35 to 47, w herein the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1.

[0317] Embodiment 49 is the method of any one of embodiments 35 to 48, wherein the activator of eukaryotic adenylyl cyclase is forskolin.

[0318] Embodiment 50 is the method of any one of embodiments 35 to 49, wherein the retinoid is Ch 55.

[0319] Embodiment 51 is the method of any one of embodiments 35 to 50, wherein the DOT1L inhibitor is EPZ004777.

[0320] Embodiment 52 is the method of any one of embodiments 35 to 51, wherein the TGF-P inhibitor is RepSox.

[0321] Embodiment 53 is the method of any one of embodiments 35 to 52, wherein the culture medium in (a) and / or (b), is supplemented with valproic acid, CHIR-99021. tranylcypromine, forskolin, Ch 55. EPZ004777, and RepSox.

[0322] Embodiment 54 is the method of any one of embodiments 35 to 53, wherein the culture medium in (a) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

[0323] Embodiment 55 is the method of any one of embodiments 35 to 54, wherein the culture medium in (b) is supplemented with valproic acid. CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

[0324] Embodiment 56 is the method of any one of embodiments 35 to 55, further comprising selecting for the induced pluripotent P. capensis stem cells.

[0325] Embodiment 57 is the method of embodiment 56, wherein the selecting for induced pluripotent P. capensis cells comprises treatment with doxycycline and antibiotic selection and wherein the transfected chemically reprogrammed P. capensis cells are resistant to the antibiotic used for selection.

[0326] Embodiment 58 is the method of embodiment 57, wherein the antibiotic selection comprises treatment with hygromycin or puromycin.

[0327] Embodiment 59 is the method of embodiment 58, wherein the antibiotic selection comprises treatment with hygromycin, wherein the cells are treated every two days with hygromycin, and wherein the treatment lasts ten days.

[0328] Embodiment 60 is the method of embodiment 58, wherein the antibiotic selection comprises daily treatment with puromycin for five days.

[0329] Embodiment 61 is the method of any one of embodiments 35 to 60, w herein the primary7P. capensis cells are P. capensis fibroblasts.

[0330] Embodiment 62 is the method of embodiment 61, wherein the P. capensis fibroblasts are female P. capensis fibroblasts.

[0331] Embodiment 63 is an induced pluripotent P. capensis stem cell produced by the method of any one of claims 35 to 61.

[0332] Embodiment 64 is a method of differentiating the induced pluripotent P. capensis stem cell of claim 63 into endoderm, mesoderm, or ectoderm.

[0333] Embodiment 65 is a method of forming an embryoid body from the induced pluripotent P. capensis stem cell of claim 63.

Claims

CLAIMSWhat is claimed is:

1. A method of generating induced pluripotent Procavict capensis stem cells comprising:(a) culturing primary P. capensis cells in a culture medium supplemented with anHD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and a TGF-0 inhibitor to generate pre-induced P capensis cells; and(b) generating the induced pluripotent P capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P capensis cells with at least OCT4ISOX2IKLF4ICMYC, and SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes and culturing the transfected pre-induced P capensis cells in a culture medium supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, a DOT1L inhibitor, and aTGF-P inhibitor.

2. The method of claim 1 further comprising transfecting the pre-induced P capensis cells with an antibiotic resistance gene.

3. The method of claim 1. wherein the OCT4ISOX2IKLF4ICMYC is from Loxodonta africana.

4. The method of claim 1 comprising transfecting the pre-induced P. capensis cells with OCT4ISOX2IKLF4ICMYCILIN28A and SV40 T-antigen or an shRNA targeting TP53 retrogenes.

5. The method of claim 4, wherein the OCT4ISOX2IKLF4ICMYCILIN28A is from Loxodonta africana.

6. The method of claim 1 comprising transfecting the pre-induced P. capensis cells with OCT4ISOX2IKLF4ICMYCILIN28A / NANOG and SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes.

7. The method of claim 5, wherein the OCT4 / SOX2 / KLF4 / CMYC / LIN28A / NANOG is from Loxodonta africana.

8. The method of claim 1, wherein the cells are transfected with an SV40 T-antigen or an shRNA targeting TP53 and / or TP53 retrogenes in Elephas maximus.

9. The method of claim 1, wherein the pre-induced P capensis cells are transfected with a single stranded RNA replicon comprising OCT4ISOX2IKLF4ICMYC, and optionally an antibiotic resistance gene.

10. The method of claim 1, wherein the primed P. capensis cells are transfected with a plasmid comprising OCT4ISOX2IKLF4ICMYC.

11. The method of claim 1. wherein the culture medium in (a) and / or (b) is supplemented with from about 0. 1 to about 1 mM of an HDAC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, from about 2 to about 10 pM of a DOT1L inhibitor, and from about 0.5 to about 4 pM of a TGF-P inhibitor.

12. The method of claim 1, wherein the HDAC inhibitor is valproic acid.

13. The method of claim 1. wherein the GSK-3 inhibitor is CH1R-99021.

14. The method of claim 1, wherein the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1.

15. The method of claim 1, wherein the activator of eukaryotic adenylyl cyclase is forskolin.

16. The method of claim 1. wherein the retinoid is Ch 55.

17. The method of claim 1, wherein the DOT1L inhibitor is EPZ004777.

18. The method of claim 1, wherein the TGF-P inhibitor is RepSox.

19. The method of claim 1, wherein the culture medium in (a) and / or (b), is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin. Ch 55, EPZ004777, and RepSox.

20. The method of claim 1, wherein the culture medium in (a) is supplemented with valproic acid. CHIR-99021, tranylcypromine, forskolin. Ch 55, EPZ004777, and RepSox.

21. The method of claim 1, wherein the culture medium in (b) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

22. The method of claim 1, further comprising selecting for the induced pluripotent P capensis stem cells.

23. The method of claim 22, wherein the selecting for induced pluripotent P. capensis cells comprises treatment with doxycycline and antibiotic selection and wherein the transfected chemically reprogrammed P capensis cells are resistant to the antibiotic used for selection.

24. The method of claim 23, wherein the antibiotic selection comprises treatment with hygromycin or puromycin.

25. The method of claim 24, wherein the antibiotic selection comprises treatment with hygromycin, wherein the cells are treated every two days with hygromycin. and wherein the treatment lasts ten days.

26. The method of claim 24, wherein the antibiotic selection comprises daily treatment with puromycin for five days.

27. The method of claim 1, wherein the primary P. capensis cells are P. capensis fibroblasts.

28. The method of claim 27, wherein the P. capensis fibroblasts are female P capensis fibroblasts.

29. A method of generating induced pluripotent Procavia capensis stem cells comprising:(a) culturing primary P. capensis cells transfected with OCT4, SOX2. KI.1'4. CMY C, and GLIS1, and optionally an antibiotic resistance gene in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, a DOTIL inhibitor, and a TGF-|3 inhibitor to generate primed P. capensis cells;(b) generating pre-induced P. capensis cells from the primed P. capensis cells by transfecting the primed P capensis cells with OCT4. SOX2, KL F4, C YC. and NANOG and culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, a DOTIL inhibitor, and a TGF-J3 inhibitor; and(c) generating the induced pluripotent P. capensis stem cells from the pre-induced P. capensis cells by transfecting the pre-induced P. capensis cells with OCT4. SOX2, KI 4 4. CMYC, NANOG, LIN28A, and SV40 and culturing the transfected preinduced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, a D0T1L inhibitor, and a TGF-0 inhibitor.

30. The method of claim 29, further comprising transfecting the primary' P. capensis cells with OCT4, SOX2, KLF4, CMYC, and GLIS1 and optionally an antibiotic resistance gene.

31. The method of claim 30, wherein the primary P. capensis cells are transfected with a single stranded RNA replicon comprising OCT4, SOX2, KLF4, CMYC, GLIS1, and optionally an antibiotic resistance gene.

32. The method of claim 29, wherein the primed P. capensis cells are transfected with a plasmid comprising OCT4, SOX2, KLF4, CMYC, and NANOG.

33. The method of claim 29, wherein the pre-induced P. capensis cells are transfected with a plasmid comprising OCT4, SOX2, KLF4, CMYC, NANOG, LIN28A, and SV40.

34. The method of any one of claims 29 to 33, wherein the culture medium in (a), (b), and / or (c) is supplemented with from about 0. 1 to about 1 mM of an HD AC inhibitor, from about 10 to about 25 pM of a GSK-3 inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukaryotic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, from about 2 to about 10 pM of a DOT1L inhibitor, and from about 0.5 to about 4 pM of a TGF-P inhibitor.

35. The method of any one of claims 29 to 33, wherein generating pre-induced P. capensis cells from the primed P. capensis cells comprises:(1) transfecting the primed P. capensis cells with OCT4, SOX2. KLF4, C YC. and NANOG,'(2) culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOTI L inhibitor, a TGF-P inhibitor, and a ROCK inhibitor; and(3) culturing the transfected primed P. capensis cells in a culture medium devoid of a ROCK inhibitor supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT IL inhibitor, a TGF-P inhibitor, and an agent to induce transcription of transfected genes.

36. The method of claim 35, wherein the method comprises culturing the transfected primed P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a D0T1L inhibitor, a TGF-P inhibitor, and a ROCK inhibitor on a feeder layer.

37. The method of any one of claim 29 to 33, wherein the generating the induced pluripotent Procavia capensis stem cells comprises:(1) transfecting the pre-induced P. capensis cells with OCT4, SOX2, KLF4, CMYC, and NANOG’.(2) culturing the transfected pre-induced P capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and a ROCK inhibitor; and(3) culturing the transfected pre-induced P. capensis cells in a culture medium devoid of a ROCK inhibitor supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, a TGF-P inhibitor, and an agent to induce transcription of transfected genes.

38. The method of claim 37, wherein the method comprises culturing the transfected pre-induced P. capensis cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a DOT1L inhibitor, aTGF-P inhibitor, and a ROCK inhibitor on a feeder layer.

39. The method of claim 37, wherein the agent to induce transcription of transfected genes is doxycycline.

40. The method of claim 37, wherein the ROCK inhibitor is Y-27632.

41. The method of claim 29, wherein the HD AC inhibitor is valproic acid.

42. The method of claim 29, wherein the GSK-3 inhibitor is CHIR-99021.

43. The method of claim 29, wherein the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1.

44. The method of claim 29, wherein the activator of eukaryotic adenylyl cyclase is forskolin.

45. The method of claim 29, wherein the retinoid is Ch 55.

46. The method of claim 29, wherein the DOT1L inhibitor is EPZ004777.

47. The method of claim 29, wherein the TGF-P inhibitor is RepSox.

48. The method of claim 29, wherein the culture medium in (a), (b), and / or (c) is supplemented with valproic acid. CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

49. The method of claim 29, wherein the culture medium in (a) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

50. The method of any one of claims 29 to 33, wherein the culture medium in (b) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin, Ch 55, EPZ004777, and RepSox.

51. The method of any one of claims 29 to 33, wherein the culture medium in (c) is supplemented with valproic acid, CHIR-99021, tranylcypromine, forskolin. Ch 55, EPZ004777, and RepSox.

52. The method of any one of claims 29 to 33, wherein the primary P. capensis cells are P. capensis fibroblasts.

53. The method of claim 52, wherein the P. capensis fibroblasts are female P. capensis fibroblasts.

54. The method of any one of claims 29 to 33, wherein step (a) comprises culturing primary P. capensis cells transfected with OCT4, 80X2. KLF4, CMYC, and GLIS1 , and an antibiotic resistance gene in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, a D0T1L inhibitor, and a TGF-P inhibitor to generate primed P. capensis cells.

55. The method of any one of claims 29 to 33, further comprising selecting for the induced pluripotent / 3capensis stem cells.

56. The method of claim 55, wherein the selecting comprises culturing the cells w ith an antibiotic for which the antibiotic resistance gene was transfected in (a).

57. The method of claim 56, wherein the antibiotic resistance gene is puromycin- resistance and wherein the antibiotic is puromycin.

58. An induced pluripotent P capensis stem cell produced by the method of claim 29.

59. An induced pluripotent P. capensis stem cell expressing at least OCT4. SOX2, KI 4 '4. CMYC, NANOG, LIN28A, and SV40.

60. An induced pluripotent P. capensis stem cell expressing at least OCT4, SOX2, KLF4, CMYC, NANOG, LIN28A, and SV40, wherein the stem cell is generated by transfection with at least OCT4. SOX2, KLF4, CMYC, NANOG, LIN28A, and SV40 and chemical reprogramming.

61. A method of differentiating the induced pluripotent P. capensis stem cell of any one of claims 58 to 60 into endoderm, mesoderm, or ectoderm.

62. A method of forming an embryoid body from the induced pluripotent P. capensis stem cell of any one of claims 58 to 60.

63. An induced pluripotent / capensis stem cell produced by the method of any one of claims 1 to 28.

64. A method of differentiating the induced pluripotent P. capensis stem cell of claim 63 into endoderm, mesoderm, or ectoderm.

65. A method of forming an embryoid body from the induced pluripotent P. capensis stem cell of claim 63.

66. The method of claim 1, wherein the method comprises transfecting the pre-induced P. capensis cells with at least OCT4ISOX2IKLF4ICMYC and SV40 T-antigen.

67. The method of claim 1, wherein the method comprises transfecting the pre-induced P. capensis cells with at least OCT4ISOX2IKLF4ICMYC, an shRNA targeting TP53, and TP53 retrogenes.

68. The method of claim 1. wherein the method comprises transfecting the pre-induced P. capensis cells with at least OCT4ISOX2IKLF4ICMYC and an shRNA targeting TP53.

69. The method of claim 1, wherein the method comprises transfecting the pre-induced P. capensis cells with at least OCT4ISOX2IKLF4ICMYC and an shRNA targeting TP53 retrogenes.

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