WO2025106897A1 - Methods and compositions for creating induced pluripotent stem cells - Google Patents

Abstract

The disclosure is directed to methods of generating induced pluripotent Afrotheria species stem cells by chemically reprogramming primary Afrotheria species cells. In certain embodiments, the methods also include selecting for the induced pluripotent elephant stem cells. Further, the cells are transfected with at least (i) C4, (ii) CS, (iii) C6, and (iv) SV40LT.

Description

METHODS AND COMPOSITIONS FOR CREATING INDUCED PLURIPOTENT STEM CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application 63/599,633, filed on November 16, 2023, and U.S. Provisional Application 63/561,013, filed on March 4, 2024, all of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] This disclosure relates to generating induced pluripotent elephant stem cells from primary elephant cells, including methods of generating the cells, media and protocols used for the generation, as well as differentiation of these cells. This disclosure also relates to generating induced pluripotent Afrotheria species stem cells from primary Afrotheria species cells, including methods of generating the cells, media and protocols used for the generation.

REFERENCE TO SEQUENCE LISTING SUBMITTING ELECTRONICALLY

[0003] This application contains a sequence listing, which is submitted electronically. The contents of the electronic sequence listing (069296. 13WO1 Sequence Lisitng.xml; size: 16911 bytes; and creation date of November 15, 2024) is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0004] Isolating pluripotent stem cells from mammals may require invasive procedure including harvesting embryos. As such, it is often more desirable and easier to transform somatic cells into pluripotent stems, i.e., to generate induced pluripotent stem cells (iPSCs). [0005] iPSCs are commonly generated by transfecting somatic cells with genes associated with stem cells such as OCT4. SOX2, NA NOG. KI.1 '4. C-MYC, I.IN28. and GLIS1. iPSCs may also be generated by chemically reprogramming somatic cells. Chemical reprogramming involves culturing the somatic cells in cell culture media containing chemical inducers of pluripotency. Examples of protocols allegedly suitable for chemical reprogramming are disclosed in U.S. Patent No. 9,982,237 and WO 2017/091943.

[0006] According to Kumar et al.. World J Stem Cells. 7(2): 315-328 (2015), using transduction, it has been possible to generate iPSCs using somatic cells from buffalo, cattle, dogs, goats, horses, pigs, rabbits, and sheep. iPSCs from bats have also been generated.

Thus far, the generation of iPSCs for other mammals, such as whales, has proven to be more difficult. In particular, it has not been possible to generate iPSCs from elephant somatic cells. [0007] What is needed is a protocol that allows for the generation of iPSCs from elephant somatic cells or other Afrotheria species somatic cells.

SUMMARY OF THE INVENTION

[0008] This disclosure is directed to induced pluripotent elephant stem cells, methods of generating such cells, and culture media that may be used to generate induced pluripotent stem cells such as induced elephant pluripotent stem cells or induced pluripotent Afrotheria species stem cells. In addition, the disclosure provides for methods of differentiating these induced pluripotent elephant stem cells. Furthermore, this disclosure is directed to methods of generating induced pluripotent Afrotheria species stem cells (such as e.g, cells of a species in the clade Paenungulata (e.g., elephants or rock hyrax)).

[0009] One aspect of the disclosure is directed to methods of generating induced pluripotent elephant stem cells that include: (a) chemically reprogramming elephant cells by culturing primary elephant cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor; and (b) transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent (such as e.g. shRNA) targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent elephant stem cells. In certain embodiments, the methods also include selecting for the induced pluripotent elephant stem cells.

[0010] In some embodiments, the cells are transfected with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT. In other embodiments, the cells are transfected with (i) C4, (ii) C5, (iii) C6. and (iv) an agent (e.g., shRNA) targeting TP53 and/or TP53 retrogenes. In alternate embodiments, the cells are transfected with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and an agent (e.g., shRNA) targeting TP53 and/or TP53 retrogenes.

[0011] In some embodiments, the methods include culturing the elephant cells in a culture medium 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-|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 eukary otic adenylyl cyclase, from about 0.5 to about 3 pM of a retinoid, and from about 2 to about 10 pM of a DOT1L inhibitor. [0012] In other embodiments, the method includes transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium 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-(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, and from about 2 to about 10 pM of a DOT IL inhibitor.

[0013] The transfection may include changing the culture medium every two days and/or introducing a selection marker, such as, e.g., antibiotic resistance.

[0014] In an embodiment of the methods where the methods include selecting, the selecting for induced pluripotent elephant stem cells includes treatment with doxycycline and antibiotic selection and wherein the transfected chemically reprogrammed elephant cells are resistant to the antibiotic used for selection. The antibiotic selection may include treatment with hygromycin or puromycin. In certain 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 other embodiments, the antibiotic selection comprises daily treatment with puromycin for five days.

[0015] The selecting may also include culturing the cells in a medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-0 inhibitor, a monoamine oxidase inhibitor, an activator of eukar otic adenylyl cyclase, a retinoid, and a DOT IL inhibitor. In certain embodiments, 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-|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, and from about 2 to about 10 pM of a DOT1L inhibitor. In some embodiments, the methods include changing the medium every two days and culturing for at least 150 days.

[0016] In certain embodiments, selection is achieved using a feeder layer. In other embodiments, selection is achieved without use of a feeder layer. The step of selecting can also include culturing the cells on a feeder layer in a medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, and a DOT1L inhibitor. In some embodiments, 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 DOT1L inhibitor. In certain embodiments, the feeder layer is an mouse embiyonic fibroblast (MEF) feeder layer.

[0017] In other embodiments, the selecting further includes culturing the cells on an antibiotic resistant feeder layer after culturing the cells on the feeder layer with antibiotic selection. The antibiotic selection can include treatment with hygromycin or puromycin. In one embodiment, the antibiotic selection includes treatment with hygromycin, whereby the cells are treated every two days with hygromycin. and whereby the treatment lasts ten days. In other embodiments, the antibiotic selection includes treatment with puromycin daily for five days.

[0018] Another aspect of the disclosure is directed to methods of generating induced pluripotent Afrotheria species stem cells that include: (a) chemically reprogramming Afrotheria species cells by culturing primary Afrotheria species cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF- inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, and a DOT1L inhibitor; transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4. (ii) C5. (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF- inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT IL inhibitor to generate induced pluripotent Afrotheria species stem cells; and optionally selecting for induced pluripotent Afrotheria species stem cells.

[0019] In one embodiment, the method includes culturing the primary Afrotheria species cells in a culture medium 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.

[0020] In another embodiment, the method includes transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes T in a culture medium 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.

[0021] In certain embodiments, the transfecting includes changing the culture medium every two days. In other embodiments, the transfecting includes introducing a selection marker. In one embodiment, the selection marker is antibiotic resistance. In certain embodiments, the selecting for induced pluripotent Afrotheria species stem cells includes treatment with doxycycline and antibiotic selection and wherein the transfected chemically reprogrammed Afrotheria species cells are resistant to the antibiotic used for selection. In certain embodiments, the antibiotic selection includes treatment with hygromycin or puromycin. In further embodiments, the antibiotic selection includes treatment with hygromycin, whereby the cells are treated every two days with hygromycin, and whereby the treatment lasts ten days. In further embodiments, the antibiotic selection includes treatment with puromycin daily for five days.

[0022] In one embodiment, the selecting for induced pluripotent Afrotheria species stem cells further includes culturing the cells in a 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 DOT IL inhibitor. 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.

[0023] The culture conditions in the methods may vary. In one embodiment, the method includes changing the medium every two days and culturing for at least 150 days. In another embodiment, the selecting further includes culturing the cells on a feeder layer in a medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF- inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor. In yet 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- 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 DOT1L inhibitor. In one embodiment, the feeder layer is an mouse embry onic fibroblast (MEF) feeder layer. [0024] In other embodiments, the selecting further includes culturing the cells on an antibiotic resistant feeder layer after culturing the cells on the feeder layer with antibiotic selection. For example, in certain embodiments, the antibiotic selection includes treatment with hygromycin or puromycin. In another embodiment, 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 one embodiment, the antibiotic selection includes treatment with puromycin daily for five days.

[0025] In one embodiment, the HD AC inhibitor is valproic acid. In another embodiment, the GSK-3 inhibitor is CHIR-99021. In a further embodiment, the TGF- inhibitor is RepSox. In an alternate embodiment, the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1. In a further embodiment, the activator of eukaryotic adenylyl cyclase is forskolin. In an alternate embodiment, the retinoid is Ch 55. In an alternate embodiment, the DOT1L inhibitor is EPZ004777.

[0026] In one embodiment, the method includes culturing Afrotheria species cells in a culture medium supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2- PCPA) HC1, forskolin, Ch 55, and EPZ004777. In another embodiment, the method includes transfecting the chemically reprogrammed Afrotheria species cells with at least C4, C5, C6 and SV40LT in a culture medium supplemented with valproic acid. CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin. Ch 55, and EPZ004777. In an alternate embodiment, the selecting for induced pluripotent Afrotheria species stem cells includes culturing the cells in a medium supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1. Ch 55, and EPZ004777.

[0027] In certain embodiments, the method includes transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT. In other embodiments, the method includes transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6. and (iv) an agent targeting TP53 and/or TP53 retrogenes. In certain embodiments, the agent is shRNA that targets TP53. In other embodiments, the agent is shRNA that targets TP53 retrogenes.

[0028] The methods of generating induced pluripotent Afrotheria species stem cells can be used with any Afrotheria species cell. In one embodiment, the Afrotheria species is a cell of a species in the clade Paenungulata. In another embodiment, the cell is an elephant cell or a rock hyrax cell. In an alternate embodiment, the elephant cell is an Elephas maximus cell. [0029] In the media used in the methods, the HD AC inhibitor can be valproic acid, the GSK-3 inhibitor can be CHIR-99021, the TGF-P inhibitor can be RepSox, the monoamine oxidase inhibitor can be tranylcypromine (2-PCPA) HC1, the activator of eukaryotic adenylyl cyclase can be forskolin, the retinoid can be Ch 55, and/or the DOT IL inhibitor can be EPZ004777. In certain embodiments, a culture medium supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin, Ch 55, and EPZ004777 is used.

[0030] Another aspect of the disclosure is directed to induced pluripotent elephant stem cells. In certain embodiments, the cells are produced by the methods of the disclosure. One embodiment is an induced pluripotent elephant stem cell expressing at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes, wherein the stem cell is generated by chemically reprogramming elephant cells and transfecting the cells to express at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes. Another embodiment is an induced pluripotent elephant stem cell expressing at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes, wherein the stem cell is generated by chemically reprogramming elephant cells. In certain embodiments, the expression of at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes is greater than the expression of at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in elephant cells that are not chemically reprogrammed.

[0031] Yet another aspect of the disclose is directed to cell culture media for reprogramming cells (such as e.g., elephant somatic cells). In one embodiment, the cell culture medium is a 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. In other embodiments, the cell culture medium is a basal 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 DOT IL inhibitor.

[0032] In certain embodiments, the culture medium 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 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. In one embodiment, the HDAC inhibitor is valproic acid. In another embodiment, the GSK-3 inhibitor is CHIR-99021. In yet another embodiment, the TGF-P inhibitor is RepSox. In an alternate embodiment, the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1. In a further embodiment, the activator of eukaryotic adenylyl cyclase is forskolin. In certain embodiments, the retinoid is Ch 55. In other embodiments, the DOT1L inhibitor is EPZ004777. In certain embodiments, the culture medium is supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2- PCPA) HC1, forskolin, Ch 55, and EPZ004777. The disclosure also includes a kit containing the cell culture medium and one or more vectors comprising at least (i) C4. (ii) C5. (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes.

[0033] Other embodiments of the disclosure are directed to uses of the induced pluripotent elephant stem cells. One aspect of the disclosure is directed to methods of differentiating the induced pluripotent elephant cell into endoderm, mesoderm, or ectoderm. Another aspect of the disclosure is directed to methods of forming an embryoid body from the induced pluripotent elephant cell.

[0034] In certain embodiments, the induced pluripotent elephant stem cells are generated from cells obtained from Elephas maximus. In other embodiments, the induced pluripotent elephant stem cells are generated using a protocol described in the examples.

[0035] Other features and advantages of the invention will be apparent from the detailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0001] 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. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary’ fee.

[0036] FIG. 1 shows an image of ClEmMEn wild-type morphology.

[0037] FIG. 2 shows an image of cell morphology' after the cells passaged 2~3 times via selection of colonies (EVOS 10X).

[0038] FIG. 3A (Brightfield merged with green fluorescence channel) and FIG. 3B (Brightfield only) show images of the cells at 48 hours after transfection with C4 along with GFP-expressing shRNA plasmid targeting TP53 retrogene.

[0039] FIG. 4 shows an image of cells transfected with C4 TP53shRNA2 after hygromycin selection on Laminin521 coated plates (EVOS 10X). [0040] FIG. 5 shows that an image of cells demonstrating that C4shRNA2 has better iPSC like morphology on a MEF feeder layer (EVOS 10X).

[0041] FIG. 6 shows an image of cells transfected with C4iSV40 after re-selection using 0.5 pg/ml puromycin (EVOS 10X).

[0042] FIG. 7 shows the results of the embryoid body formation assay using Aggrewell for the positive control (hiPSC) and the negative control (only chemical B cocktail).

[0043] FIG. 8 shows the results of the embryoid body formation assay using Aggrewell for induced pluripotent elephant stem cells.

[0044] FIG. 9 shows the markers associated with germ layer specification and pluripotency network.

[0045] FIG. 10A shows the results of testing for PAX mRNA after the embryoid body formation assay for the negative control and induced pluripotent elephant stem cells.

[0046] FIG. 10B shows the results of testing for PAX mRNA after the embryoid body formation assay for the negative control and induced pluripotent elephant stem cells.

[0047] FIG. 11 A-J show various culture conditions for reprogramming attempts. A vast majority⁷ of reprogramming attempts resulted in no morphological change, cell death, or cell senescence. Some protocols yielded cells that were partially reprogrammed. FIG. 11 A-J show the reprogramming protocols for: TFome screens with libraries of individual TFs screened in combination (FIG. 1 1A); C2 MET cell line (FIG. 1 IB); KEmFEn TF-based polycistronic reprogramming cell line (FIG. 11C); TEmFEp TSC cell line (FIG. 1 ID); C3- loxC4-TP53shRNA4 cells (FIG. HE); Lenti-viral attempts (FIG. 1 IF); Sendai virus attempts (FIG. 11G); pre-iPSCs with either chemical cocktail A, B, or C (0.5 mM VPA. lOpM CHIR, lOpM Repsox, 5pM Tranly (Tranylcypromine (2-PCPA) HC1), lOpM Forskolin) (FIG. 11H); emiPSCs (FIG. I ll) and emiPSCs with stacked transgene expression (FIG. 11 J).

[0048] FIG. 12A-J show cell morphologies for partial reprogramming. Partially reprogrammed cells showed morphological features that were reminiscent of intermediate reprogramming morphologies. All cells shown at I OX magnification, scale bar = 200pM. Small colonies created during TFome screening (FIG. 12A). TEmFEp TSC cells (FIG. 12B). cKEmFEn piPSC cells (FIG. 12C). C2 MET cells (FIG. 12 D). C3-loxC4-TP53shRNA4 cells (FIG. 12E). Chemical cocktail A cells (FIG. 12F). Chemical cocktail B cells textbfh (FIG. 12G); chemical cocktail C cells (FIG. 12H); Lenti-viral reprogramming attempt cell clusters (FIG. 121); Sendai virus reprogramming attempt cell clusters (FIG. 12 J).

[0049] FIG. 13A-D show MA plots for partial reprogramming. All plots show significantly up-regulated genes, significantly down-regulated genes, and non-significant change. FIG. 13 A show a MA plot for C2 MET. FIG. 13B shows a MA plot for KEmFEn piPSC. FIG. 13C show a MA plot for TEmFEp TSC. FIG. 13D show a MA plot for C3-loxc4- TP53shRNA4.

[0050] FIG. 14A-D show MA plots for partial reprogramming. All plots show significantly up-regulated genes, significantly down-regulated genes, and non-significant change. FIG. 14A shows a MA plot for ClEmMEn pre-iPSC. FIG. 14B shows a MA plot for ClEmMEn pre-iPSC A. FIG. 14C shows a MA plot for ClEmMEn pre-iPSC B. FIG. 14D shows a MA plot for ClEmMEn pre-iPSC C.

[0051] FIG. 15 shows principal component analysis of partial and full reprogramming. PC A analysis compares all primary cell populations, partial reprogramming populations, and full reprogramming populations for the first two principal components (PCI, PC2).

[0052] FIG. 16A and 16B show shRNAs targeting Elephas maximus TP53 and its retrogene expansions. A set of four shRNAs were designed to target TP53 and/or its 29 corresponding retrogenes (RTGs). The shRNAs against the full length TP53 mRNA sequence (FIG. 16A) and 19 TP53 RTGs (FIG. 16B) including TP53-RTG-4 and TP53-RTG-28 mRNA sequences and measured fold change relative to an empty shRNA vector for 24, 48, and 72 hours.

[0053] FIG. 17A-C show TFome screen TF enrichment. TFome screening on emECs resulted in cells that stained positive for a mammalian TRA-1-60 or SSEA1 cell surface cell marker. These cells were FACS-sorted and sequenced to determine enrichment of TFs used to produce these cells. Relative TF enrichment is shown for a set of TFs identified from human iPSCs that stained for SSEA1 (FIG. 17 A) and TRA-1-60 and TF identified from human ESCs for SSEA1 (FIG. 17B).

[0054] FIG. 18A-I show the derivation of/'/ maximus induced pluripotent stem cells. FIG. 18A is an illustration of the reprogramming strategy involved. emEC - E. maximus endothelial or epithelial cells; emPRC - E. maximus partially reprogrammed cells; eleOSKM/NL - loxodonta africana POU5F1, SOX2, KL 4. CMYC, I.IN28A. ax NANOG transgenes; shRNAzpss - shRNAs targeting TP53 and its retrogenes. FIG. 18B-FA11 scale bars = 200pM. magnification = 10X. FIG. 18B shows a brightfield (BF) image of E. maximus primary endothelial cells (emEC). FIG. 18C shows a BF image of partially reprogrammed (pre-iPSC) cells (emPRC). FIG. 18D shows a BF image of E. maximus induced-pluripotent stem cells. (emiSPCs). FIG. 18E shows immuno-fluorescent detection of NANOG I SOX2 (AF488) / HOECHST separately and merged. FIG. 18F shows immuno- fluorescent detection of OCT41 SOX2 (AF488) / HOECHST separately and merged. FIG. 18G shows a MA plot of RNA-seq data illustrating the transcriptional differences between elephant endothelial cells (emEC) and pluripotent stem cells (emiPSC). Key canonoical pluripotency genes are labeled. FIG. 18H shows a PCA plot comparing emECs (ClEmMEn WT), emPCRs (ClEmMEn pre-iPSC), and emiPSCs (C l-loxC4/5/6-SV40 and C l-loxC4- TP53shRNA2). FIG. 181 shows RNA-seq and ATAC-seq signals of OCT4 (POU5F1) for emECs, emPRCs, and emiPSCs. Show n are peak calls of one representative sample.

[0055] FIG. 19A-E show characteristics of E. maximus induced pluripotent stem cells. FIG. 19A show's karyotyping staining of emiPSC nuclei show 56/56 chromosomes. FIG. 19B shows doubling time for emiPSC cell lines and hiPSCs as originally derived in 2007. Statistical tests show all emiPSCs are not statistically significantly slow er grow ing that originally derived hiPSCs. FIG. 19C shows expression of the canonical primed pluripotency markers THYl. FIG. 19D shows expression of the canonical naive pluripotency marker TBX3. All emiPSC lines significantly higher expression than WT controls (p < 0.01). FIG. 19E shows a MA plot showing a large set of canonical naive and primed pluripotency markers for Cl-loxC4-SV40. A vast majority of naive markers are up-regulated, and most primed markers are down-regulated.

[0056] FIG. 20A-E show the differentiation potential of E. maximus induced pluripotent stem cells. In FIG. 20 A, all scale bars are 200 pm, magnification = 10X. FIG. 20A shows immunofluorescence (IF) microscopy images of embryoid bodies (EBs) formed by emiPSC line Cl-loxC5-SV40 co-stained with antibodies detecting the expression of lineage-specific markers PAX6 (ectoderm) and GATA4 (mesoderm). FIG. 20B shows IF microscopy images of EBs formed by emiPSC line Cl-loxC5-SV40 stained with an antibody detecting the expression of lineage-specific markers FOXA2 (endoderm). FIG. 20C show s a representation of canonical germ-layer differentiation marker genes in EBs formed from each emiPSC line. emPRCs, and emECs. (LEFT) Endoderm markers, (RIGHT) ectoderm markers, and (BOTTOM) mesoderm markers. FIG. 20D shows expression of early differentiation markers PAX6 (ectoderm), GATA4 (mesoderm), and PRDM 1 (endoderm) following trilineage differentiation in emiPSC line Cl-loxC5-SV40. Statistical t tests show ** = p < 0.01 and * = p < 0.05. FIG. 20E shows microscopic images of hematoxylin-eosin-stained sections of tumor tissue after injection of emiPSCs (to form teratoma) into the upper hindleg of immuno-compromised mice. Teratomas formed from each emiPSC line resulted in different cell types being developed (marked by color in sub-panels).

[0057] FIG. 21A and 21B show distinct characteristics of elephant induced pluripotent stem cells. FIG. 21 A shows a comparison of canonical pluripotency gene expression in emiPSCs compared to iPSCs from other mammals. FIG. 21B show's the results of a principal component analysis of emiPSCs compared to iPSCs from other mammals. Data points clustered by approximate rate of developmental clock as described in Lazaro, J, et al. , Stem Cells, 2023. [0058] FIG. 22 A-C show teratomas. Tumors were extracted from hind legs of immunocompromised mice post-emiPSC injection. All tumors were surgically removed after 5.5 weeks and measured as pictured. FIG. 22A shows a tumor from Cl-loxC4TP53shRNA2. FIG. 22B shows a tumor from Cl-loxC5-SV40. FIG. 22C shows a tumor from Cl-loxC6- SV40.

[0059] FIG. 23A-F show embryoid bodies for cell lines stained for early differentiation markers. Immunofluorescence (IF) microscopy images of embry oid bodies (EBs) formed by emiPSC lines. All cells shown at 10X magnification, scale bar = 200 pm. FIG. 23A shows Cl-loxC4-SV40 stained for PAX6 (ectoderm) and GATA4 (mesoderm). FIG. 23B shows Cl-loxC4-SV40 stained for FOXA2 (endoderm). FIG. 23C shows C l-loxC6-SV40 stained for PAX6 (ectoderm) and GATA4 (mesoderm). FIG. 23D shows Cl-loxC6-SV40 stained for FOXA2 (endoderm). FIG. 23E shows Cl-loxC4-TP53shRNA2 stained for PAX6 (ectoderm) and GATA4 (mesoderm). FIG. 23F shows Cl-loxC4-TP53shRNA2 stained for FOXA2 (endoderm).

[0060] FIG. 24A-D show MA plots for emiPSCs. MA plots highlighting core regulatory genes that control cell grow th rate that are often strongly downregulated in iPSCs from other species. FIG. 24A shows an MA plot for Cl-loxC4-SV40. FIG. 24B shows an MA plot for Cl-loxC5-SV40. FIG. 24C shows an MA plot for Cl-loxC6-SV40. FIG. 24D shows an MA plot for Cl-loxC4-TP53shRNA2.

[0061] FIG. 25A and FIG. 25B show emiPSC lines reinforced with additional transgene expression. emiPSC lines were reinforced with additional copies of loxC4 (OSKM) and NANOG PiggyBac overexpression cassettes. Cells were then measured for relative expression changes via RT-qPCR for primer pairs flanking exon-intron regions of SOX2 (FIG. 25 A) and NANOG (FIG. 25B) to ensure that measured expression increases were endogenous and not simply transgene expression.

[0062] FIG. 26A-E shows the results of TFome TF-enrichment screening of emECs (KErnFEn and TEmFEp) and emMSC (E. maximus mesenchymal stromal cells - SEmFMSC). FIG. 26 A show's colonies with cell death. FIG. 26B show s the appearance of senescent colonies. FIG. 26C-E show' relative enrichment of TFs identified from human iPSCs that stained for SSEA1 (FIG. 26C) and TRA-1-60 (FIG. 26D) and TFs identified from human ESCs for SSEA I . (FIG. 26E). TFome screening was performed on emECs (KErnFEn and TEmFEp) and emMSC (E. maximus mesenchymal stromal cells - SEmFMSC) on a small set of simple reprogramming conditions. Cells w ere cultured with Essential 8 (E8) medium and mouse embryonic fibroblast (MEF) feeder on gelatin-coated plate or E8 or mTeSRl medium on Matrigel-coated plates. These attempts all resulted in either cell death (FIG. 26 A) or heterogenous appearance of senescent colonies (FIG. 26 B). These cell populations were FACS-sorted and sequenced to determine enrichment of TFs used to produce these cells. All cells shown at 10X magnification, scale bar = 200 //M. Relative TF enrichment is shown for a set of TFs identified from human iPSCs that stained for SSEA1 (FIG. 26C) and TRA-1-60 (FIG. 26D) and TFs identified from human ESCs for SSEA1 (FIG. 26E). Subsequent rounds of screening with these refined sets of TFs did not yield improvement.

[0063] FIG. 27 shows a summaiy of viral reprogramming attempt conditions. emECs were treated with viral transductions with both Sendai virus and Lenti virus under standard reprogramming conditions defined by these methods. Different MOIs and standard plating conditions were tested.

[0064] FIG. 28A and FIG. 28B show Morphological progression of viral reprogramming attempts. FIG. 28A shows Lenti-viral treatment reprogramming timeline and morphological results. FIG. 28B shows Sendai virus treatment reprogramming timeline and morphological results. In both cases, senescent colonies emerged that died after attempted transfer. All images taken at scale bar = 200pM.

[0065] FIG. 29 A to 29F show culture conditions for TF-based, chemical, and hybrid reprogramming attempts. A vast majority of reprogramming attempts resulted in no morphological change, cell death, or cell senescence. Some protocols yielded cells that were partially reprogrammed. FIG. 29A shows the reprogramming protocol for C2 MET cell line. FIG. 29B shows the reprogramming protocol for TEmFEp XEN cell line. FIG. 29C shows KEmFEn TF-based polycistronic reprogramming cell line. FIG. 26D shows the reprogramming protocol for C3-loxC4-TP53shRNA4 cells. FIG. 29E shows pre-iPSCs with either chemical cocktail A, B, or C; and finally, FIG. 29F shows the successful emiPSC protocol.

[0066] FIG. 30A and 30B show shRNAs targeting E. maximus TP53 and its retrogene expansions. A set of four shRNAs were designed to target TP53 and/or its twenty -nine corresponding retrogenes (RTGs). The shRNAs was tested against the full length TP53 mRNA sequence (FIG. 30A) and 19 TP 53 RTGs (FIG. 30B) including TP53-RTG-4 and TP53-RTG-28 mRNA sequences and measured fold change relative to an empty shRNA vector for 24, 48. and 72 hours.

[0067] FIG. 31A to 31F show the cell morphologies for partial reprogramming for: a. C2 MET cells (FIG. 31A); TEmFEp XEN cells (FIG. 31. B); KEmFEn piPSC cells (FIG. 31C); C3-loxC4-TP53shRNA4 cells (FIG. 3 ID); chemical cocktail A cells (FIG. 3 IE); chemical cocktail B cells (FIG. 3 IF); Chemical cocktail C cells (FIG. 31G); Partially reprogrammed cells showed morphological features that were reminiscent of intermediate reprogramming morphologies. All images shown at scale bar = 200pM.

[0068] FIG. 32A to FIG. 32D show MA plots for partial reprogramming for C2 MET (FIG. 32A), KEmFEn piPSC (FIG. 32B), TEmFEp XEN (FIG. 32C), and C3-loxc4-TP53shRNA4 (FIG. 32D). All plots show significantly up-regulated genes, significantly down-regulated genes, and non-significant change.

[0069] FIG. 33A to FIG. 33D show MA plots for partial reprogramming for ClEmMEn pre-iPSC (FIG. 33A), ClEmMEn pre-iPSC A (FIG. 33B), ClEmMEn pre-iPSC B (FIG. 33C). and ClEmMEn pre-iPSC C (FIG. 33D). All plots show significantly up-regulated genes, significantly down-regulated genes, and non-significant change.

[0070] FIG. 34 shows principal component analysis of partial and full reprogramming. PCA analysis compares all primary⁷ cell populations, partial reprogramming populations, and full reprogramming populations for the first two principal components (PC 1 , PC2). Successful reprogramming attempts noted in bold.

[0071] FIG. 35A and FIG. 35B show the results of NANOG primary structure phylogenetic analysis. NANOG has unique evolutionary behavior compared to other pluripotency-related genes. FIG. 35A shows base-specific conservation scores for NANOG, KLF4, OCT4, and SOX2. NANOG overall shows lower conservation across the length of the sequence, and it has more sequence with species- and clade-specific indels compared to KI. I A OCT4, and SOX2. FIG. 35B shows UCSC genome browser tracks showing the position of NANOG, KLF4, OCT4, and SOX2 in the hg38 human genome alongside annotated repeat elements, or transposable elements. NANOG has substantially more repeat regions in its exonic sequence compared to KLF4. OCT4, and SOX2, which may explain its rapid evolution, particularly of large indels across species.

[0072] FIG. 36A-36H show derivation of E. maximus induced pluripotent stem cells. FIG. 36A show s an illustration of the tw o reprogramming strategies used for reprogrammed elephant cells. Transgene-only reprogramming does not require and intermediate pre-iPSC step. emEC - E. maximus endothelial or epithelial cells; emPRC - E. maximus partially reprogrammed cells; eleOSKM/NL - Loxodonta africana or E. maximus POU5F1, SOX2, KLF4, CMYC, LIN28A, and NANOG transgenes; shRN Az/wa - shRNAs targeting TP53 and its retrogenes; SV40 - SV40 T-antigen; eleHRASG12V - a mutant E. maximus HRAS gene. b.-g. All scale bars = 200//M. FIG. 36B shows brightfield (BF) image of . maximus primary endothelial cells (emEC). FIG. 36c shows BF image of partially reprogrammed (pre-iPSC) cells (emPRC). FIG. 36D shows BF image of E. maximus chemically reprogrammed induced-pluripotent stem cells (emiSPCs). FIG. 36E shows (TOP) Immuno-fluorescent detection of NANOG I SOX2 (AF488) / HOECHST separately and merged. FIG. 36E shows immuno-fluorescent detection of OCT4 I SOX2 (AF488) / DAPI separately and merged for chemically reprogrammed. FIG. 36F shows a PCA plot comparing emECs (ClEmMEn WT), emPCRs (ClEmMEn pre-iPSC), and emiPSCs (Cl-loxC4/5/6-SV40 and Cl-loxC4- TP53shRNA2). FIG. 36G shows a MA plot of RNA-seq data illustrating the transcriptional differences between elephant endothelial cells (emEC) and pluripotent stem cells (emiPSC) for chemically reprogrammed iPSCs Key canonical pluripotency genes are labeled. FIG. 36H shows RNA-seq and ATAC-seq signals of OCT4 (POU5F1) emECs and emiPSCs. Read tracks are representative of two replicates.

[0073] FIG. 37A-37D show immunofluorescent staining controls for pluripotency markers. All scale bars = 200//M. NANOG and OCT4 antibodies are custom elephant amino-acid sequence optimized, SOX2 antibody is commercial due to extremely high sequence overlap. FIG. 37A shows immuno-fluorescent detection of NANOG I SOX2 (AF488) I HOECHST separately and merged in ClEmMEn WT cells. FIG. 37B shows immuno-fluorescent detection of OCT41 SOX2 (AF488) / HOECHST separately and merged in C lEmMEn WT cells. FIG. 37C shows immuno-fluorescent detection of NANOG I SOX2 (AF488) / HOECHST separately and merged in human iPSC cells. FIG. 37D shows immuno- fluorescent detection of OCT4 / SOX2 (AF488) / HOECHST separately and merged in human iPSC cells.

[0074] FIG. 38A-38D show the results of NANOG protein structure phylogenetic analysis. NANOG has unique evolutionary behavior compared to other pluripotency-related genes. FIG. 38 A shows a phylogenetic tree with branch lengths representing NANOG evolutionary⁷ rate along a fixed consensus mammalian phylogeny. Bars show each species' NANOG sequence as it appears in the alignment. The alignment visualization highlights the nonconserved region in the center of the NANOG protein alignment and the large species-specific insertion only found in the Asian elephant. FIG. 38B shows alpha-fold overlay of structure predictions of protein structure for seven disparately related species: human, bat, armadillo, whale, hyrax, elephant (African), elephant (Asian). FIG. 38C shows overlayed fold predictions at the DNA binding domain for the same seven species. FIG. 38D shows E. maximus NANOG has an additional exon not observed in any other species analyzed. E. maximus NANOG is overlayed with Loxodonta africana NANOG and the extra exon region is also shown.

[0075] FIG. 39 shows core endogenous pluripotency marker comparison between emiPSC derivation methods. Reprogramming with either two-step chemical methods or one-step transgene-only methods yield different profiles of endogenous pluripotency gene expression. Chemical cocktail methods result in higher endogenous KLF4 gene expression, while transgene-only methods result in far higher endogenous expression of OCT4 and LIN28A. [0076] FIG. 40A-C show emiPSC lines reinforced with additional transgene expression. FIG. 40A shows the protocol to reinforce the emiPSC lines. FIG. 40B shows endogenous NANOG expression. FIG. 40C shows endogenous SOX2 expression. emiPSC lines were reinforced with additional copies of loxC4 (OSKM) and NANOG PiggyBac overexpression cassettes via a serial transfection. Cells were then measured for relative expression changes after 7+ days via RT-qPCR for primer pairs flanking exon-intron regions of NANOG (FIG. 40B) and c. the coding sequence and 3’ UTR of SOX2 (FIG. 40C) to ensure that measured expression increases were endogenous and not simply transgene expression.

[0077] FIG 41A-D show MA plots for Cl-loxC4-SV40 cell line markers after withdrawal of DOX for 10 days for core pluripotency makers (FIG. 41 A), additional pluripotency markers (FIG. 41B), canonical naive and primed markers (FIG. 41C), and core cell-cycle regulatory markers (FIG. 4 ID). All plots show significantly up-regulated genes, significantly down-regulated genes, and non-significant change.

[0078] FIG. 42A-42C show the results of whole genome sequencing of emiPSCs. All emiPSC lines described were sent for WGS with Illumina DNA sequencing. Results representative of sequencing results for all lines. FIG. 42A shows a karyogram depicting loci of potential mutation across elephant genome. FIG. 42B shows a count of potential mutations in each chromosome. FIG. 42C shows the size of observed potential indels.

[0079] FIG. 43A-C shows characteristics of A. maximus induced pluripotent stem cells. FIG. 43A shows the doubling time for emiPSC cell lines and hiPSCs as originally derived in 2007. Statistical tests show that one of the emiPSC lines is significantly (p < 0.01) faster grow ing that originally derived hiPSCs, all other growth data not significant. FIG. 43B is a MA plot depicting a large set of canonical naive and primed pluripotency Cl-loxC6- TP53shRNA2. FIG. 43C shows the RNA-seq and ATAC-seq signals of NANOG, KLF4, THY J, and SOX2.

[0080] FIG. 44 shows Oxford Nanopore long-read RNA sequencing with RNA004 kit technology. Single-molecule sequencing data reveals thousands of novel E. maximus transcripts, gffcompare assigns each assembled transcript a class code, which represents the degree and type of novelty when compared to a reference transcriptome GCF_024166365.l_mEleMaxl_primary_haplotype_genomic.gtf. The class code definitions are included in the third column. Each transcript was assembled from a minimum of one sample. The number of transcripts discovered matching each classification code is shown in the second column. The ‘S’ classification is not included due to the low number of assembled transcripts with this code. The number in the second column denotes the number of transcripts assembled that are contained in one of four samples C2-emC4-SV40 (C2C5, two replicates), C2-emC6-SV40 (C2C17, two replicates).

[0081] FIG. 45A and 45B long-read NANOG alignments. Long-read NANOG alignments from C2-emC4-SV40 (C2C5 r2) and C2-emC6-SV40 (C2C17 rl) shown in the Integrative Genomics Viewer. Each RNA molecule’s associated polyA tail length is written to the left in green. FIG. 45A shows reads aligning to the first four predicted exons of NANOG. FIG. 45B shows reads aligning to the fifth predicted exon of NANOG.

[0082] FIG. 46 shows principal component analysis short-read and long-read sequencing for emiPSC lines. PCA analysis compares primary cell populations, partial reprogramming populations, and full reprogramming populations for PCI and PC3 for both short-read and long-read RNA sequencing.

[0083] FIG. 47A-D show upregulation of early differentiation markers in EBs over time: PAX6 (Ectoderm) (FIG. 47 A); GATA4 (Mesoderm) (FIG. 47B); FOXA2 (Endoderm) (FIG. 47C); CDX2 (Endoderm) (FIG. 47D). Expression of early differentiation markers were measured over the course of EB formation for three emiPSC cell lines. Data shown for RT- qPCR fold change compared to ClEmMEn WT cells relative to GADPH expression.

[0084] FIG. 48A-48D show the differentiation potential of E. maximus induced pluripotent stem cells. All scale bars = 200/zM. FIG. 48A shows immunofluorescence (IF) microscopy images of stained embryoid bodies (EBs) aggregates using the AggreWell method on Day 3 formed by emiPSC line Cl-loxC6-SV40 co-stained with antibodies detecting the expression of lineage-specific markers NOG (ectoderm), CDX2 (mesoderm), and CTNNB1 (endoderm). FIG. 48B shows a graphical representation of canonical germ-layer differentiation marker genes in EBs formation chemically reprogrammed emiPSCs at Day 3 of EB formation. (LEFT) Ectoderm markers, (MIDDLE) endoderm markers, and (RIGHT) mesoderm markers at Log2 fold-change relative to starting emiPSC line for two biological replicates. FIG. 48C shows a graphical representation of TBX6 anA HANDl (mesoderm). CHRD and IRF6 (ectoderm), and CTNNB1 and FOXA1 (endoderm) following tri -lineage differentiation in emiPSC lines using StemDiff reagents for Cl-loxC6-SV40. Statistical double-sided Student t-tests show ** = p < 0.01 and * = p < 0.05. FPKM: Fragments Per Kilobase of transcript per Million mapped reads normalized read count based on gene length and the total number of mapped reads. FIG. 48D shows IF microscopy images of emiPSC cell lines C l-loxC6-SV40 (TOP) and Cl-loxC6-shRNA2 (BOTTOM) differentiated for 4 days via E. maximus NGN2 over-expression, stained with primary antibody SYNE [0085] FIG. 49 shows expression of early differentiation makers for chemically reprogrammed cells at Day 1 of EB formation using the AggreWell EB formation method. [0086] FIG. 50A and 50B show immunofluorescent staining controls for early differentiation markers. FIG. 50A shows immunofluorescence (IF) microscopy images of embryoid bodies (EBs) formed by emiPSC line Cl-loxC6-SV40 stained with only secondary antibodies anti-rabbit Texas-Red and anti-mouse AlexaFluor 488. FIG. 50B shows IF microscopy images of 2D-EBs after plating formed by emiPSC line C2-emC6-SV40 stained with only secondary antibodies anti-rabbit Texas-Red and anti-mouse AlexaFluor 488. All scale bars = 200//M.

[0087] FIG. 51 shows expression of early differentiation makers for chemically reprogrammed cells via tri-lineage differentiation.

[0088] FIG. 52A and 52B shows immunofluorescent staining controls for neurodifferentiation assays. FIG. 52A shows immunofluorescence (IF) microscopy images of neuro-differentiation of emiPSC line Cl-loxC6-SV40 via NGN2-ov erexpression stained with only secondary antibodies anti-rabbit Texas-Red and anti-mouse AlexaFluor 488. FIG. 52B shows IF microscopy images of an hiPSC cell line differentiated for 4 days via A. maximus NGN2 overexpression, stained with primary antibody TAY -anti-mouse, with secondary antibody anti-mouse AlexaFluor 488, and DAPI. All scale bars = 200//M

[0089] FIG. 53A-53G show distinct characteristics of elephant induced pluripotent stem cells. RNA-seq data of multiple species is compared. FIG. 53A show a principal component analysis of emiPSCs compared to ESCs of many mammals from multiple species. fESC: formative-like ESC, 2C-ESC: 2C-like ESC. FIG. 53B shows UMAP clustering of emiPSCs and iPSCs from naked mole rat (NMR), human, and mouse. FIG. 53C shows up-regulated differentially expressed genes (DEGs) between cancer-resistant iPSCs. FIG. 53D shows down-regulated differentially expressed genes (DEGs) betw een cancer-resistant iPSCs. FIG. 53E shows the results of a GO analysis of pathways different between cancer-resistant and non-cancer-resistant iPSC lines. FIG. 53F shows the results of an analysis o ARFRAS genes across species that have interactions with TP53 and other core regulatory genes. Specific genes of interest shown below⁷ in same order as above. FIG. 53F shows an analysis of aging- related genes across species.

[0090] FIG. 54A-F show potential teratoma formation. Potential teratomas formed from emiPSC hind-leg injection into immunocompromised mice hind-leg regions. Tumors extracted (TOP), H&E cross-sections (MIDDLE), and professional double-blind histological analysis of cell types of example cross-sections (BOTTOM). FIG. 54A-C show⁷ images of tumors surgically removed after 5.5 weeks pictured for Cl-loxC4-TP53shRNA2 (FIG. 54A), Cl-loxC5-SV40 (FIG. 54B), and Cl-loxC6-SV40 (FIG. 54C). FIG. 54D and FIG. 54E show tumor H&E cross-sections 8 weeks post-injection for cell lines: Cl pre-iPSC (FIG. 54D), and Cl-loxC6-SV40 (FIG. 54E). FIG. 54F shows tumor H&E cross-sections 4 weeks postinjection for a tumor generated from edited Mus musculus ESC cell line (mmDKO24). Pervasive necrosis in this sample was noted.

[0091] FIG. 55A and B show MA plots for emiPSC LIF and TP53 retrogenes genes. FIG. 55A shows LIF retrogene expression for Cl-loxC6-TP53shRNA2. FIG. 55B shows TP53 retrogene expression for Cl-loxC6-TP53shRNA2.

[0092] FIG. 56A and FIG. 56B show MA plots for emiPSC core regulatory’ genes for Cl- loxC6-SV40 andCl-loxC6-TP53shRNA2, respectively.

[0093] FIG. 57A-57G show characteristics of Procavia capensis (Rock hyrax) induced pluripotent stem cells. FIG. 57A-57C show phase contrast images of Procavia capensis fibroblasts (FIG. 57 A). P. capensis pre-iPSCs (pcPRCs) (FIG. 57B), and /*, capensis iPSCs (pciPSCs) (FIG. 57C). FIG. 57D shows the results of a karyotype analysis of pciPSCs having Pi Tty- four chromosomes. FIG. 57E shows immunofluorescent imaging for OCT4 and SOX2 in pciPSCs. FIG. 57F and FIG. 57G 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. 57F) as well as additional primed and naive pluripotency markers (FIG. 57G). All scale bars = 200pM.

DETAILED DESCRIPTION

[0094] This disclosure is based on the discovery that induced pluripotent elephant stem cells can be generated using a unique step-wise protocol as described herein. Specifically, the disclosure is based on the discovery that elephant iPSCs can be generated using a combination of chemical reprogramming of primary' elephant cells and subsequent transfection of these cells with at least (i) C4, (ii) C5, (hi) C6. and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes.

[0095] Accordingly, this disclosure provides for culture media that can be used in the step- wise protocol and the induced pluripotent elephant stem cells produced by this protocol. The disclosure also provides for methods for generating induced pluripotent elephant stem cells that include chemically reprogramming primary elephant cells with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-f> inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor and then transfecting the reprogrammed cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes to generate the induced pluripotent elephant stem cells. During the transfection, the cells are exposed to an HDAC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOTIL inhibitor.

[0096] In certain embodiments, methods include a two-step process of chemical-media induction and colony selection followed by overexpression of elephant transcription factors OC74. SOX2. K/.F5. MYC ± NANOG, and LIN28A.

[0097] This disclosure is also based on the discovery that the unique step-wise protocol as described herein can be used not only to generate elephant iPSCs but also induced pluripotent Afrotheria species stem cells (such as e.g. induced pluripotent stem cells of the clade Paenungulata such as such e.g. elephants (Elephas maximus) or rock hyrax Procavia capensis). Specifically, the disclosure is based on the discovery that Afrotheria species iPSCs can be generated using a combination of chemical reprogramming of primary Afrotheria species cells and subsequent transfection of these cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes.

[0098] Accordingly, this disclosure provides for methods for generating induced pluripotent Afrotheria species stem cells that include chemically reprogramming primary Afrotheria species cells with an HDAC inhibitor, a GSK-3 inhibitor, a TGF-0 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT IL inhibitor and then transfecting the reprogrammed cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes to generate the induced pluripotent Afrotheria species stem cells (e.g. clade Paenungulata). During the transfection, the cells are exposed to an HDAC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT IL inhibitor.

[0099] In certain embodiments, methods include a two-step process of chemical-media induction and colony selection followed by overexpression of Afrotheria species transcription factors OC74. 80X2. KI JO. MYC ± NANOG. and LIN28A.

[0100] 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.

I. Definitions

[0101] 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.

[0102] 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. [0103] 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. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as 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.

[0104] 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.

[0105] 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. [0106] 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.

[0107] 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.

[0108] 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 applicability 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.”

[0109] 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-human embryonic stem cells, pluripotent stem cells, multipotent stem cells, amniotic stem cells, placental stem cells, or adult stem cells. 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, or iPS cell, has the induced capacity to differentiate, under appropriate conditions, to a cell phenotype derived from each of the endoderm, mesoderm, and ectoderm germ layers.

[0110] 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 embryo 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. [0111] As used herein, the phrase '‘somatic elephant cell” refers to any cell from an elephant that is not a germ cell, a reproductive cell, or a stem/progenitor cell.

[0112] As used herein, the phrase “induced pluripotent elephant stem cell” or “elephant iPSC” or “eiPSC” refers to any pluripotent stem cell that the been generated from an elephant somatic cell.

[0113] As used herein, the phrase '‘primary elephant cell” refers to any cell from any elephant cell that is not pluripotent, including germ cells and non-pluripotent cells. In certain embodiments, a primary elephant cell can be a somatic elephant cell.

[0114] As used herein, the phrase “somatic Afrotheria species cell” refers to any cell from an Afrotheria species that is not a germ cell, a reproductive cell, or a stem/progenitor cell.

[0115] As used herein, the phrase “induced pluripotent Afrotheria species stem cell” or “Afrotheria species iPSC” or “aiPSC” refers to any pluripotent stem cell that the been generated from an Afrotheria species somatic cell.

[0116] As used herein, the phrase “primary Afrothena species cell” refers to any cell from any Afrotheria species cell that is not pluripotent, including germ cells and non-pluripotent cells. In certain embodiments, a primary⁷ Afrotheria species cell can be a somatic Afrotheria species cell.

[0117] 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.

[0118] As used herein, the term '‘consisting of’ excludes any element, step, or ingredient not specified in the claim element.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

IL Culture media for seneratins induced pluripotent stem cells

[0124] One aspect of the disclosure is directed to culture media that can be used to generate induced pluripotent stem cells. In one embodiment, the culture media are used to generate induced pluripotent elephant stem cells from primary elephant cells or somatic elephant cells. In certain embodiments, the induced pluripotent elephant stem cells are generated from cells obtained from E. maximus. In another embodiment, the culture media are used to generate induced Afrotheria species stem cells from primary Afrotheria species cells or somatic Afrotheria species cells. In certain embodiment, the Afrotheria species cells are from the clade Paenungulata such as e.g., elephants or rock hyrax.

[0125] The culture media are basal culture media that have been supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-[3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase (a cAMP agonist), a retinoid, and a DOT1L inhibitor. [0126] 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 butyrate, and phenylbutyrate. In certain embodiments, the media contain from about 0. 1 to about 1 mM of the HD AC inhibitor.

[0127] In one embodiment, the GSK-3 inhibitor is CHIR-99021 (6-((2-((4-(2,4- Dichlorophenyl)-5-(4-methyl-lH-imidazol-2-yl) pyrimidin-2yl)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.

[0128] One embodiment of suitable TGF-P inhibitor for use in the media is RepSox (2-(3- (6-Methylpyridin-2-yl)-lH-pyrazol-4-yl)-l,5-naphthyridine). Other exemplary TGF-P inhibitors include, but are not limited to. SB431542 (4-(4-(benzo[d][l,3]dioxol-5-yl)-5- (25yridine-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)- IH-pyrazole- 1 -carbothioamide), and tranilast. In some embodiments, the cell culture medium is supplemented with from about 0.5 to about 4 pM of the TGF-P inhibitor.

[0129] A variety of monoamine oxidase inhibitor inhibitors may be used in the media. In one embodiment, monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1. 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.

[0130] Similarly, a variety of activators of eukary otic adenylyl cyclase may be used in the media. Suitable examples include, but are not limited to, forskolin ([(3R,4aR,5S,6S,6aS,10S,10aR,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- isobuty 1-1 -methylxanthine. In one embodiment, the activator of eukary otic 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 eukaryotic adenylyl cyclase.

[0131] 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-[ 1-hy droxy-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.

[0132] In one embodiment, the DOTL1L 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 DOTL1L 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)phenyl)urea). The culture media may be supplemented with from about 2 to about 10 pM of the DOT IL inhibitor.

[0133] 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 DOT1L 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 DOTIL inhibitor.

[0134] In other embodiments, the basal culture medium is supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HCL forskolin, Ch 55, and EPZ004777. 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 oO. l 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, and from about 2 to about 10 pM of EPZ004777.

[0135] Another aspect of the disclosure is directed to kits for generating induced pluripotent stem cells comprising the cell culture media of the disclosure and chemical reprogramming factors. In one embodiment, the kit includes the cell culture medium and one or more vectors comprising at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes. III. Methods of generating induced pluripotent Afrotheria species (e.g., elephant or rock hyrax) stem cells

[0136] The disclosure also provides methods for generating induced pluripotent elephant stem cells that include chemically reprogramming primary elephant cells and then transfecting the reprogrammed cells to generate the induced pluripotent elephant stem cells. [0137] A variety of primary elephant cells may be used in the methods of the disclosure. In some embodiments, the primary elephant cells are somatic elephant cells. In other embodiments, the somatic elephant cells are endothelial cells, epithelial cells, or fibroblasts such as fetal fibroblasts, adult fibroblasts, and skin fibroblasts. In one embodiment, the primary elephant cells are endothelial cells. In some embodiments, the primary elephant cells are Elephas maximus cells.

[0138] The disclosure further provides methods for generating induced pluripotent Afrotheria species stem cells that include chemically reprogramming primary' Afrotheria species cells and then transfecting the reprogrammed cells to generate the induced pluripotent Afrotheria species stem cells. The methods can be used to generate induced pluripotent stem cells from any Afrotheria species. In certain embodiments, the induced pluripotent stem cells are generated from clade Paenungulata cells (e.g, elephants or rock hyrax).

[0139] A variety of primary Afrotheria species cells may be used in the methods of the disclosure. In some embodiments, the primary Afrotheria species cells are somatic Afrotheria species cells. In other embodiments, the somatic Afrotheria species cells are endothelial cells, epithelial cells, or fibroblasts such as fetal fibroblasts, adult fibroblasts, and skin fibroblasts. In one embodiment, the primary Afrotheria species cells are endothelial cells. In certain embodiments. Afrotheria species is rock hyrax or elephant (e.g., Elephas maximus or Loxodonta africana).

[0140] The methods of the disclosure include transfection to knockdow n and/or knockout expression of the TP53 gene and/or TP53 retrogenes for cell growth as such knockdown and/or knockout is thought to aid reprogramming. Accordingly, in certain embodiments, the methods include:

(a) chemically reprogramming primary elephant cells by culturing the primary elephant cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT IL inhibitor:

(b) transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent elephant stem cells; and optionally (c) selecting for induced pluripotent elephant stem cells.

[0141] In alternate embodiments, the methods include:

(a) chemically reprogramming primary elephant cells by culturing the primary elephant cells in a culture medium supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin, Ch 55, and EPZ004777; and

(b) transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6. and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2- PCPA) HC1, forskolin, Ch 55, and EPZ004777 to generate induced pluripotent elephant stem cell; and optionally

(c) selecting for induced pluripotent elephant stem cells.

[0142] In other embodiments, the methods include:

(a) chemically reprogramming primary elephant cells by culturing the primary elephant cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT IL inhibitor; and

(b) transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent elephant stem cell.

[0143] In further embodiments, the method includes: transfecting chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-J3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent elephant stem cells; and optionally selecting for induced pluripotent elephant stem cells.

[0144] The methods of the disclosure may also be used generate induced pluripotent stem cells from other mammals. In certain embodiments, the methods of the disclosure can be used to generate induced pluripotent stem cells from any primary stem cells.

[0145] In some embodiments, the methods can be used to generate induced pluripotent Afrotheria species stem cells. Thus, in certain embodiments, the methods include: (a) chemically reprogramming primary Afrotheria species cells by culturing the primary Afrotheria species cells in a 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 D0T1L inhibitor;

(b) transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a 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 DOT IL inhibitor to generate induced pluripotent Afrotheria species stem cells; and optionally

(c) selecting for induced pluripotent Afrotheria species stem cells.

[0146] In alternate embodiments, the methods of generating induced pluripotent Afrotheria species stem cells include:

(a) chemically reprogramming primary Afrotheria species cells by culturing the primary Afrotheria species cells in a culture medium supplemented with valproic acid, CHIR- 99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin, Ch 55, and EPZ004777; and

(b) transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5. (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HCL forskolin, Ch 55, and EPZ004777 to generate induced pluripotent Afrotheria species stem cell; and optionally

(c) selecting for induced pluripotent Afrotheria species stem cells.

[0147] In further embodiments, the methods of generating induced pluripotent Afrotheria species stem cells include:

(c) chemically reprogramming primary⁷ Afrotheria species cells by culturing the primary Afrotheria species cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor. a TGF-[3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor; and

(d) transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-J3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent Afrotheria species stem cell. [0148] In further embodiments of the disclosure, the method of generating induced pluripotent Afrotheria species stem cells includes: transfecting chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent Afrotheria species stem cells; and optionally selecting for induced pluripotent Afrotheria species stem cells.

(a) chemically reprogramming primary Afrotheria species (e.g., elephant or rock hyrax) cells

[0149] The methods of the disclosure require chemically reprogramming primary elephant cells by culturing the cells in a culture medium supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT IL inhibitor. Alternatively, the methods of the disclosure require chemically reprogramming primary Afrotheria species cells by culturing the cells in a culture medium supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, and a DOTIL inhibitor.

[0150] In one embodiment, the method includes culturing the cells in a basal culture medium supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a TGF-|3 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 culture medium may further be supplemented with serum. In other embodiments, the culture medium is serum-free.

[0151] 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 HDAC 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-(3 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 D0T1L inhibitor.

[0152] 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-(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, and from about 2 to about 10 pM of a DOT IL inhibitor.

[0153] 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-P inhibitor is RepSox. In a further embodiment, the monoamine oxidase inhibitor is tranylcypromine (2- PCPA) HC1. In another embodiment, the activator of eukaryotic adenylyl cyclase is forskolin. In a further embodiment, the retinoid is Ch 55. In an alternate embodiment, the DOT1L inhibitor is EPZ004777.

[0154] Any of the HD AC inhibitors, the GSK-3 inhibitors, the TGF-P inhibitors, the monoamine oxidase inhibitors, the activators of eukaryotic adenylyl cyclase, the retinoids, and the DOT1L inhibitors described in II. above can be used to chemically reprogram the primary elephant cells or the primary Afrotheria species cells.

[0155] In certain embodiments, the culture medium is supplemented with valproic acid, CHIR-99021, RepSox, trany lcypromine (2-PCPA) HC1, forskolin, Ch 55, and/or EPZ004777. In other embodiments, the culture medium is supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HCL forskolin. Ch 55, and EPZ004777.

[0156] Prior to chemically reprogramming the cells, the primary elephant cells or primary Afrotheria species cells may be isolated or expanded. The primary elephant or primary' Afrotheria species cells may also be cryopreserved.

[0157] In certain embodiments, the chemically reprogramming includes passaging the cells for at least 2-3 passages. The passaging may involve media exchange.

(b) transfecting chemically reprogrammed elephant/Afrotheria species cells

[0135] After the primary elephant cells are reprogrammed, they are transfected with at least C4 (elephant/other species OCT4. SOX2, KI.1-4. C-MYC), C5 (elephant/other species OCT4, 80X2. KIJ '4. C-MYC, LIN28d), C6 (elephant/other species OC74. SOX2, KIX4. C-MYC, LIN28a, NANOG) and SV40LT and/or an agent targeting TP53 and TP53 retrogenes. In some embodiments, the cells transfected with at least C4 (elephant/other species OCT4. $0X2, KI.F4. C-MYC), C5 (elephant/other species OCT4. $0X2, KI. 4. C-MYC. LIN28a), C6 (elephant/other species OCT4, $0X2, KI.F4. C-MYC, LIN28a, NANOG) and SV40LT and an agent targeting TP53 and TP53 retrogenes. In other embodiments, the cells are transfected with at least C4 (elephant/other species OCT4. $0X2, KI.14. C-MYC), C5 (elephant/other species OCT4. SOX2, KLF4. C-MYC, LlN28a), C6 (elephant/other species OCT4, $0X2, KLF4, C-MYC, LIN28a, NANOG) or an agent targeting TP53 and TP53 retrogenes.

[0136] Similarly, after the primary Afrotheria species cells are reprogrammed, they are transfected with at least C4 (Afrotheria species/other species OCT4, SOX2, KLF4, C-MYC), C5 (Afrotheria species/other species OCT4, SOX2, KLF4. C-MY C, LIN28a), C6 (Afrotheria species/other species OCT4, 0X2, KLF4, C-MYC, LIN28a, NANOG) and SV40LT and/or an agent targeting TP53 and TP53 retrogenes. In some embodiments, the cells transfected with at least C4 (Afrotheria species/other species OCT4, $0X2, KLF4, C-MYC), C5 (Afrotheria species/other species OCT4, $0X2, KLF4, C-MYC, LIN28a), C6 (Afrotheria species/other species OCT4, $0X2, KLF4, C-MYC, LIN 28a, NANOG) and SV40LT and an agent targeting TP53 and TP53 retrogenes. In other embodiments, the cells are transfected with at least C4 (Afrotheria species/other species OCT4, $0X2, KLF4, C-MYC), C5 (Afrotheria species/other species OCT4, $0X2, KLF4. C-MYC, LIN28a), C6 (Afrotheria species/other species OCT4, $0X2, KLF4, C-MYC, LIN 28a, NANOG) or an agent targeting TP 53 and TP 53 retrogenes. [0137] As used herein, an agent targeting TP53 is any agent that is capable of knocking down or knocking out TP53, such as e.g., shRNA or CRISPR. In certain embodiments, the agent is shRNA targeting TP53. In other embodiments, the agent is shRNA that targets TP53 retrogenes. In other embodiments, the agent is CRISPR. In certain embodiments, instead of using CRISPR or a shRNA targeting TP53 and TP53 retrogenes, TP53 is knocked out with editing tools.

[0138] In certain embodiments, C4 is elephant OCT4, $0X2, KLF4, cMyc (Yamanaka factors), C5 is elephant OCT4, $0X2, KLF4, cMyc and LIN28a, and C6 is elephant OCT4. $0X2, KLF4, C-MYC. NANOG. LIN28a. Without being bound by theory, SV40LT is included in the transfection to overcome the cell cycle surveillance, as well as the shRNA against elephant TP53 and/or TP53 retrogenes. Furthermore, without being bound by theory', SV40LT has an important role in suppressing the TP53 pathw ay and increasing cell growth, which is why the factor was included in the transfection.

[0139] In one embodiment, C4 is elephant OCT4, $0X2, KLF4, C-MYC, C5 is elephant OCT4, $0X2, KLF4, C-MYC, LIN28a, and/or C6 is elephant OCT4, $0X2, KLF4, C-MYC, LIN28a, NANOG. In another embodiment, C4 is Elephas maximus OCT4, $0X2, KLF4, C- MYC, C5 is Elephas maximus OCT4, $0X2. KLF4, C-MY C, LIN28a, and/or C6 is Elephas maximus 0CT4, SOX 2. KI.F4. C-MYC, LIN28a, NANOG. In an alternate embodiment, C4 is Loxodonta africana OCT4, SOX2, KLF4, C-MYC, C5 is Loxodonta africana OCT4, SOX2, KI.1'4. C-MYC, LIN28a, and/or C6 is Loxodonta africana OCT4, SOX2, KLF4, C-MYC, LIN28a, NANOG. In yet another embodiment, C4 is Afrotheria species OCT4, SOX2, KLF4, C-MYC, C5 is Afrotheria species OCT4. SOX2, KLF4, C-MYC. LIN28a, and/or C6 is Afrotheria species OCT4, SOX2, KLF4, C-MYC, LIN28a, NANOG. In yet another embodiment, C4 is any mammalian OCT4, SOX2, KLF4, C-MYC, C5 is any mammalian OCT4. SOX2, KLF4, C-MYC, LIN28a, and/or C6 is any mammalian OCT4, SOX2, KLF4, C- MYC, LIN28a, NANOG.

[0140] In certain embodiments, commercially available transfection systems, such as the Neon transfection system (Invitrogen MPK100265), are used.

[0141] In one aspect, the transfection of the chemically reprogrammed elephant cells includes transfecting the above-referenced genes in the presence of a 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. In certain embodiments, a basal culture medium (such as e.g., DMEM or Knockout DMEM) is 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 DOT IL inhibitor. The culture medium may contain serum or be serum-free.

[0142] In certain embodiments, the methods include transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a 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 to generate induced pluripotent elephant stem cells.

[0143] In other embodiments, the methods include transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6. and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a 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 to generate induced pluripotent elephant stem cells.

[0144] In further embodiments, the methods include transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT in a 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 to generate induced pluripotent elephant stem cells. [0145] In alternate embodiments, the methods include transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF- inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent elephant stem cells. In one embodiment, shRNA targets TP53. In another embodiment, the shRNA targets TP53 retrogenes.

[0146] In another aspect, the transfection of the chemically reprogrammed Afrotheria species cells includes transfecting the above-referenced genes in the presence of a 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 DOT IL inhibitor. In certain embodiments, a basal culture medium (such as e.g.. DMEM or Knock-out DMEM) is supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-|3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT IL inhibitor. The culture medium may be serum or serum-free.

[0147] In certain embodiments, the methods include transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent Afrotheria species stem cells.

[0148] In other embodiments, the methods include transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-J3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent Afrotheria species stem cells.

[0149] In further embodiments, the methods include transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6. and (iv) SV40LT in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent Afrotheria species stem cells. [0150] In alternate embodiments, the methods include transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent Afrotheria species stem cells. In one embodiment, shRNA targets TP53. In another embodiment, the shRNA targets TP53 retrogenes.

[0151] In one embodiment, the culture medium during transfection is supplemented with from about 0. 1 to about 1 mM, alternatively about 0.5 mM, alternatively at least 0.5 mM of an HDAC inhibitor. In another embodiment, the culture medium during transfection 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 during transfection is supplemented with from about 0.5 to about 4 pM, alternatively about 2 pM. alternatively at least 2 pM of a TGF-(3 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 during transfection 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 during transfection 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 during transfection is supplemented with from about 2 to about 10 pM, alternatively about 5 pM, alternatively at least 5 pM of a DOT IL inhibitor.

[0152] In another embodiment, the culture medium during transfection 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 0.5 to about 4 pM of a TGF-(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, and from about 2 to about 10 pM of a DOTIL inhibitor.

[0153] In one embodiment, the HDAC inhibitor is valproic acid. In another embodiment, the GSK-3 inhibitor is CHIR-99021. In an alternate embodiment, the TGF-[3 inhibitor is RepSox. In a further embodiment, the monoamine oxidase inhibitor is tranylcypromine (2- PCPA) HC1. In another embodiment, the activator of eukaryotic adenylyl cyclase is forskolin. In a further embodiment, the retinoid is Ch 55. In an alternate embodiment, the DOT1L inhibitor is EPZ004777. [0154] Any of the HDAC inhibitors, the GSK-3 inhibitors, the TGF-0 inhibitors, the monoamine oxidase inhibitors, the activators of eukaryotic adenylyl cyclase, the retinoids, and the DOT1L inhibitors described in II. above can be used during the transfection of chemically reprogramed primary elephant cells or the transfection of chemically reprogrammed primary Afrotheria species cells.

[0155] In certain embodiments, the culture medium during transfection is supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin, Ch 55, and/or EPZ004777. In other embodiments, the culture medium during transfection is supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin. Ch 55, and EPZ004777.

[0156] In one embodiment, the method includes transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium 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 0.5 to about 4 pM of a TGF- 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.

[0157] In another embodiment, the method includes transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium 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 0.5 to about 4 pM of a TGF-(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, and from about 2 to about 10 pM of a DOT IL inhibitor.

[0158] The transfecting may include changing the culture medium every two days. In some embodiments, where desired a selection marker, such as antibiotic resistance (to e g., hygromycin or puromycin) is also transfected.

[0159] The transfection may be carried in the presence of absence of a feeder layer. In certain embodiments, the transfecting is earned 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. (c) selecting for induced pluripotent Afrotheria species/elephant stem cells

[0153] After the induced pluripotent Afrotheria species e.g., clade Paenungulata such e.g., elephant or rock hyrax) stem cells are generated, they may be isolated. Tn certain embodiments, isolation is achieved by culturing the induced pluripotent elephant stem cells under conditions that favor the induced pluripotent elephant stem cells. In other embodiments, isolation is achieved by culturing the induced pluripotent Afrotheria species stem cells under conditions that favor the induced pluripotent Afrotheria species stem cells. Thus, in certain embodiments, the methods of the disclosure also include selecting for the induced pluripotent stem cells. In other embodiments, the methods of the disclosure may omit the selection step.

[0154] In some embodiments, the selecting for induced pluripotent elephant stem cells includes introducing a selection marker during transfection. In other embodiments, the selecting for induced pluripotent Afrotheria species stem cells includes introducing a selection marker during transfection. In one embodiment, the selection marker is antibiotic resistance, such as. e.g, resistance to hygromycin or puromycin.

[0155] Accordingly, in one embodiment, the selecting for induced pluripotent elephant stem cells includes treatment with doxycycline and antibiotic selection and wherein the transfected chemically reprogrammed elephant cells are resistant to the antibiotic used for selection. In another embodiment, the selecting for induced pluripotent Afrotheria species stem cells includes treatment with doxycycline and antibiotic selection and wherein the transfected chemically reprogrammed Afrotheria species cells are resistant to the antibiotic used for selection.

[0156] In some embodiments, the induced pluripotent elephant stem cells are resistant to hygromycin or puromycin. In these embodiments, the selection includes treatment with hygromycin or puromycin. In one embodiment, the antibiotic selection includes treatment with hygromycin, whereby the cells are treated every two days with hygromycin, and whereby the treatment lasts ten days. In another embodiment, the antibiotic selection includes treatment with puromycin daily for five days.

[0157] During the selecting, the induced pluripotent elephant stem cells are cultured in culture (e.g., basal) medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, and a DOT1L inhibitor. In some embodiments, selection for the induced plunpotent elephant stem cells includes culturing the cells on a feeder layer, such as. e.g., an mouse embryonic fibroblast (MEF) feeder layer. In other embodiments, selection for the induced pluripotent elephant stem cells includes culturing the cells under feeder-free conditions (z.e., in the absence of a feeder layer).

[0158] In other embodiments, the induced pluripotent Afrotheria species stem cells are resistant to hygromycin or puromycin. In these embodiments, the selection includes treatment with hygromycin or puromycin. In one embodiment, the antibiotic selection includes treatment with hygromycin, whereby the cells are treated every two days with hygromycin, and whereby the treatment lasts ten days. In another embodiment, the antibiotic selection includes treatment with puromycin daily for five days.

[0159] During the selecting, the induced pluripotent Afrotheria species stem cells are cultured in culture (e.g. basal) medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, and a DOT1L inhibitor. In some embodiments, selection for the induced pluripotent Afrotheria species stem cells includes culturing the cells on a feeder layer, such as, e.g.. an mouse embryonic fibroblast (MEF) feeder layer. In other embodiments, selection for the induced pluripotent Afrotheria species stem cells includes culturing the cells under feeder-free conditions (i.e., in the absence of a feeder layer).

[0160] In one embodiment, the culture medium during selection is supplemented with from about 0. 1 to about 1 mM, alternatively about 0.5 mM. alternatively at least 0.5 mM of an HDAC inhibitor. In another embodiment, the culture medium during selection 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 during selection is supplemented with from about 0.5 to about 4 pM. alternatively about 2 pM, alternatively at least 2 pM of a TGF-0 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 during selection 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 during selection 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 during selection is supplemented with from about 2 to about 10 pM, alternatively about 5 pM, alternatively at least 5 pM of a DOT1L inhibitor.

[0161] In another embodiment, the culture medium during selection 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 0.5 to about 4 pM of a TGF-(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, and from about 2 to about 10 pM of a DOT IL inhibitor.

[0162] 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-(3 inhibitor is RepSox. In a further embodiment, the monoamine oxidase inhibitor is tranylcypromine (2- PCPA) HC1. In another embodiment, the activator of eukaryotic adenylyl cyclase is forskolin. In a further embodiment, the retinoid is Ch 55. In an alternate embodiment, the DOT1L inhibitor is EPZ004777.

[0163] In certain embodiments, the culture medium during selection is supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin, Ch 55, and/or EPZ004777. In other embodiments, the culture medium during selection is supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin, Ch 55. and EPZ004777.

[0164] Any of the HDAC inhibitors, the GSK-3 inhibitors, the TGF-P inhibitors, the monoamine oxidase inhibitors, the activators of eukaryotic adenylyl cyclase, the retinoids, and the DOT1L inhibitors described in II. above may be used to select for the induced pluripotent elephant stem cells or the induced pluripotent Afrotheria species stem cells. [0165] In certain embodiments, the selecting includes culturing the induced pluripotent elephant stem cells under feeder-free conditions, followed by culturing the cells on a feeder layer.

[0166] In other embodiments, the selecting includes culturing the induced pluripotent elephant 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 elephant stem cells on a feeder layer along with doxycycline.

[0167] In further embodiments, the selecting includes culturing the induced pluripotent elephant 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 elephant stem cells on a feeder layer along with doxycycline, followed by culturing the induced pluripotent elephant cells on a feeder layer resistant to the antibiotic used for the selection and the antibiotic used for selection (such as e.g., hygromycin or puromycin).

[0168] In one embodiment, the induced pluripotent elephant stem cells are resistant to two antibiotics (such as e.g., hygromycin and puromycin). In this embodiment, the induced pluripotent elephant stem cells are first cultured in medium supplemented with doxycycline. The cells are then exposed to treatment with the antibiotic. If the antibiotic is hygromycin, the cells are treated every two days and the treatment lasts ten days. If the antibiotic is puromycin. the cells are treated daily with puromycin for five days. After antibiotic treatment, the cells are then cultured on a feeder layer in the presence of doxycycline. Alternatively, the cells are then cultured under feeder-free conditions.

[0169] In another embodiment, the induced pluripotent elephant stem cells are resistant to two antibiotics (such as e.g., hygromycin and puromycin). In this embodiment, the induced pluripotent elephant stem cells are first cultured in medium supplemented with doxycycline. The cells are then exposed to treatment with the antibiotic. If the antibiotic is hygromycin, the cells are treated every two days and the treatment lasts ten days. If the antibiotic is puromycin, the cells are treated daily with puromycin for five days. After antibiotic treatment, the cells are then cultured on a feeder layer in the presence of doxycycline. Alternatively, the cells are then cultured under feeder-free conditions. After this culture, the cells are then cultured on an antibiotic resistant feeder layer. The cells are then exposed to treatment with the antibiotic. If the antibiotic is hygromycin, the cells are treated every two days and the treatment lasts ten days. If the antibiotic is puromycin, the cells are treated daily with puromycin for five days.

[0170] In certain embodiments, for each of the steps used to select for the induced pluripotent elephant stem cells a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-|3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor as described herein is used. [0171] In alternate embodiments, the selecting includes culturing the induced pluripotent Afrotheria species stem cells under feeder-free conditions, followed by culturing the cells on a feeder layer.

[0172] In some embodiments, the selecting includes culturing the induced pluripotent Afrotheria 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 Afrotheria species stem cells on a feeder layer along with doxycycline. [0173] In other embodiments, the selecting includes culturing the induced pluripotent Afrotheria 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 Afrotheria species stem cells on a feeder layer along with doxycycline, followed by culturing the induced pluripotent Afrotheria species cells on a feeder layer resistant to the antibiotic used for the selection and the antibiotic used for selection (such as e.g., hygromycin or puromycin). [0174] In an embodiment, the induced pluripotent Afrotheria species stem cells are resistant to two antibiotics (such as e.g, hygromycin and puromycin). In this embodiment, the induced pluripotent Afrotheria species stem cells are first cultured in medium supplemented with doxycycline. The cells are then exposed to treatment with the antibiotic. If the antibiotic is hygromycin, the cells are treated every two days and the treatment lasts ten days. If the antibiotic is puromycin, the cells are treated daily with puromycin for five days. After antibiotic treatment, the cells are then cultured on a feeder layer in the presence of doxycycline. Alternatively, the cells are then cultured under feeder-free conditions.

[0175] In an alternate embodiment, the induced pluripotent Afrotheria species stem cells are resistant to two antibiotics (such as e.g., hygromycin and puromycin). In this embodiment, the induced pluripotent Afrotheria species stem cells are first cultured in medium supplemented with doxycycline. The cells are then exposed to treatment with the antibiotic. If the antibiotic is hygromycin. the cells are treated every two days and the treatment lasts ten days. If the antibiotic is puromycin, the cells are treated daily with puromycin for five days. After antibiotic treatment, the cells are then cultured on a feeder layer in the presence of doxycycline. Alternatively, the cells are then cultured under feeder- free conditions. After this culture, the cells are then cultured on an antibiotic resistant feeder layer. The cells are then exposed to treatment with the antibiotic. If the antibiotic is hygromycin, the cells are treated every two days and the treatment lasts ten days. If the antibiotic is puromycin, the cells are treated daily with puromycin for five days.

[0176] In further embodiments, for each of the steps used to select for the induced pluripotent Afrotheria species stem cells a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT IL inhibitor as described herein is used. [0177] In some embodiments, the selection includes changing the medium every two days and culturing the cells for up to 150 days, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90. at least 100, at least HO. at least 120, at least 130, at least 140, or at least 150 days.

[0178] In certain embodiments, an isolation step is used in place of the selection step. For example, in some embodiments, the cells are isolated using conventional techniques, such as e.g., cell sorting, density gradient centrifugation, and flow cytometry. IV. iPSCs a. Elephant iPSCs

[0179] The disclosure also provides for induced pluripotent elephant stem cells that are generated from primary elephant cells. In certain embodiments, the induced pluripotent elephant stem cells are generated using any of the methods described herein.

[0180] One embodiment of the disclosure is an induced pluripotent elephant stem cell expressing (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and TP53 retrogenes. wherein the stem cell is generated by chemically reprogramming a primary elephant cell.

[0181] Another embodiment of the disclosure is an induced pluripotent elephant stem cell expressing (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes. wherein the stem cell is generated by chemically reprogramming a primary elephant cell and transfecting the reprogrammed elephant cell to express (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes.

[0182] In one embodiment, the induced pluripotent elephant stem cells of the disclosure express (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT. In another embodiment, the induced plunpotent elephant stem cells of the disclosure express (i) C4. (ii) C5. (iii) C6, and (iv) an agent targeting TP53 and/or TP53 retrogenes. In yet another embodiment, the induced pluripotent elephant stem cells of the disclosure express (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and an agent targeting TP53 and/or TP53 retrogenes.

[0183] In certain embodiments, the expression of (i) C4, (ii) C5, (iii) C6, and (iv) an agent targeting TP53 and/or TP53 retrogenes in the induced pluripotent elephant stem cells is greater than the expression of (i) C4, (ii) C5, (iii) C6, and (iv) an agent targeting TP53 and/or TP53 retrogenes in primary elephant cells that are not chemically reprogrammed. b. Afrotheria species iPSCs

[0184] The disclosure also provides for induced pluripotent Afrotheria species stem cells that are generated from primary Afrotheria species cells using any of the methods described herein. One embodiment of the disclosure is an induced pluripotent Afrotheria species stem cell expressing (i) C4, (ii) C5, (iii) C6. and (iv) SV40LT and/or an agent targeting TP53 and TP53 retrogenes, wherein the stem cell is generated by first chemically reprogramming a primary' Afrotheria species cell.

[0185] Another embodiment of the disclosure is an induced pluripotent Afrotheria species stem cell expressing (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes, wherein the stem cell is generated by first chemically reprogramming a primary Afrotheria species cell and then transfecting the reprogrammed Afrotheria species cell to express (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes.

[0186] In one embodiment, the induced pluripotent Afrotheria species stem cells of the disclosure express (i) C4, (ii) C5, (iii) C6. and (iv) SV40LT. In another embodiment, the induced pluripotent Afrotheria species stem cells of the disclosure express (i) C4, (ii) C5, (iii) C6, and (iv) an agent targeting TP53 and/or TP53 retrogenes. In yet another embodiment, the induced pluripotent Afrotheria species stem cells of the disclosure express (i) C4, (ii) C5, (iii) C6. and (iv) SV40LT and an agent targeting TP53 and/or TP53 retrogenes.

[0187] In certain embodiments, the expression of (i) C4, (ii) C5, (iii) C6, and (iv) an agent targeting TP53 and/or TP53 retrogenes in the induced pluripotent Afrotheria species stem cells is greater than the expression of (i) C4, (ii) C5, (iii) C6, and (iv) an agent targeting TP53 and/or TP53 retrogenes in primary Afrotheria species cells that are not chemically reprogrammed.

IV Uses of iPSCs

[0188] The disclosure also includes using the elephant iPSCs. In particular, the elephant 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 multiplex editing. In further embodiments, the elephant iPSCs can be used to generate chimeras, embryos, germ cell progenitors, and gametes.

[0189] The disclosure further includes using the Afrotheria species iPSCs. In particular, the Afrotheria species 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 multiplex editing. In further embodiments, the Afrotheria species iPSCs can be used to generate chimeras, embryos, germ cell progenitors, and gametes.

[0190] In certain embodiments, the elephant iPSCs or Afrotheria species iPSCs are differentiated into neurons, neuronal precursors, and/or cardiomyocyte precursors. In some embodiments, the differentiation factors used in Example 6 are used.

EXAMPLES

[0191] The invention is now described with reference to the following Examples. These

Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0192] 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.

EXAMPLE 1 - Protocol to Generate Elephant iPSC

[0193] Induced pluripotent elephant stem cells were generated using the protocol described in this example. Prior to devising the protocol in this Example, the protocols for chemical reprogramming are disclosed in U.S. Patent No. 9,982,237 and WO 2017/091943 were considered and determined not to be sufficient to all for generation of iPSC from somatic cells from elephants.

Culture media

[0194] As used throughout this protocol “ESC medium'’ is an ESC culture medium in 500 ml containing the following: 390 ml of KnockOut DMEM (Gibco 10829-018); 50 ml or 10% knockout serum replacement (Invitrogen); 50 ml or 10% fetal bovine serum (Hy clone); 5 ml or 1% GlutaMAX (1%; Gibco 35050079); 5 ml or 1% nonessential amino acids (Invitrogen); 500 pl or 55 pM 2-mercaptoethanol (55 mM (1000X); Gibco 21985023); and 50 ng/ml bFGF (20 pg/ml; heat stable; Life technologies PHG0369).

[0195] Furthermore as used throughout this protocol “B cocktail” contains the following: 0.5 mM of VPA (EtOH; Selleckchem S3944); 5 pM of CHIR-99021 (DMSO; Selleckchem S1263); 2 pM of RepSox (DMSO; Selleckchem S7223); 10 pM of Tranylcypromine (2- PCPA) HC1 (DMSO; Selleckchem S4246); 20 pM of Forskolin (DMSO; Selleckchem S2449); 1 pM of Ch 55 (DMSO; Tocris 2020); and 5 pM of EPZ004777 (DMSO;

Selleckchem S7353).

[0196] The MEF culture media used in this protocol contains 15% FBS, 1% Amp/Pen, 1% Non-essential AA and DMEM/Glutamax (Gibco 10569).

Step 1: Chemical reprogramming

[0197] Elephant somatic cells were reprogrammed. Specifically, 150K ClEmMEn p5 wild-type cells were plated on six wells in 30% FBS/1% antibiotic/antimycotic Zl % Non- essential AA/ EGM2 media with Laminin521 coating (5 pg/ml; Gibco A29248). The morphology of the ClEmMEn wild-tj pe cells is shown in FIG. 1 (the culture conditions are shown in Table 1-5). Twenty-four hours after seeding 150,000 cells, the B cocktail or ESC medium with small molecules was added. The medium was changed every two days.

[0198] Once colonies reached sufficient size, they were hand-picked and passaged 2-3 times. The cell morphology of hand-picked colonies after 2-3 passages is shown in FIG. 2.

Step 2: Transfection using Neon Transfection System

[0199] After chemical reprogramming, the reprogrammed cells were transfected using the Neon Transfection System (Thermo Fisher Scientific). The Neon Transfection System Kit, which was used for transfection, includes the following: 30 mL Resuspension Buffer R, 30 mL Resuspension Buffer T, 2 x 150 mL Electrolytic Buffer E2, 96 x 100 pL Neon™ Tips, 20 Neon™ Electroporation Tubes.

[0200] Prior to transfection, the PB transposons were gathered, the transposases plasmids were maintained on ice, and transfection reagents were maintained at room temperature. The Neon Electroporation tubes were assembled and 3 ml of Buffer E2 was added into each tube. B cocktail in ESC media (with 2 pM PFT and 10 pM RI) was placed on Laminin521 coated 6 well plates in the incubator for cells after transfection. The transposases plasmid, transposon plasmid, and Buffer R were combined. For the transfections, the conditions in Tables 1-1 to 1 -3 were used.

[0201] The cells were trypsinized (0.05% Trypsin at 37°C for 5~8 minutes), collected, counted (750K cells/reaction) and centrifuged at 0.2 g for 4 minutes. The cells were then resuspended in 300 pl of Buffer R. Then 75 pL of the cell suspension was added to piggyBac complexes according to the tables above. Using 100 pl Neon Transfection tips, the samples were electroporated at 1600V/10ms/3 pulses and the cells were directly plated to preloaded 6 well (4.) 24 hours after transfection, the medium was refreshed. The cells were then cultured for more than 150 days with medium changed every two days. FIG. 3A (Brightfield merged with green fluorescence channel) and FIG. 3B (Brightfield only) shows the cells at 48 hours after transfection with C4 along with GFP-expressing shRNA plasmid targeting TP53 retrogene. Images were taken at 10X magnification.

Step 3: Selection for Induced Pluripotent Stem cells

[0202] After transfection, the transfected cells were isolated. A variety of different protocols may be used to isolate the transfected cells. In this example antibiotic selection was used to select for the induced cells.

Step 3 A: Doxycycline treatment start along with antibiotic selection [0203] Treatment with 2 pg/ml doxycycline (Sigma D5207) started once the cells reached 80% confluency. During the treatment B cocktail/ESC were replaced with Doxycycline every two days. For antibiotic selection, the following conditions were used: (1) hygromycin 50 pg/ml for C4TP53shRNA2 every two days 5 times (10 days); or puromycin 0.5 pg/ml for C4/5/6 iSV40LT daily 5 times (5 days). After selection, media was changed every two days (B cocktail/ESC media on 5 pg/ml Laminin521). FIG. 4 shows C4 TP53shRNA2 after hygromycin selection on Laminin521 coated plate (EVOS 10X).

Step 3B: Culture on MEF feeder layer

[0204] An MEF feeder layer culture was prepared by coating a six well plate with 0. 1% Gelatin (Stem cell technologies 07903) at 37 °C for minimum 15 minutes. The plate was then washed with DPBS and 500K-1M cells/well in MEF media were plated. The next day, 150K cells/well on MEF layer were split using the conditions in Table 1-4, giving rise to the following populations: C4shRNA2 p9; C4iSV40LT plO; C5iSV40LT plO; and C6iSV40LT p9.

[0205] B cocktail/ESC was changed with doxycycline every two days. C4shRNA2 was observed to have a better iPSC like morphology on MEF feeder layer (FIG. 5). At the stage. mRNA level of reprogramming markers was not detected. However, since cells without integration may grow faster and consume nutrients, further antibiotic selection was carried out.

Step 3C: Re-selection on antibiotic resistant MEF feeder layer

[0206] For the re-selection, DR4 MEF, irradiated (Gibco A34966), which is antibiotic resistant, was used. To begin re-selection, an mouse embryonic fibroblast (MEF) feeder layer culture was prepared by coating a six well plate with 0.1% Gelatin (Stem cell technologies 07903) at 37 °C for minimum 15 minutes. The plate was then washed with DPBS and 500K.-1M cells/well in MEF media were plated. The next day. the cells were split into 150K cells/well on the MEF layer using the protocol in Table 1-4, giving rise to the following populations: C4shRNA2 pl 1; C4iSV0LT pl2; C5iSV40LT pl2; C6iSV40LT pl 1. Once the cell confluency reached 80%, antibiotic selection was started. The following treatments were used to antibiotic selection: hygromycin 50 pg/ml for C4TP53shRNA2 every two days 5 times (10 days); or puromycin 0.5 pg/ml for C4/5/6 iSV40LT daily 5 times (5 days). FIG. 6 shows transfected cells with C4iSV40 after re-selection using 0.5 pg/ml puromycin.

EXAMPLE 2 - Embryoid Body Formation

[0207] To confirm that the protocols in Example 1 and disclosed herein indeed generated induced pluripotent elephant stem cells, the cells produced in Example 1 were cultured in under conditions sufficient for embryoid body formation.

[0208] Specifically, induced pluripotent elephant stem cells were cultured for five days in the ESC medium described above supplemented with cocktail B (containing valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin, Ch 55, and EPZ004777). As a negative control, primary elephant cells were chemically reprogramed by culturing the cells for five days in the ESC medium described above supplemented with cocktail B. As a positive control, human iPSCs (hiPSCs) were also cultured under mTeSRplus medium. To confirm embryoid body formation, the expression of PAX6 mRNA and GATA6 mRNA were also assessed. Embryoid body formation was assayed using Aggrewell. The results for the negative control (only chemical B cocktail) and positive control (hiPSC) are shown in FIG. 7. The results for the induced pluripotent elephant stem cells are shown in FIG. 8.

[0209] Germ layer specification is indicated by expression of TBXT, PAX6, and GATA6 and pluripotency is indicated by expression of OCT4. NANOG, and SOX2 (FIG. 9).

Accordingly, the cells after the embryoid body formation assay were tested for PAX6 mRNA (FIG. 10A) and GATA6 mRNA (FIG. 10B). Compared to the negative control, all the induced pluripotent elephant stem cells tested in the embryoid body formation protocol showed significant increase in PAX6 mRNA and GATA6 mRNA. This testing confirms embryoid formation from the cells.

EXAMPLE 3 - Partial emiPSC Reprogramming Attempts

[0210] Elephant induced pluripotent stem cells were created only after an exhaustive span of attempts with cunent standard reprogramming methods were tried and failed. First, reprogramming of Elephas maximus cells w as attempted via the use of over-expression of standard ‘Yamanaka reprogramming factors’: OCT4, SOX2, KLF4, w MYC (OSKM) in a variety of formats. This included transgene expression via episomal (Junying Yu, J. el al., Science, 324(5928):797-801, 2009), lentivirus (Chang, C.W., et al., Stem Cells, 27(5): 1042- 1 49, 2009), Sendai-virus (Yang, W. et al., ipse reprogramming from human peripheral blood using sendai virus mediated gene transfer. 2008), and PiggyBac (Woltjen, K et al.. Nature, 458(7239):766-770, 2009) in a variety of different variations with transcription factor (TF) sequences from either mouse or human (FIG. 11 A-J). These transgene expression methods were also tested in tandem with the over-expression of shRNAs that target TP53, SV40 T-antigen, NANOG, and/or LIN28A. While some of these methods yielded some potential morphological differences from primary cells at different parts of the process, ultimately all of these types of attempts failed as a result of either cell death, senescence, or no observed change compared to primary starting cell types (FIGS. 12A-J, 13A-D, 14A-D, and 15).

[0211] In addition to transgene-dependent methods, a growing set of chemical reprogramming methods that do not require the use of transgenes was also explored. A set of published cocktail sets (Hou P. P. et al. Science, 341(6146):651-654, 2013; Guan, JY et al. Nature. 605(7909):325-331, 2022; Chen, X. et al. Nature Cell Biology, 25(8): 1146-1156, 2023) that was customized and made variations upon (FIG. 181) was tested. In addition, other chemical cocktails to accomplish this task were also tested. In summary, these approaches also resulted in either cell death, senescence, or no observed change, but in a small set of experiments also appeared to yield encouraging morphological changes (FIGS. 12A-J, 13A-D, 14A-D, and 15).

[0212] The process of creating emiPSCs required the testing of many established reprogramming methods that have been tried and tested on other species. While many of these attempts resulted in no morphological change to the starting cell population, cell population senescence, and cell death, some protocols were able to produce cells that appeared partially on their way to iPSC characteristics (FIG. 11 A-J, FIG. 12A-J, FIG. 13A-D, FIG. 14A-D and FIG. 15). Briefly, it was attempted to (1) screen broad new sets of human TF combinations with the human TFome (FIG. 11 A), (2) create emiPSCs from polycistronic PiggyBac mouse OSKML and SV40T antigen vectors (FIG. 11C), (3) induce cellular reprogramming via complex chemical treatment progressions supplemented with episomal transgene expression on two cell lines (one male, one female) (FIG. 1 IB. 1 ID), (4) create emiPSCs from polycistronic PiggyBac elephant OSKM/NL and SV40T antigen or TP53 shRNA vectors (FIG. 1 IE), (5) create emiPSCs via standard viral reprogramming methods utilizing human transgenes (FIG. 1 IF, 11G), (6) create emiPSCs via chemical induction alone (FIG. 11H).

[0213] One of the first attempts to reprogram these notoriously difficult cells was to question whether additional reprogramming TFs that may not yet be described in the literature were missing. To this end, computational tools to NGS data from human iPSCs and ESCs was applied see Tables 3-1 to 3-3 below). Table 3-1 Literature Sets

LITERATURE SETS ELEPHANT TFs (LITERATURE)

Table 3-2 Computations Sets

MOGRIFY IRENE

Table 3-2 Computations Sets

Table 3-3 Computations Sets

CELLCARTOGRAPHER SCREENING ROUND 1 SCREENS

Table 3-3 Computations Sets

CELLCARTOGRAPHER SCREENING ROUND 2 SCREENS

[0214] These combinations were tested (FIG. HA) with these sets of TFs and enriched populations of cells that exhibited potential up-regulation of cell surface pluripotency markers (FIG. 17). While some interesting enrichment was found in these samples, testing of these refined combinations did not yield further success; cells in these protocols tended to form small, tight colonies at the beginning of the experiment and then these colonies stagnated (FIG. 12A). It was speculated at this point whether or not elephant TF amino acid sequences are different enough that human TF proteins may not work as they are supposed to in these cells, but as most TF-binding domains of TFs tend to be conserved, there is still no conclusive data on this topic. It was also of concern that since all these TFs were on individual expression cassettes, that a screen may miss requirements where all members of a reprogramming set are required.

[0215] Accordingly, reprogramming of emECs with polycistronic PiggyBac expression Yamanaka factors (FIG. 11C) was attempted. The female endothelial cell line that was attempted to be reprogrammed solely with trans-gene overexpression via PiggyBac yielded a different morphology than the other two (FIG. 12C), but alas still did not show high pluripotency marker expression, aside from SOX2 (FIG. 13B). While the morphology' of these colonies was loosely on track, the populations grew very slow and still lacked key characteristics of iPSCs. In parallel, a similar method that used elephant specific OSKM/NL amino acid sequences and shRNAs to modulate the expression of TP53 and its retrogenes (FIGs. 1 IE, 16A, and 16B) was also tested. While it could not be validated that the shRNAs were repressing either the canonical TP53 sequence and its retrogenes (TP53shRNA4) or just most retrogenes (TP53shRNA2) (FIG. 16A+D), this transgene-only approach was still not effective at creating emiPSCs (FIG. 12E, FIG. 13D).

[0216] Next, testing methods that combined both transgene and complex media formulations (FIG. 1 IB. 1 ID) were attempted. These attempts used complex chemical treatments, supplemented with episomal reprogramming factors over-expression. After similar treatments, the female line showed marker profiles and morphology⁷ characteristic of cells in the trophoblast stem-cell-like state (FIG. 12B, 13C), and the male line showed marker profiles and morphology roughly characteristic of cells undergoing the mesenchymal to epithelial transition (MET) (FIG. 12D, 13 A).

[0217] Finally, attempts to reprogram elephant cells with the same chemical protocol demonstrated for mice were interesting in both a morphological (FIG. 11F-H) and growth perspective. A number of different variations of chemical cocktails were tested: A (0.5 mM VP A, lOpM CHIR, 20pM Repsox, lOpM Tranly (Tranylcypromine (2-PCPA) HC1), lOpM Forskolin, 1 RAR agonist); B (0.5 mM VP A, 15pM CHIR, 2pM Repsox, lOpM Tranly, 20pM Forskolin, 2 RAR agonist); and C (0.5 mM VP A, lOpM CHIR, lOpM Repsox, 5pM Tranly, lOpM Forskolin). While these features were exciting, molecular analysis revealed that these cells still did not have expression of core pluripotency factors (FIG. 14A-D). Furthermore, while this result was puzzling, given the morphology, growth, and expression of secondary pluripotency factors, adding PiggyBac, polycistonic Yamanaka factors using the endogenous Loxodonta africana sequences and modulators of TP53 and/or its retrogenes to these cells (FIG. 111. 11 J) was pursued. Chemical cocktail B was the chosen cocktail to pursue most TF-ov erexpression experiments. While the NGS data suggested that chemical cocktail C (FIG. 15) w as indeed closer to final emiPSC molecular composition, it grew? slower that cells derived from cocktail B, and that with a repeated process with refined colony -picking strategies, chemical cocktail B ultimately produced the cells referred herein as ’C I EmMEn pre-iPSC’.

[0218] Principal component analysis of all partial success cell lines and final success cell lines paints a very interesting picture (FIG. 15). Surprisingly, the pure polycistronic transgene expression method that utilized SV40T as opposed to TP53shRNAs clustered very closely to the hybrid chemical/transgene methods and indeed not far off from true emiPSCs. Similarly, purely chemical methods appeared to do a lot of heavy lifting to make emiPSCs but could not alone finish the job. Even more striking was the case of C3-loxc4- TP53shRNA4 cells, which appeared to create some morphological changes and had many of the needed components to create emiPSCs yet appears to have barely moved the needle towards emiPSCs and apparently in the wrong direction.

EXAMPLE 4 - Generation of Asian elephant pluripotent stem cells

[0219] It was the integration of these two ty pes of approaches shown in Example 3 that ultimately yielded successful production of Elephas maximus induced pluripotent stem cells (emiPSCs). Specifically, emiPSCs were derived by first partially reprogramming primary endothelial cells (ernECs) to an intermediate ‘pre-iPSC’ state (emPRC) with a derivative chemical reprogramming protocol based on prior work (Hou P. P. et al. Science,

341(6146): 651-654, 2013) and then using elephant-specific trans gene (Elephant AA sequence homology percentages to human Yamanaka factors are: OCT4 - 88.4%, SOX2 - 98. 1%, KLF4 - 88.6%, MYC - 91.1 %) over-expression to complete the reprogramming process (FIG. 18A). While encouraging changes to morphology compared to primary cells was observed through chemical treatment alone (FIG. 18B, 18C), it was not until transgene introduction, that this process was completed, and a canonical stem cell morphology and growth rate was observed (FIG. 18D). However, even this final transition was difficult to establish, as transgene amino acid sequences from mouse and human were not fruitful. Furthermore, no meaningful change was observed in either grow th, morphology or molecular composition until a final component was added - a transgene to interfere with TP53. Introducing transgenes to over-express SV40 T-antigen or an shRNA that targeted RNA only from TP53 retrogenes and not the full-length TP53 gene (FIG. 1 A, B) ultimately⁷ resulted in success. Interestingly, in the reverse order, when primary⁷ cells were treated to induce transgenes before the treatment with the chemical cocktail, cells quickly senesced and died in all tested conditions.

[0220] Upon clear establishment of correct stem cell morphology, a full stem cell characterization was pursued. The cells were demonstrated to express core pluripotency proteins via immunofluorescence (IF) (FIG. 18E, 18F) and it was confirmed via RNAseq that these core genes and others are indeed present in four derivative emiPSC lines (FIG. 18G). Interestingly, upon examination of the transcriptome of the emiPSC lines, emPRCs, and ernECs, nearly 90% of the transcriptome differences were accomplished with the chemical cocktail alone (FIG. 18H). Finally, OCT4 (POU5F1) epigenetic and transcriptomic read mapping were examined to determine if perhaps the chemical pre-treatment yielded increased regions of open chromatin upstream of the OCT4 locus (FIG. 181). It appears that this indeed was not the case, confirming that the chemical cocktail does a lot of work morphing the general epigenetic landscape of these cells (FIG. 14A-D, FIG. 15), but does not open chromatin upstream of OCT4. EXAMPLE 5 - Testing of Asian elephant pluripotent stem cells

[0221] The Asian elephant pluripotent stem cells obtained in Example 4 were further tested to confirm that they are indeed pluripotent. For this testing, the following methods were used.

[0222] Once the expression of core pluripotency markers was established, the basic grow th and chromosomal normality of the cells was evaluated. To do this, a simple nuclei isolation, lysis, and chromosomal counting methodology was performed to confirm that the cells indeed had 56/56 expected chromosomes (FIG. 19A). Next, the doubling time of the set of emiPSCs on mouse embryonic feeder cells (MEFs) (FIG. 19B) was measured and it was determined that the doubling time for each line was approximately slightly over 2 days, which seemed reasonable given the 2-year elephant gestation period.

[0223] Next, gene expression of genes canonically expressed in primed or naive iPSCs was examined to determine whether the cells were more primed or naive. While the cells had morphology⁷ more generally associated with naive cells in other species and have negligible spontaneous differentiation observed (FIG. 19D), expression of canonical markers could shed more light on this. For primed marker THY1. very highly significant down-regulation across emiPSC lines (FIG. 19C), and highly significant up-regulation of TBX3 (FIG. 19D) was observed. However, across the board with most naive cell markers, upregulation of emiPSCs compared to emECs (FIG. 19E) was observed. There are indeed some canonical primed markers seen significantly upregulated (ZIC2 and GRHL2). but it is known that primed and naive pluripotency markers vary some across species. Considering all these indications, it was reasoned that these cells are in a more naive than primed state.

[0224] Given that the cell lines demonstrated expected molecular and phenotypic qualities of stem cells and a normal karyotype, the differentiation potential of these cell lines was examined next. First, an embryoid body (EB) formation experiment was performed. It was demonstrated that all of these cell lines form EBs in 5-7 days and that they contain cells that express early differentiation markers from each of the three germ layers - PAX6 (ectoderm), GATA4 (mesoderm), and FOXA2 (endoderm) (FIG. 20A). Since they consistently formed these EBs (note that emPRCs were never capable of creating Ebs), NA sequencing on these populations was performed to look for the presence of additional early -differentiation markers (FIG. 20B). While many of these were indeed present and clearly upregulated compared to wild type cells, as with naive versus primed pluripotency markers, species differences should be expected.

[0225] After seeing the presence of many of the expected early differentiation markers in EBs, tri-lineage differentiation assays w ere performed to verify the presence of marker genes in the expected lineages. To this end, up-regulation of early differentiation markers in these differentiated cells compared to both their originating emiPSC line and ernECs (FIG. 20D) was observed.

[0226] Finally, given that the iPSC lines could form EBs and begin differentiation into the three germ layers, it was tested whether these lines could form teratomas. To do this. emiPSCs were provided for hind-leg injections into immuno-compromised mice for 4-6 weeks. It was observed that the lines were indeed able to form teratomas, and that furthermore, a variety of differentiated cell types were observed in the teratomas formed from each cell line. Interestingly, while the differences between the emiPSC lines was relatively minor, more variety of cell types in the cell line that contained an shRNA against TP53 retrogenes, than the lines which required SV40 T-antigen was observed.

[0227] Comparative transcriptomics. Upon establishment of the iPSC lines and their differentiation capacity, a molecular comparison to stem cells from a diverse set of other mammalian stem cells was conducted. First, core pluripotency and naive pluripotency genes from humans, marmosets, mouse, rabbits, cattle, and elephants were investigated (FIG. 21A). It was found that while some of the expression of core pluripotency genes OCT4, SOX 2. NANOG, and LIN28A was much lower in some of the emiPSC lines than all others, expression of some naive factors such as TFAP2C, KLF4. vcA MYC was higher than most. Furthermore, the general trend seemed to align well with the other large mammals - cattle and rhino. A principal component analysis of the overall transcriptome confirmed this (FIG. 21B). Furthermore, based on these natural clustering of species, elephants can be classified as being in the zone of intermediate developmental clock according to analyses in Lazaro, J, et al., Stem Cells, 2023.

[0228] Additional emiPSC Characterization. The emiPSC lines share a vast majority of molecular, morphological, and phenotypic features, however there are minor differences. These are exemplified with small variations to teratoma size and composition (FIGs. 22 A- C). embryoid body features (FIGs. 23A-F), and features related to growth (FIG. 24A and FIG. 24B). Intriguingly, while most iPSCs from other well-studied organisms exhibit a strong down-regulation of TP53, CDKN1A, CDKN1B, CDKN2A, and CDKN2B, emiPSCs appear to do the opposite; in most cases these genes are either up-regulated or unchanged compared to primary cells.

[0229] Reinforced reprogramming transgene expression. While the emiPSCs passed the test in a multitude of molecular and functional tests, it was curious that NANOG expression was non-significantly upregulated in the emiPSCs. This breaks cannon for stem cells created from other species, however. To address this point, another round of nucleofection with Piggy Bae genes was performed to attempt to boost NANOG expression in these cells by adding extra copies of both elephant Yamanaka factors (OSKM) and NANOG individually (FIG. 25 A, FIG. 25B). To affirm that endogenous NANOG expression was increased (as opposed to simply transgene expression), RT-qPCR with primers that bridge intron-exon gaps of mRNA that the transgenes do not have was performed. The results demonstrate that endogenous NANOG (and endogenous SOX2) expression were indeed upregulated in all lines, however the functional implications of these cell line changes remain under study.

[0230] The testing in Examples 3-5 show a successful protocol for generating induced pluripotent stem cells in elephants. The protocol, which required complex chemical media formulations to partially reprogram primary cells and elephant-specific transgenes to fully reprogram, can create emiPSCs with variable sets of transgenes that each exhi bit slightly different characteristics. Furthermore, cells were validated their karyotypic integrity, measured their high growth rate at negligible spontaneous differentiation rates, and determined that these cells are probably naiver than primed in nature.

[0231] The emiPSC lines were then functionally characterized and it was demonstrated that they were able to form EBs and differentiated into the three germ layers. Next, it was demonstrated that these lines are also capable of producing teratomas with diverse cell typepopulations within, proving that they do indeed possess high differentiation potential.

Finally, these cells were compared to iPSCs from a diverse set of other mammal iPSCs, and it was shown that these cells seem to cluster closest to other large-massed mammals.

Protocols used in Examples 3-5

[0232] The following protocols were used to generate the data discussed in Examples 3-5. [0233] RNA sequencing. RNA extraction was performed using the RNeasy Plus Universal Mini Kit (Qiagen 73404). After RNA preparation, RNA was sent directly to NovoGene for external QC and sample sequencing. For ATAC-seq sample, a frozen cell pellet was sent to NovoGene and all assays, library prep, and sequencing was performed by NovoGene.

[0234] NGS analysis. RNA-seq analysis was performed using the Expression Analysis in RNASeq workflow on the Form Bio platform. Reads are trimmed using TrimGalore, to remove low quality (qual < 25) ends of reads and remove reads < 35bp. Trimmed reads are aligned to a reference genome using STAR2 (default) or HiSAT. BAMs from the same sample generated by multiple runs are merged using Samtools. The abundance of transcripts and genes are assessed using FeatureCount to generate raw gene counts, StringTie to generate FPKM and Salmon to generate raw transcript counts. Sample comparisons and differential gene/transcript expression analysis are performed using EdgeR, DESeq2 and IsoformSwitchAnalyzeR. ATAC-seq analysis is performed in a similar workflow. Specifically, reads are trimmed using TrimGalore, to remove low-quality (qual < 25) ends of reads and remove reads < 35bp. This workflow can be run with native open-source tools (NOST) or with Parabricks. With NOST. trimmed reads are aligned to a reference genome using BWA mem or Minimap2. BAMs from the same sample generated by multiple runs are merged using Samtools. Alignment quality is assessed using FastQC, Samtools, and Bedtools. With Parabricks, trimmed reads are aligned, duplicate reads are marked, and alignment quality’ is accessed using fq2bam. Quality metrics are summarized with MultiQC. [0235] Comparative transcriptomics. Public RNA-seq data for human, marmoset, mouse, rabbit, cattle, and rhinoceros were downloaded from NCBI Short Read Archive (SRA). GRCh38, mCalJal.2.pat.X, GRCm38, OryCun2, BosTau9. NRM-Dsumatrensis-vl as human, marmoset, mouse, rabbit, cattle, and rhinoceros reference genome were used, respectively. The count matrices from all species were subsequently merged by homologous gene sets that were downloaded from BioMart. Technical biases across datasets were then minimized by RUVg function with top 5,000 empirical controls in RUVSeq Bioconductor package (vl.28.0).

[0236] Cellular reprogramming. Primary Elephas maximus endothelial cells (emPRC) were used as the starting cell line for reprogramming. These cells are maintained in 30% FBS/1% antibiotic/antimycotic /!% Non-essential AA/ EGM-2 media (Lonza) with Laminin521 coating (5 zg/ml; Gibco A29248). Once the cells became approximately 70% confluent, a chemical cocktail medium was used for partial reprogramming to emPRCs - KO DMEM (Gibco 10829-018) + 10% KOSR (Invitrogen) + 55 //M 2-mercaptoethanol (55 mM (1000X); Gibco 21985023) + 50 ng/ml bFGF (20 //g/ml; heat stable; Life technologies PHG0369) + 0.5 mM VPA (EtOH; Selleckchem S3944) + 5 zM CHIR-99021 (DMSO; Selleckchem S1263) + 2 /M RepSox (DMSO; Selleckchem S7223) + 10 //M Tranylcypromine (2-PCPA) HC1 (DMSO; Selleckchem S4246) + 20 /M Forskolin (DMSO; Selleckchem S2449) + 1 //M Ch 55 (DMSO; Tocns 2020) + 5 //M EPZ004777 (DMSO; Selleckchem S7353). The medium was changed every two days until small emPRC colonies were observed. Once colonies reached sufficient size, they were hand-picked and mechanically passaged 2-3 times. Once a 2-3 million emPRCs 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 OCT41 SOX21 KLF41 cMyc (C4) or L. africana OCT4ISOX2IKLF4lcMydLIN28a (C5) ox L. africana OCT4ISOX2IKLF4lcMydLIN28alNANOG (C6)] and [SV40 T-antigen or an shRNA targeting TP53 retrogenes in Elephas maximus], Cells were recovered, selected with mammalian selection markers hygromycin and puromycin, and induced for 1 month until full emiPSC (Elephas maximus iPSC) morphology, grow th, and molecular signature were observed.

[0237] Immunofluorescent staining. EBs were fixed using 4% v/v paraformaldehyde (15710, Electron Microscopy Sciences, Hatfield, PA, USA) for 20 minutes, washed three times and permeabilized with 0.5% Triton X-100 (Sigma- Aldrich, Saint Louis, MO), for 20 minutes. Samples were washed three times and blocked with 5% BSA. 0.05% Triton for 1 hour and incubated with primary antibodies diluted in 1% BSA, 0.05% Triton-XlOO overnight at 4 °C. Samples were washed three times and incubated with secondary antibodies for 1 hour at 37 °C, then washed three times and counter-stained with Hoechst 33342 (Hl 399, Invitrogen, Carlsbad, CA, USA) for 10m at RT, washed three times and mounted with Vectashield (Vector Labs., H100010A). Micrographs were acquired with a Zeiss AxioObserver.5 LED fluorescent microscope. High Performance microscopy camera Axiom 705 mono R2. Objective LD A-Plan 10X/0.25 Phi. emiPSCs were stained with custom elephant-specific antibodies for NANOG and OCT4 with TXR-anti-rabbit (Invitrogen T- 2767) and SOX2 (Invitrogen MAI-014) with AF488-anti-mouse (Invitrogen A-21202). EBs were stained with for GATA4 with a custom elephant-specific antibody, PAX6 (Invitrogen MAI-109), and FOXA2 (Novus NB100-1263). Secondary antibodies used were TXR-anti- rabbit (Invitrogen T-2767), TXR-anti-goat (Invitrogen PAI-28662), and AF488-anti-mouse (Invitrogen A-21202).

[0238] Embryoid body formation. Embryoid bodies (EBs) were formed using either AggreWell 400 plates (StemCell 34450) or ultralow- attachment ninety-six well plates (Coming 4515). Plates were pre-treated with Anti-Adherence Rinsing Solution, after which approximately 400 cells/microwell for AggreWell 400 or (5k/well) for 96-well plates in the described reprogramming medium above were added and centrifuged at 100g for 3m. Plated cells were left undisturbed for 24 hours at 37 °C with 5% CO2 and 95% humidity. After 24 hours, medium w as changed every 48 hours with either AggreWell EB Formation Medium (StemCell 05893) or reprogramming medium without disturbing the cells, prior to IF and imaging.

[0239] Tri-lineage differentiation. 50K emiPSCs were plated onto a Laminin521 coated twelve well plates and treated for 10 days with the STEMdiff Trilineage Differentiation Kit (StemCell 05230).

[0240] Chromosomal Isolation and Counting. Chromosomal isolation and counting were performed by incubating cells in for 3 hours at 37 °C. Afterwards, cells were resuspending cells in 0.075M KC1 solution at 37 °C for 8 minutes. Next, cells were resuspended in 1 ml of fixative and gently mixed and incubated at RT for 10 minutes. Cells were then centrifuged at 900rpm for 8 minutes and resuspended in a fixative at RT for 10 minutes. This fixation step was repeated twice. Finally, cells were mounted onto a slide with dye for imaging.

[0241] Teratoma 200k - IM emiPSCs were injected into the hindleg of immunocompromised mice. Legs were observed daily for 6 weeks, after which tumor masses were extracted. Teratoma sections were evaluated by Histowiz.

EXAMPLE 6: Derivation of Elephant Induced Pluripotent Stem Cells [0242] This example describes the generation of Elephas maximus induced pluripotent stem cells (emiPSCs) using novel reprogramming methods. A multidimensional reprogramming protocol: a chemical-based approach followed by core reprogramming transcription factor overexpression was used. The key pluripotency molecular features of the emiPSCs as well as their ability to differentiate were characterized. A genomics-based approach was used to compare the emiPSCs to stem cells of other mammals and showed that the features of the emiPSCs are most like those of rhinoceroses and cattle, two other large land mammals. Finally, the molecular features of elephant, naked mole rat, human, and mouse iPSCs that have implications in cancer-related gene pathways were compared.

Methods

[0243] RNA sequencing. RNA extraction using the RNeasy Plus Universal Mini Kit (Qiagen 73404) was performed. After RNA preparation, RNA was sent directly to NovoGene for external QC and sample sequencing.

[0244] ATAC sequencing. For ATAC-seq sample, libraries were prepared with a ActiveMotif kits (ActiveMotif 53150) following instructions from the manufacturer. Completed libraries were QC’ed via TapeStation and sequenced on Illumina NextSeq and Illumina NovaSeq platforms.

[0245] Direct RNA sequencing. Total RNA was prepared for sequencing using the direct-RNA kit SQK-RNA004 (Oxford Nanopore Technologies) for ten elephant iPSC samples. Basecalling was performed twice with Dorado version 0.7.2 with the ma004_130bps_sup v5.0.0 model. The flags -estimate-poly-a -modified-bases-threshold 0.0 was employed and separately called for m6a modifications with -modified-bases-models ma004_130bps_sup v5.0.0_m6A vl and pseudouridine modifications with -modified-bases- models ma004_130bps_sup v5.0.0_pseU vl. Base called sequences were converted to fastq files with samtools version 1.20. Flags -T was employed to maintain modification tags. Reads were aligned to the E. maximus GCF_024166365.l_mEleMaxljrimary_haplotype_genomic.fna reference genome from NCBI RefSeq with minimap2 version 2.28. Flags -ax splice -t 16 -uf kl4 -y was employed based on the developer’s recommendations.

[0246] Transcriptomics analysis, using an Expression Analysis RNASeq workflow was performed. Reads were trimmed using TrimGalore (VO.6.7). to remove low quality (qual < 25) ends of reads and remove reads < 35bp. Trimmed reads are aligned to a reference genome using STAR (default) or HiSAT2. BAMs from the same sample generated by multiple runs were merged using Samtools. The abundance of transcripts and genes were assessed using FeatureCount to generate raw gene counts, StringTie to generate FPKM and Salmon to generate raw transcript counts. Sample comparisons and differential gene/transcript expression analysis were performed using EdgeR, DESeq2 and IsoformSwitchAnalyzeR. Variant calling was called with DeepVariant and Freebayes. MA plots were generated with DESeq2 (v3.18) and ggpubr (v0.6.0).

[0247] Epigenetics analysis. Reads were trimmed using TrimGalore (VO.6.7), to remove low-quality (qual < 25) ends of reads and remove reads < 35bp. Trimmed reads are aligned to a reference genome using minimap2 version 2.28. Duplicate reads were marked using Picard MarkDuplicates. BAMs from the same sample generated by multiple runs are merged using Samtools. Alignment quality was assessed using FastQC, Samtools, and Bedtools. Peak calling was run with MACS3. Alignments are converted to BigWIG files. This deepTool computeMatrix was used to calculate scores per genome region and prepares an intermediate file that is used with plotHeatmap and plotProfiles.

[0248] Comparative transcriptomics. Comparative transcriptomics Public RNA-seq data for human, marmoset, mouse, rabbit, cattle, and rhinoceros was downloaded from NCBI Short Read Archive (SRA) (ERP141318) and aligned to each reference genome as described above. GRCh38, mCalJal.2.pat.X, GRCm38, OryCun2, BosTau9, NRM-Dsumatrensis-vl was used as human, marmoset, mouse, rabbit, cattle, and rhinoceros reference genome, respectively. The count matrices from all species were subsequently merged by homologous gene sets that were downloaded from BioMart. Technical biases across datasets were then minimized by RUVg function with top 5,000 empirical controls in RUVSeq Bioconductor package (vl.28.0). RNA-seq data of ESCs and ICMs were also downloaded from NCBI SRA (ERP141318. SRP034545, SRP277253, ERP141318, SRP031504, SRP218834, ERP006823, SRP045318, SRP077283, SRP121791, SRP199053, SRP361145, SRP377779, SRP274355, SRP067094, SRP218834, SRP358136, SRP408055, SRP412732, SRP277256, SRP361483, and aligned to reference genomes: GRCh38 (human), Mmul_10 (Rhesus), BosTau9 (Cow), Sscrofal l. l (Pig), ASM130575vl (horse and wild horse), NRM- Dsumatrensis-vl (Rhinoceros), OryCun2 (rabbit), GRCm38 (mouse), and mRatBN7.2 (rat). Similarly, the count matrices were merged by BioMart homologous genes, and normalized by RUVg function as described above. RNA-seq of iPSCs and their parental somatic cells of naked-mole rat (DRP003111). human (SRP150642), and mouse (SRP116326) were merged by BioMart homologous genes and normalized with SCTransform by regressing species. Up- and down -regulated genes in iPSCs to parental somatic cells were identified by DESeq2 with 2-fold change and p<0.05 cutoff. Gene Ontology' enrichment w as analyzed by GOstats (v2.68.0) Bioconductor package.

[0249] Chemically-aided iPSC reprogramming. Primary E. maximus endothelial cells (emECs) were used as the starting cell line for reprogramming. These cells are maintained in 30% FBS/1% antibiotic/antimycotic /1% Non- essential AA/ EGM-2 media (Lonza) with Laminin521 coating (5 wg/ml: Gibco A29248). Once the cells became approximately 70% confluent, a chemical cocktail medium was used for partial reprogramming to emPRCs - KO DMEM (Gibco 10829-018) + 10% KOSR (Invitrogen) + 55 //M 2-mercaptoethanol (55 mM (1000X); Gibco 21985023) + 50 ng/ml bFGF (20 zzg/mk heat stable; Life technologies PHG0369) + 0.5 mM VPA (EtOH; Selleckchem S3944) + 5 /M CHIR-99021 (DMSO; Selleckchem S1263) + 2 /zM RepSox (DMSO; Selleckchem S7223) + 10 zM Tranylcypromine (2-PCPA) HC1 (DMSO; Selleckchem S4246) + 20 /zM Forskolin (DMSO; Selleckchem S2449) + 1 zM Ch 55 (DMSO; Tocris 2020) + 5 zM EPZ004777 (DMSO;

Selleckchem S7353). The medium was changed every' two days until small emPRC colonies were observed. Once colonies reached sufficient size, they were hand-picked and mechanically passaged 2-3 times. Once 2-3 million emPRCs were available, they were nucleofected with plasmids encoding genome-integrating (via PiggyBac), inducible, polycistronic transgene expression cassettes. These cassettes contained one of [Loxodonta africana OCT4^/SOX2/KLF4/CMYC (C4) or L. africana OCT4/SOX2/KLF4/CMYCEIN28A (C5) or L. africana OCT4/SOX2/KLF4/CMYC/LIN28A/ NANOG (C6)] and [SV40 T- antigen or an shRNA targeting TP 53 retrogenes in E. maximus}. The cells were recovered, selected with mammalian selection markers hygromycin and puromycin, and induced for 1 month until full emiPSC morphology⁷, growth, and molecular signature was observed.

Afterw ards, the cells w ere transferred onto mouse embryonic feeder cells (MEFs) for longterm maintenance and propagation (ThermoFisher A34966).

[0250] Embryoid body formation. Embryoid bodies (EBs) were formed using two basic methods: (1) an AggreWell based method and (2) a simple aggregate expansion and plating method. For method ‘(1)’ either AggreWell 400 plates (StemCell 34450) or ultra-low' attachment ninety-six well plates (Coming 4515). Plates were pre-treated with Anti- Adherence Rinsing Solution, after which approximately 400 cells/microwell for AggreWell 400 or (5k/well) for 96-well plates in the described reprogramming medium above were added and centrifuged at 100g for 3 minutes. Plated cells were left undisturbed for 24 hours at 37°C with 5% CO2 and 95% humidity. After 24 hours, medium was changed every’ 48h with either AggreWell EB Formation Medium (StemCell 05893) or reprogramming medium without disturbing the cells, prior to IF and imaging. For method ‘(2)’, emiPSCs w ere passaged as single cells using Accutase for 15 minutes and then plated into a low-attachment cell culture plate in DMEM/F12 (Invitrogen) + 10% Knock-out Serum Replacement (KOSR) + Y-27632 for 24 hours. Cells were cultured in this media for 5 days, carefully not to disrupt aggregates on media replacement. Afterw ards, aggregates were collected and plated onto standard Gelatin-coated plastic-bottom or glass-bottom plates for an additional 12 days in DMEM (Invitrogen) + 10% FBS (Gibco) + 1% L-glutamine.

[0251] Tri-lineage differentiation. 50.000 emiPSCs were plated onto a Laminin521 coated twelve well plates and treated for 5 days (Endoderm), 7 days (Mesoderm), and 10 days (Ectoderm) with STEMdiff Trilineage Differentiation Kit (StemCell 05230) as per the manufacturer's specifications.

[0252] Dual SMAD inhibition. Dual SMAD inhibition was performed as previously described in Chambers et al.. Nature Biotechnology. 27(3):275-280. 2009. Briefly. emiPSCs were re-plated onto a dish at approximately 10,000-25,000 cells/cm² Once the emiPSCs were nearly -confluent, they w ere treated with 10 mM TGF-P inhibitor SB431542 (Tocris) and 500 ng/ml of Noggin (R&D). At Day 3 of differentiation, an increasing amount of N2 media (25%, 50%, 75%) was added to the emiPSCs medium media every 2 days to Day! 1.

[0253] Neurogenin-2 (NGN2) neuronal differentiation. Neuronal induction was performed approximately as described in Zhang et al., Neuron, 78(5):785-798, 2013, with some modifications to adapt for given the emiPSC perpetual induced expression of reprogramming factors until differentiation initiated. Specifically, as opposed to lentiviral or PiggyBac-based E. maximus NGN2 (em.NGN2) delivery, plasmids to express these transgenes transiently w ere built and the protocol was only performed until Day 4. In summary’, emiPSCs w ere cultured as normal, nucleofected with transiently expressed NGN2 and plated in emiPSC medium on Matrigel-coated plates. On the first day after transfection, N2 medium was applied as described in Zhang et al. for three additional days.

[0254] Cardiomyocyte-like cell differentiation. Cardiomyocyte differentiation was performed for 34 days. On Day -5, emiPSCs were plated at a cell density’ of 0.8 million cells in a Matrigel double-coated well of a 12-well plate in the pluripotent grow th media supplemented with 10 //M Y-27632 for 24 hours. On day 0, the cells were treated with 12//M CHIR99021 (Selleckchem SI 263) in RPMI medium supplemented with B27 without insulin for 24 hours which was then changed to fresh RPMI/B27 without insulin. After 2 days 5 //M of IWP2 was added for an additional two days. From day 7 cells were moved into an RPMI/B27 medium (Gibco 17504044) and cultured for an additional 27 days.

[0255] Immunofluorescent staining. Samples were fixed using 4% v/v paraformaldehyde (15710, Electron Microscopy Sciences, Hatfield, PA, USA) for 20 minutes, washed three times and permeabilized with 0.5% Triton X-100 (Sigma- Aldrich), for 10 minutes for monolayer of cells or 30 minutes for EBs. Samples were washed three times and blocked with 5% BSA, 0.05% Triton for 1 hour and incubated with primary antibodies diluted in 1% BSA, 0.05% Triton-XlOO overnight at 4°C. Samples were washed three times and incubated with secondary antibodies for Ih at 37°C, then washed three times and counter-stained with Hoechst 33342 (H1399, Invitrogen, Carlsbad, CA, USA) or DAPI for 10 minutes at RT, washed three times and mounted with Vectashield (Vector Labs., H100010A). Micrographs were acquired with a Nikon AX confocal Ti2 microscope system Objective PLAN APO 20x OFN25 DIC N2 and a Plan Fluor 40x Oil DIC H N2, and the software NIS-Elements AR 5.41.02. Since elephants often have a considerably diverged amino acid sequence compared to human, mouse, and other studied mammal sequences, made and validated elephant amino-acid-sequence-specific antibodies for IF staining were made and validated. In some cases where the selected epitope of the custom-designed antibody overlapped perfectly with antibodies targeting the same region, commercial antibodies were substituted after testing positive controls in human iPSCs. emiPSCs were stained with custom elephant-specific antibodies for NANOG and OCT4 with TXR- anti-rabbit (Invitrogen T-2767) and SOX2 (Invitrogen MAI-014) with AF488-anti-mouse (Invitrogen A-21202). EBs were stained for PAX6 custom elephant-specific antibody, FOXF1 (AB168383, Abeam), CTNNB1 (Invitrogen MAI -301 and 71-2700), TUBBS (MA1- 118, Invitrogen), CDX2 (Invitrogen MA5-35215) and NOG (Invitrogen CF500116). Trilineage differentiations were also stained with AFP (Invitrogen MA5- 14666), BMP 2 (Invitrogen MA5-38457), and IRF6 (Abeam AB275609). Dual SMAD inhibition assays were stained with elephant-specific antibodies for PAX6 and FOXG1 (Invitrogen PAI -9043) and TUBB3 (Invitrogen MAI- 118). Neurons derived via NGN2 were stained with SYN1 (Invitrogen MA5-31919). Secondary antibodies used were TXR-anti-rabbit (Invitrogen T- 2767), TXR-anti -mouse (Invitrogen T-6390), AF488-anti-rabbit (Invitrogen Al 1055) and AF488-anti-mouse (Invitrogen A-21202).

[0256] Chromosomal Isolation and Counting. Chromosomal isolation and counting were performed by incubating cells for 3 hours at 37°C. Afterwards, cells were resuspended in 0.075M KC1 solution at 37C for 8 minutes. Next, cells were resuspended in 1 ml of fixative and gently mixed and incubated at RT for 10 minutes. Cells were then centrifuged at 900 rpm for 8 minutes and resuspended in a fixative at RT for 10 minutes. This fixation step was repeated twice. Finally, cells were mounted onto a slide with dye for imaging.

[0257] Sendai virus reprogramming attempts. E. maximus endothelial and epithelial cells were transduced with Sendai virus (ThermoFisher Scientific, A16517) following manufacturer’s instructions, with an MOI of 5:5:3 and an MOI of 10:10:6. Different cell densities were tested, with 1 x 10⁵, 1.5 x 10⁵ and 3 x 10⁵ cells transduced per reaction, with the addition of 5//g/mL of protamine sulfate (Sigma, P3369-10G). Cells were seeded on 3T3- J2 cells (StemCell Technologies: 100-0353) and Geltrex (ThermoFisher Scientific, A1413302).

[0258] Lentivirus reprogramming attempts. E. maximus endothelial and epithelial cells were transduced with Lentivirus (Sigma, SCR5451) following manufacturer's instructions, with an MOI of 2, 5, 10, and 20. A total of 1 x 10⁵ and 1.5 x 10⁵ cells were transduced per reaction, with the addition of 5/zg/mL of protamine sulfate (Sigma, P3369- 10G). Cells were seeded on 3T3-J2 cells (StemCell Technologies: 100-0353) and Geltrex (ThermoFisher Scientific, A1413302).

[0259] Lentivirus reprogramming (Sigma, SCR5451) with an MOI of 10 was also tested in E. maximus endothelial and epithelial cells, following manufacturer’s instructions, with the addition of transcription factors OCT4, LIN28A, and NANOG (Cell omics, PLV-10012- 50, PLV-10015-50, and PLV-10075-50 respectively), each at an MOI of 10. A total of 1 x 10⁵ and 1.5 x 10⁵ cells were transduced per reaction, with the addition of 5 rg/mL of protamine sulfate (Sigma, P3369-10G). Cells were seeded on 3T3-J2 cells (StemCell Technologies: 100-0353) and Geltrex (ThermoFisher Scientific, A1413302).

[0260] Teratoma. 200,000-1,000,000 emiPSCs were injected into the hindleg of immunocompromised mice. Legs were observed daily for 5.5 weeks, after which tumor masses were surgically extracted. Teratoma sections were evaluated by professional histologists at Histowiz.

Asian elephant reprogramming

[0261] After a comprehensive screen of more traditional reprogramming approaches failed, elephant induced pluripotent stem cells were generated using chemical-based pluripotency media with selected colony expansion followed by overexpression of key pluripotency transcription factors OSKM(LN) and oncogene SV40LT and/or short hairpin RNAs (shRNAs) against elephant TP53. Multiple attempts with current standard reprogramming methods were tried, and failed, and resulted in no, or incomplete, reprogramming. First, reprogramming of E. maximus cells was atempted by over-expressing original ‘Yamanaka reprogramming factors’: OCT4. SOX 2. KLF4, and MYC (OSKM) in different vector formats. This included transgene expression via episomal, Lentivirus, Sendai-virus, and PiggyBac in a variety of different combinations with transcription factor (TF) sequences from either mouse or human. These transgene expression methods were also tested in tandem with the overexpression of shRNA that target TP53, and over-expression of SV40 T-antigen, NANOG, and/or LIN28A. While some approaches yielded potential morphological differences compared to primary cells at different parts of the process, ultimately all atempts failed as a result of either cell death, senescence, or no observed morphological changes compared to starting primary parental lines (see FIGs. 26 to 34).

[0262] In addition to transgene-dependent methods, chemical reprogramming methods that do not require the use of transgenes were also explored. A set of published chemical cocktails (Hou et al.. Science, 341(6146):651-654, 2013; Guan et al., Nature, 605(7909):325- 331, 2022; and Chen et al., Nature Cell Biology, 25(8): 1146-1156, 2023) that was customized by varying chemical constituents in those cocktails was tested. These approaches resulted mostly in cell death, senescence, or no observ ed change, but in a small set of experiments also appeared to yield encouraging morphological changes.

[0263] Integration of these two types of approaches finally resulted in the first generation of E. maximus induced pluripotent stem cells (emiPSCs). More specifically, emiPSCs were derived by first partially reprogramming primary endothelial cells (emECs) to an intermediate ‘pre-iPSC’ state (emPRC) with a derivative chemical reprogramming protocol based on prior work by Hou et al., Science, 341 (6146): 651-654, 2013, and then using elephant-specific transgene (Elephant AA sequence homology percentages to human Yamanaka factors are: OCT4 - 88.4%, SOX2 - 98.1%, KLF4 - 88.6%, MYC - 91.1%) (FIG. 35A and FIG. 35B) overexpression to complete the reprogramming process (FIG. 36A). While observing encouraging morphological changes compared to primary cells through chemical treatment alone (FIG. 36B, FIG. 36C, and FIG. 29E), the more canonical stem cell morphology was observed only after introduction of pluripotency transgenes (FIG. 36D, FIG. 29F). Furthermore, no change in morphology relative to the pre-iPSCs was observed until the TP53 expression was modulated. This was achieved by over-expression of SV40 Large T-antigen or an shRNA that only targeted RNA from TP53 retrogenes and not the full-length TP53 gene (FIG. 30 A and FIG. 30B). Interestingly, when primary cells were treated to induce transgenes before the treatment with the chemical cocktail, cells quickly senesced and underwent apoptosis in all tested conditions. [0264] Next, and to follow this progress, a reprogramming method that does not require an intermediate step or complicated chemical formulations was developed. While canonical transgene-only methods were tried, it was noted that much of the epigenetic landscape of the primary’ cells needed to be altered with chemicals before the transgenes were able to complete the reprogramming process (FIG. 36F). Thus, a method in which a mutated HRAS gene (HRASG12V) can be used to remove some epigenetic barriers by mitigating antagonism between transcription and DNA replication was explored. It was found that with the same transgenes used in the chemical method, HRASG12V could be used in simpler media conditions to reprogram emECs directly to emiPSCs as well.

[0265] Upon establishing a canonical stem cell morphology⁷ for both reprogramming methods (FIG. 36D), a full stem cell characterization was pursued. First, it was demonstrated that emiPSCs express core pluripotency proteins via immunofluorescence (IF) (FIG. 36E, FIG. 37). Next, it was confirmed via RNA-seq that these core genes and others are indeed present in four denvative emiPSC lines (FIG. 36G). Interestingly, upon a further examination of the transcriptome of the emiPSC lines, emPRCs, and emECs, nearly 90% of the transcriptome differences were accomplished with the chemical cocktail alone (FIG. 36 F). Finally. OCT4 POU5F1) epigenetic and transcriptomic read-mapping was examined to determine if perhaps the chemical pre-treatment yielded increased regions of open chromatin upstream of the OCT4 locus. The chemical cocktail was found to accomplished the majority⁷ of the modulation for the epigenetic landscape of these cells (FIG. 33A-33D and 34) yet was not sufficient to modulate chromatin accessibility upstream of OCT4.

[0266] Interestingly, highly significant upregulation of NANOG expression in the emiPSCs was not observed. This low NANOG expression pattern is unusual for iPSCs of any species studied to date. Of all core pluripotency’ genes, NANOG is the most genetically’ divergent across species (FIG 35A, 35B, and 37A-D). This is interesting because of OCT4, SOX2, KFL4, and NANOG, NANOG has show n to have the most implication in cancer and cancer stem cells. E. maximus has an extra exon at the end of NANOG, whose function is currently unknown, and while the DNA binding domain seems to be structurally conserved, the rest of the protein in elephants is highly divergent from human and mouse. Despite these structural differences, increasing NANOG and other core pluripotency gene expression (FIG. 39) to quantities typically seen in other species' stem cells was still attempted. To boost NANOG expression in these cells, another round of nucleofection was performed with pluripotency genes. Extra copies of both elephant Y amanaka factors (OSKM), and NANOG individually were added (FIG. 40A-40C). To show⁷ increased expression of endogenous NANOG (not simply transgene expression), RT-qPCR was performed with primers that bridge intron-exon gaps of mRNA in NANOG and the coding sequence - 3’ UTR region of S0X2, otherwise not found in transgenes (see Table 6-4 for primers). The results demonstrate that endogenous NANOG (and endogenous 0X2) expression was boosted in the emiPSC lines. After establishing a boost to endogenous expression of NANOG and SOX2, it was assessed whether the cells could withstand the withdrawal of transgene over-expression driven by doxycycline (DOX). DOX was withdrawn for 10 days, and it was observed via RNAseq that, w hile the expression of OCT4 and SOX2 decreased, they remained significantly upregulated compared to parental cells (FIG. 41 A). Further, the expression profile of other core pluripotency markers and cell-cycle regulatory markers remained mostly unchanged (FIG. 41B-D), thus DOX from downstream differentiation studies was withdrawn.

Asian Elephant pluripotent stem cells

[0267] Once the expression of core pluripotency markers was established, the basic growth and chromosomal normality of the cells w as evaluated. A simple nuclei isolation, lysis, and chromosomal counting methodology was performed to confirm that the cells indeed had 56/56 of the expected chromosome number for all lines. Whole-genome sequencing (FIG. 42A-42C) was also performed, and it was found that, w hile the cell lines may have evolved some SNPs and indels, few- candidate indels resulted in a frameshift. Next, the doubling time of the set of emiPSCs on mouse embryonic feeder cells (MEFs) (FIG. 43 A) was measured and it was determined that the doubling time for each line was approximately 2 days, which seemed reasonable given the 22-month elephant gestation period.

[0268] Next, expression of genes canonically expressed in primed versus naive iPSCs w as examined to determine w hether either ty pe of the emiPSCs was more primed or naive. While it was observed that the chemically reprogramming strategy resulted in relatively naive morphology with negligible spontaneous differentiation, cells reprogrammed with transgenes only exhibited a more classically primed morphology (FIG. 36D). How ever, since morphology is often a lower-confidence metric, and due to a true positive control at present (an elephant embryonic stem cell line), the expression of canonical naive and primed stem cell markers (FIG. 43B) was examined. Significant upregulation of many naive genes such as TFAP2C, AXIN2, and TFCP2L1 in all emiPSC lines, as well as upregulation of canonically primed markers such as GRHL2 and ZIC2 in all emiPSCs was observed.

[0269] To map the gene regulatory framework of these cells. RNA-seq and ATAC-seq data for key markers NANOG. KLF4, THY1 , and SOX2 (FIG. 43C) was examined.

Analysis of both SOX2 RNA and open chromatin showed similar patterns for all emiPSCs and show ed relatively low RNA expression, but a clear pattern of open chromatin in the 5’ region of the gene compared to a mostly empty read region in emECs. KLF4 was intriguing because, although it is expressed in endothelial cells, it was upregulated highly in the chemically reprogrammed cells, but down-regulated in the transgene-only reprogrammed cells; the open-chromatin pattern from ATAC-seq supported this finding. In contrast, another stem cell marker that is present in both endothelial cells and stem cells ( HY!) displayed the opposite trend. Finally, NANOG had very low expression in both cell types, with two important observations: first, the extra exon of E. maximus NANOG is expressed (this pattern observed when there is endogenous expression); second, chemically reprogrammed cells have slightly lower expression of this gene, but a much clearer, unique open chromatin pattern.

[0270] Additional analysis of the RNA via direct long-read sequencing with nanopore sequencing also uncovered a large number of potential splice variants that w ere not annotated by automated annotation of the EleMaxl genome (FIG. 44, Table 6-1). This analysis further elucidated NANOG transcript expression (FIG. 45A and 45B) — namely, that it appears that the fifth annotated exon of NANOG could be transcribed separately as is the 4-exon transcript due to the presence of poly-A tails seen after both the 4th and 5th exons. NANOG expression and transcript architecture will require more in-depth studies. The principal component analysis patterns of emEC. emPCR, and emiPSC cell lines for both long- and short-read sequencing (FIG. 46) was further confirmed.

[0271] Following up on this in-depth analysis of many stem cell markers, the differentiation potential of the emiPSCs was established next. To start, a simple Aggrewell-based approach was attempted and four early differentiation markers in EBs (PAX6, GATA4. FOXA2, and CDX2) -wece measured (FIG. 47A-D). Based on these preliminary experiments, multiple EB formation methods, tri-lineage differentiation assays, and directed differentiation to neuronal precursor cells (see Chambers et al. , Nature Biotechnology 27(3):275-280, 2009), neurons (Zhang et al., Neuron, 78(5): 785-798, 2013), and cardiomyocyte-like cells were established. First, two different EB formation assays - a short Aggrewell-based method for 1-3 days (exemplified with chemically reprogrammed emiPSCs in FIG. 48A, FIG. 48B. FIG. 49) and a longer method with a 5-8 day aggregate formation step, followed plating and 2D expansion for 12 more days were performed. All the cell lines form EBs within 1-3 days, and they contained cells that express early differentiation markers from each of the three germ layers (FIG. 48A. 48B, 50A, 50B). Specifically. CDX2 (mesoderm and sometimes extraembryonic tissues and non-neural ectoderm), NOG (ectoderm), and CTNNBJ (endoderm and sometimes other germ layers) were detected in early time point aggregates. At later time points after replating (and in tri-lineage differentiation), TUBB3 (ectoderm), FOXF1 (mesoderm), CTNNB1, wAAFP (endoderm) were detected.

[0272] Next tri-lineage differentiation assays were performed to determine the presence of marker genes in their respective lineages. Up-regulation of early differentiation markers was observed in these differentiated cells compared to both their originating emiPSC line via RNAseq (FIG. 48C and FIG. 51) and IF staining. These expression changes were also generally consistent with the differentiation patterns observed in EB formation.

[0273] Finally, directed differentiations of the emiPSCs into neurons, neural precursors, and cardiomyocyte precursors was performed. Chemically-reprogrammed emiPSCs were differentiated into neurons via transient over-expression of E. maximus NGN 2 for 4 days with a variation of a robust neural differentiation protocol for human iPSCs (Zhang et al. , Neuron. 78(5): 785-798, 2013) and stained the resulting cells for STAY, a pan-neuronal cell marker (FIG. 49D, FIG. 52A, and FIG. 52B). Next, transgene-only reprogrammed emiPSCs were differentiated via dual-SMAD inhibition (Chambers et al., Nature Biotechnology, 27(3y.225- 280, 2009) for 8-11 days and the cell populations were stained for early neural differentiation marker PAX6, early differentiation marker FOXG1, and later neuronal marker TUBB3 (FIG. 52A, 52B). To create cardiomyocyte precursor cells, transgene-only reprogrammed emiPSCs were differentiated in cardiac differentiation medium for 31 days and regulation of key cardiomyocyte marker genes was observed via RT-qPCR.

Comparative Genomics

[0274] After establishing emiPSC lines and their differentiation capacity, a comparative analysis of stem cells from a diverse set of other mammalian stem cells was performed. First, the transcriptome profiles of emiPSCs were compared with those of ESCs or inner cell masses (ICMs) from various species. A principal component analysis of these transcriptomes further supported that large mammals are closer to the iPSC lines than primate or rodent ESCs/ICMs (FIG. 53 A). Based on the clustering of these eleven species, elephants can be grouped into the zone of intermediate developmental clock as described in the stem cell zoo study by Lazaro et al.. Cell Stem Cell, 2023. The same clustering of emiPSCs independent of reprogramming method was observed.

[0275] Given that emiPSC lines could form EBs and differentiate into the early three germ layers, testing was conducted to determine whether these lines could demonstrate differentiation potential in vivo. The standard assay for this is teratoma formation, but there are two complicating factors that had to considered : (1) this assay lacks certain community standards and its presence or absence in datasets is researcher-dependent; and (2), successful demonstrations of these assays in mammals that are cancer-resistant, long-lived, and/or long-gestating species have required either excessively long grow th periods or additional genetic interventionfsee Table 6-2 below).

[0276] In Table 6-2, the species are organized by gestation time in days and the average mass (kg), lifespan (yrs.), and if they have documented cancer resistance (C.R.) are listed. Table 6-2 also indicates if iPSCs have ever been derived for this species and if teratomas have been demonstrated as a measure of their pluripotency.

[0277] The teratoma assay was pursued to probe the differentiation potential of the cells and the hypothesis that it should be very difficult to create teratomas without additional genetic engineering. emiPSCs were injected into hind-leg of immuno-compromised mice and observed growth for 5.5 weeks. Formation of potential teratomas with multiple tissue ty pes for each test emiPSC cell line were detected (FIG. 54A-54C). Interestingly, while the molecular differences between the emiPSC lines was relatively minor, more variety’ of cell types in the cell line that contained an shRNA against TP53 retrogenes. than the lines which required SV40 T-antigen, was observed. At the 5.5 week mark, no growth formation with partially reprogrammed pre-iPSCs or WT emECs was observed. This evidence leads to the hypothesis that perhaps some initial emiPSC outgrowth may be occurring, but halts at this early stage. When the experiment was repeated, growth from cell line Cl-loxC6-SV40 that continued until the 8 w eek mark, in addition to some growth of the pre-iPSCs, was observed; this supports the observation that even the pre-iPSCs have some degree of pluripotency (FIG. 54E, 54E). Given these results, an additional control with a mouse ES Double ASIP/MLPH Knock-Out line (DKO24) was ran for 4 weeks. Although teratoma formation (FIG. 54F) was observed, the tumor appeared necrotic and was primarily of ectodermal composition. The lack of any' endodermal representation in these assays could be the result of high variability in these assays in addition to the biological challenge of producing cancerous growths with elephant cells see Table 6-3).

[0278] Next elephant stem cells were analyzed relative to another cancer-resistant and difficult to reprogram species, the naked mole rat (Heterocephalus glaber). Uniform Manifold Approximation and Projection (UMAP) clustering analysis revealed that elephant and naked mole rat iPSCs share a large number of molecular features that are not shared with non-cancer-resistant species, specifically with respect to TGFB and Wnt signaling pathways (FIG. 53A-53D) and organelle fusion and lipid metabolisms that are known to be less expressed in stem cells in human and mouse (FIG. 53E).

[0279] Once specific genes studied in the context of inhibiting teratoma-creating roadblocks previously studied in Heterocephalus glaber (NMR) were further examined, similar trends of 7M<S 47? -related genes (FIG. 53F), such as CDKN1A and CDKN2A (ARF) were identified. EmiPSCs may also have additional roadblocks to cancer formation in this process via strong and aty pical upregulation of CDKN2A in emiPSCs. Furthermore, while mice rely of ERAS to form tumors, NMR have a mutation in this gene and require mouse ERAS over-expression to form tumors. While the emiPSCs express this gene highly, they still struggle to form tumors. Given that gene clusters relevant to cancer and stem cell biology⁷ also overlap strongly with aging, cross-species trends with aging-related genes (FIG. 53G) were examined. Of particular interest were trends with telomerase TERT), the AMPK pathway (PRKAA 1). and sirtuins. Interestingly. SIRT1 was upregulated in each of the four species, but SIRT7. which is implicated in stem cell aging, is upregulated in NMR and elephants, and downregulated in humans and mouse stem cells.

[0280] Finally, the trends in TP 53 and LIF retrogene expression and core regulatory genes in emiPSCs compared to ernECs (FIG. 55A, 55B. 56A, and 56B) were examined. Upregulation of one TP53 retrogene was consistently observed in the emiPSCs, specifically LOC126068267 - although others may also play minor roles that have yet to characterized. Very intriguingly, in chemically-reprogrammed emiPSCs, canonical LIF expression is high and retrogene activity is low, while in the transgene-only method, a dramatic upregulation of a LIF retrogene LOC126065436 - the E. maximus homolog to LIF6 of L. africana which was previously implicated in apoptotic pathways in primary cells - was seen.

Conclusion

[0281] The testing in this example demonstrates a successful protocol for generating induced pluripotent stem cells in elephants. The emiPSC karyotypic integrity was validate, measured their high growth rate at negligible spontaneous differentiation rates was measure, and it was determined that these cells have significant upregulation of many naive pluripotency markers as well as canonical primed stem cell markers.

[0282] These emiPSC lines were then functionally characterised and it was shown that they were able to form EBs, differentiate into all the three germ layers, and undergo directed differentiation into neurons, neural precursors, and cardiomyocyte-like cells.

Immunofluorescence (for tri-lineage differentiated cells, EBs, and directed differentiation), RNAseq, and RT-qPCR data all affirmed that the expected transcripts and proteins were present in these experiments. While some potential teratoma formation some was observed, it was detailed how tumor formation with elephant cells may not be possible without additional genetic engineering.

[0283] This study describes new methods that enabled the first successful production of emiPSCs, but further optimization of these approaches could help elephant species as well as others. Specifically, further analysis is required into the implication of the TP53 pathway in road-blocking elephant pluripotency. Of particular interest is disentangling the canonical versus the retrogene TP53 contribution to determine the molecular features conferring reprogramming resistance. Also, the duration of reprogramming was long, at almost two months. This duration tends to be 5-10 days for model organisms like mouse, and over 3 weeks for large mammals including humans.

[0284] Comparative analysis across a diverse set of mammalian iPSCs and ESCs showed that elephant iPSCs seem to cluster closest to stem cells from other large-bodied mammals. However, differences between species in pluripotency marker expression and differentiation potential between suggest that more work is necessity' to determine whether reprogramming methods or species differences are driving these observations. For example, the observed initially relatively low expression of NANOG was puzzling and boosting elephant iPSCs with more OSKM and NANOG did increase the overall endogenous NANOG and SOX2 expression.

EXAMPLE 7: Procavia capensis (rock hyrax) iPSCs

[0285] Procavia capensis (rock hyrax) fibroblasts were reprogrammed with the chemical reprogramming method in Example 6. Following the protocol and timeline as described below rock hyrax fibroblasts were reprogrammed into colonies with characteristic stem cell morphology (see FIG. 57A-57C). For additional validation of key stem cell features, kary otype after reprogramming was assessed (FIG. 57D). The cells were stained for stem cell markers OCT4 and SOX2 (FIG. 57E), and the expression of core and secondary plunpotency genes (FIG. 57F and FIG. 57G). Interestingly, these pciPSCs showed similar expression patterns of secondary' pluripotency⁷ 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 KLF4, but the tested Procavia capensis (Rock hyrax) fibroblasts were not extensively characterized. While Rock hyrax does not have TP53 retrogene expansion, they do indeed have the same expansion of LIF retrogenes. Rock hyrax differentiation protocol

[0286] 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 pM Tranylcypromine (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 PiggyBac), inducible, polycistronic transgene expression cassettes. These cassettes contained one of [Loxodonta africana OCT4ISOX2IKLF4ICMYC or L. africana OCT4/SOX2/KLF4/CMY C/LIN28A or L. africana OCT4/SOX2/KLF4/CMYC/LIN28A/NANOG)] and [SV40 T-antigen or an shRNA targeting TP53 retrogenes in Elephas maximus]. Subsequently, the cells were recovered, selected with mammalian selection markers hygromycin and puromycin, and induced for 1 month until full induced pluripotent stem cells (pciPSCs) morphology, growth, and molecular signature was observed. Afterwards, the cells were transferred onto mouse embryonic feeder cells (MEFs) for long-term maintenance and propagation (ThermoFisher A34966).

[0287] 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 exemplar}', 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. EMBODIMENTS

[0288] Provided here are illustrative embodiments of the disclosed technology. These embodiments are illustrative only and do not limit the scope of the present disclosure or of the claims attached.

[0289] Embodiment 1 is a method of generating induced pluripotent Afrotheria species stem cells comprising: (a) chemically reprogramming Afrotheria species cells by culturing primary Afrotheria species cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor; (b) transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-0 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent Afrotheria species stem cells; and optionally (c) selecting for induced pluripotent Afrotheria species stem cells.

[0290] Embodiment 2 is the method of embodiment 1, wherein the method comprises culturing the primary Afrotheria species cells in a culture medium 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-(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, and from about 2 to about 10 pM of a DOT IL inhibitor.

[0291] Embodiment 3 is the method of embodiments 1 or 2, wherein the method comprises transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium 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-(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, and from about 2 to about 10 pM of a DOT IL inhibitor.

[0292] Embodiment 4 is the method of any one of embodiments 1 to 3, wherein the transfecting comprises changing the culture medium every two days.

[0293] Embodiment 5 is the method of any one of embodiments 1 to 3. wherein the transfecting comprises introducing a selection marker. [0294] Embodiment 6 is the method of embodiment 5, wherein the selection marker is antibiotic resistance.

[0295] Embodiment 7 is the method of any one of embodiments 1 to 6, wherein the selecting for induced pluripotent Afrotheria species stem cells comprises treatment with doxycycline and antibiotic selection and wherein the transfected chemically reprogrammed Afrotheria species cells are resistant to the antibiotic used for selection.

[0296] Embodiment 8 is the method of embodiment 7, wherein the antibiotic selection comprises treatment with hygromycin or puromycin.

[0297] Embodiment 9 is the method of embodiment 8, 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.

[0298] Embodiment 10 is the method of embodiment 8, wherein the antibiotic selection comprises daily treatment with puromycin for five days.

[0299] Embodiment 11 is the method of any one of embodiments 1 to 10, wherein the selecting for induced pluripotent Afrotheria species stem cells further comprises culturing the cells in a medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT IL inhibitor.

[0300] Embodiment 12 is the method of embodiment 1 1, wherein 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-(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, and from about 2 to about 10 pM of a DOT IL inhibitor.

[0301] Embodiment 13 is the method of embodiments 11 or 12, wherein the method comprises changing the medium every two days and culturing for at least 150 days.

[0302] Embodiment 14 is the method of any one of embodiments 7 to 13. wherein the selecting further comprises culturing the cells on a feeder layer in a medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-0 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor.

[0303] Embodiment 15 is the method of embodiment 14. wherein 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-(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, and from about 2 to about 10 pM of a DOT IL inhibitor.

[0304] Embodiment 16 is the method of embodiments 14 or 15, wherein the feeder layer is an mouse embryonic fibroblast (MEF) feeder layer.

[0305] Embodiment 17 is the method of any one of embodiments 14 to 16. wherein the selecting further comprises culturing the cells on an antibiotic resistant feeder layer after culturing the cells on the feeder layer with antibiotic selection.

[0306] Embodiment 18 is the method of embodiment 17, wherein the antibiotic selection comprises treatment with hygromycin or puromycin.

[0307] Embodiment 19 is the method of embodiment 18, 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.

[0308] Embodiment 20 is the method of embodiment 18, wherein the antibiotic selection comprises daily treatment with puromycin for five days.

[0309] Embodiment 21 is the method of any one of embodiments 1 to 20, wherein the HD AC inhibitor is valproic acid.

[0310] Embodiment 22 is the method of any one of embodiments 1 to 21, wherein the GSK- 3 inhibitor is CHIR-99021.

[0311] Embodiment 23 is the method of any one of embodiments 1 to 22, wherein the TGF- P inhibitor is RepSox.

[0312] Embodiment 24 is the method of any one of embodiments 1 to 23 wherein the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1.

[0313] Embodiment 25 is the method of any one of embodiments 1 to 24, wherein the activator of eukaryotic adenylyl cyclase is forskolin.

[0314] Embodiment 26 is the method of any one of embodiments 1 to 25, wherein the retinoid is Ch 55.

[0315] Embodiment 27 is the method of any one of embodiments 1 to 26. wherein the DOT1L inhibitor is EPZ004777.

[0316] Embodiment 28 is the method of any one of embodiments 1 to 20, wherein the method comprises culturing Afrotheria species cells in a culture medium supplemented with valproic acid, CHIR-99021, RepSox. tranylcypromine (2-PCPA) HC1. forskolin. Ch 55. and EPZ004777.

[0317] Embodiment 29 is the method of any one of embodiments 1 to 20 or 28, wherein the method comprises transfecting the chemically reprogrammed Afrotheria species cells with at least C4, C5, C6 and SV40LT in a culture medium supplemented with valproic acid, CHIR- 99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin, Ch 55, and EPZ004777.

[0318] Embodiment 30 is the method of any one of clams 1 to 20, 28 or 29, wherein the selecting for induced pluripotent Afrotheria species stem cells comprises culturing the cells in a medium supplemented with valproic acid, CH1R-99021. RepSox, tranylcypromine (2-PCPA) HC1, Ch 55, and EPZ004777.

[0319] Embodiment 31 is the method of any one of embodiments 1 to 30, wherein the method comprises transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4. (ii) C5. (iii) C6, and (iv) SV40LT.

[0320] Embodiment 32 is the method of any one of embodiments 1 to 30, wherein the method comprises transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5. (iii) C6, and (iv) an agent targeting TP53 and/or TP53 retrogenes.

[0321] Embodiment 33 is the method of embodiment 32, wherein the agent is shRNA that targets TP53.

[0322] Embodiment 34 is the method of embodiment 32, wherein the agent is shRNA that targets TP53 retrogenes.

[0323] Embodiment 35 is the method of any one of embodiments 1 to 34, wherein the Afrotheria species is a cell of a species in the clade Paenungulata.

[0324] Embodiment 36 is the method of embodiment 35, wherein the cell is an elephant cell or a rock hyrax cell.

[0325] Embodiment 37 is the method of embodiment 36, wherein the elephant cell is an Elephas maximus cell.

[0326] Embodiment 38. An induced pluripotent Afrotheria species stem cell produced by the method of any one of embodiments 1 to 37.

[0327] Embodiment 39 is an induced pluripotent elephant stem cell expressing at least (i) C4. (ii) C5. (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes, wherein the stem cell is generated by chemically reprogramming elephant cells and transfecting the reprogrammed cells to express at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes.

[0328] Embodiment 40 is an induced pluripotent elephant stem cell expressing at least (i) C4. (ii) C5. (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes, wherein the stem cell is generated by chemically reprogramming elephant cells.

[0329] Embodiment 41 is the induced pluripotent elephant stem cell of embodiment 40, wherein the expression of (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes is greater than the expression of (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in elephant cells that are not chemically reprogrammed.

[0330] Embodiment 42 is the induced pluripotent elephant stem cell of any one of embodiments 39 to 41. wherein the agent is shRNA that targets TP53.

[0331] Embodiment 43 is the induced pluripotent elephant stem cell of any one of embodiments 39 to 41, wherein the agent is shRNA that targets TP53 retrogenes.

[0332] Embodiment 44 is the induced pluripotent elephant stem cell of any one of embodiments 39 to 43. wherein the elephant cell is an Elephas maximus cell.

[0333] Embodiment 45 is a method of differentiating the induced pluripotent elephant cell of any one of embodiments 39 to 44 into endoderm, mesoderm, or ectoderm.

[0334] Embodiment 46 is a method of forming an embryoid body from the induced pluripotent elephant cell of any one of embodiments 39 to 44.

[0335] Embodiment 47 is a cell culture medium for reprogramming cells comprising a medium supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a TGF- inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, and a DOT IL inhibitor.

[0336] Embodiment 48 is the cell culture medium of embodiment 47 wherein the medium comprises a basal medium supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a TGF- f> inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, and a DOT IL inhibitor.

[0337] Embodiment 49 is the cell culture medium of embodiments 47 or 48. wherein the culture medium 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 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 DOT1L inhibitor.

[0338] Embodiment 50 is the cell culture medium of any one of embodiments 47 to 49 wherein the HDAC inhibitor is valproic acid.

[0339] Embodiment 51 is the cell culture medium of any one of embodiments 47 to 50, wherein the GSK-3 inhibitor is CHIR-99021.

[0340] Embodiment 52 is the cell culture medium of any one of embodiments 47 to 51, wherein the TGF-0 inhibitor is RepSox.

[0341] Embodiment 53 is the cell culture medium of any one of embodiments 47 to 52, wherein the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1. [0342] Embodiment 54 is the cell culture medium of any one of embodiments 47 to 53, wherein the activator of eukaryotic adenylyl cyclase is forskolin.

[0343] Embodiment 55 is the cell culture medium of any one of embodiments 47 to 54, wherein the retinoid is Ch 55.

[0344] Embodiment 56 is the cell culture medium of any one of embodiments 47 to 55. wherein the DOT1L inhibitor is EPZ004777.

[0345] Embodiment 57 is the cell culture medium of any one of embodiments 47 to 49, wherein the culture medium is supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1. forskolin, Ch 55. and EPZ004777.

[0346] Embodiment 58 is a kit comprising the cell culture medium of any one of embodiments 47 to 57 and one or more vectors comprising at least (i) C4, (ii) C5, (iii) C6, and

(iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes.

[0347] Further embodiment 1. A method of generating induced pluripotent elephant stem cells comprising: (a) chemically reprogramming elephant cells by culturing primary elephant cells in a 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; (b) transfecting the chemically reprogrammed elephant cells with at least (i) C4. (ii) C5. (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HDAC inhibitor, a GSK-3 inhibitor, a TGF-P inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent elephant stem cells; and optionally (c) selecting for induced pluripotent elephant stem cells.

[0348] Further embodiment 2. The method of further embodiment 1, wherein the method comprises culturing the primary elephant cells in a culture medium 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 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.

[0349] Further embodiment 3. The method of further embodiments 1 or 2, wherein the method comprises transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes T in a culture medium 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 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.

[0350] Further embodiment 4. The method of any one of further embodiments 1 to 3, wherein the transfecting comprises changing the culture medium every two days.

[0351] Further embodiment 5. The method of any one of further embodiments 1 to 3, wherein the transfecting comprises introducing a selection marker.

[0352] Further embodiment 6. The method of further embodiment 5, wherein the selection marker is antibiotic resistance.

[0353] Further embodiment 7. The method of any one of further embodiments 1 to 6, wherein the selecting for induced pluripotent elephant stem cells comprises treatment with doxycycline and antibiotic selection and wherein the transfected chemically reprogrammed elephant cells are resistant to the antibiotic used for selection.

[0354] Further embodiment 8. The method of further embodiment 7. wherein the antibiotic selection comprises treatment with hygromycin or puromycin.

[0355] Further embodiment 9. The method of further embodiment 8, 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.

[0356] Further embodiment 10. The method of further embodiment 8, wherein the antibiotic selection comprises daily treatment with puromycin for five days.

[0357] Further embodiment 11. The method of any one of further embodiments 1 to 10, wherein the selecting further comprises culturing the cells in a medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-J3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor.

[0358] Further embodiment 12. The method of further embodiment 11, wherein 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-(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, and from about 2 to about 10 pM of a DOT1L inhibitor.

[0359] Further embodiment 13. The method of further embodiments 11 or 12, wherein the method comprises changing the medium every two days and culturing for at least 150 days. [0360] Further embodiment 14. The method of any one of further embodiments 7 to 13, wherein the selecting further comprises culturing the cells on a feeder layer in a medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a D0T1L inhibitor.

[0361] Further embodiment 15. The method of further embodiment 14, wherein 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-0 inhibitor, from about 5 to about 25 pM of a monoamine oxidase inhibitor, from about 10 to about 30 pM of an activator of eukary otic 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.

[0362] Further embodiment 16. The method of further embodiments 14 or 15. wherein the feeder layer is an mouse embryonic fibroblast (MEF) feeder layer.

[0363] Further embodiment 17. The method of any one of further embodiments 14 to 16, wherein the selecting further comprises culturing the cells on an antibiotic resistant feeder layer after culturing the cells on the feeder layer with antibiotic selection.

[0364] Further embodiment 18. The method of further embodiment 17, wherein the antibiotic selection comprises treatment with hygromycin or puromycin.

[0365] Further embodiment 19. The method of further embodiment 18, 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.

[0366] Further embodiment 20. The method of further embodiment 18, wherein the antibiotic selection comprises daily treatment with puromycin for five days.

[0367] Further embodiment 21. The method of any one of further embodiments 1 to 20, wherein the HD AC inhibitor is valproic acid.

[0368] Further embodiment 22. The method of any one of further embodiments 1 to 21, wherein the GSK-3 inhibitor is CHIR-99021

[0369] Further embodiment 23. The method of any one of further embodiments 1 to 22, wherein the TGF-(3 inhibitor is RepSox.

[0370] Further embodiment 24. The method of any one of further embodiments 1 to 23 wherein the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1.

[0371] Further embodiment 25. The method of any one of further embodiments 1 to 24, wherein the activator of eukary otic adenylyl cyclase is forskolin.

[0372] Further embodiment 26. The method of any one of further embodiments 1 to 25. wherein the retinoid is Ch 55.

[0373] Further embodiment 27. The method of any one of further embodiments 1 to 26, wherein the DOT1L inhibitor is EPZ004777. [0374] Further embodiment 28. The method of any one of further embodiments 1 to 20, wherein the method comprises culturing elephant cells in a culture medium supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin, Ch 55, and EPZ004777.

[0375] Further embodiment 29. The method of any one of further embodiments 1 to 20 or 28, wherein the method comprises transfecting the chemically reprogrammed elephant cells with at least C4, C5, C6 and SV40LT in a culture medium supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin, Ch 55, and EPZ004777.

[0376] Further embodiment 30. The method of any one of clams 1 to 20, 28 or 29. wherein the selecting comprises culturing the cells in a medium supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HCL Ch 55, and EPZ004777.

[0377] Further embodiment 31. The method of any one of further embodiments 1-30, wherein the method comprises transfecting the chemically reprogrammed elephant cells with at least (i) C4, (n) C5, (in) C6, and (iv) SV40LT.

[0378] Further embodiment 31a. The method of any one of further embodiments 1-30, wherein the method comprises transfecting the chemically reprogrammed elephant cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) an agent targeting TP53 and/or TP53 retrogenes.

[0379] Further embodiment 32. The method of further embodiment 31 a. wherein the agent is shRNA that targets TP53.

[0380] Further embodiment 33. The method of further embodiment 31a, wherein the agent is shRNA that targets TP53 retrogenes.

[0381] Further embodiment 34. An induced pluripotent elephant stem cell produced by the method of any one of further embodiments 1 to 33.

[0382] Further embodiment 35. An induced pluripotent elephant stem cell expressing at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes, wherein the stem cell is generated by chemically reprogramming elephant cells and transfecting the reprogrammed cells to express C4, C5. C6 and SV40LT.

[0383] Further embodiment 36. An induced pluripotent elephant stem cell expressing at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes, wherein the stem cell is generated by chemically reprogramming elephant cells. [0384] Further embodiment 37. The induced pluripotent elephant stem cell of further embodiment 33, wherein the expression of C at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes is greater than the expression of at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in elephant cells that are not chemically reprogrammed. [0385] Further embodiment 38. A cell culture medium for reprogramming cells comprising a medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukary otic adenylyl cyclase, a retinoid, and a DOT IL inhibitor.

[0386] Further embodiment 39. The cell culture medium of further embodiment 38. wherein the medium comprises a basal medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-(3 inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOTIL inhibitor.

[0387] Further embodiment 40. The cell culture medium of further embodiments 38 or 39, wherein 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-0 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.

[0388] Further embodiment 41. The cell culture medium of any one of further embodiments 38 to 40, wherein the HD AC inhibitor is valproic acid.

[0389] Further embodiment 42. The cell culture medium of any one of further embodiments 38 to 41. wherein the GSK-3 inhibitor is CHIR-99021

[0390] Further embodiment 43. The cell culture medium of any one of further embodiments 38 to 42, wherein the TGF-0 inhibitor is RepSox.

[0391] Further embodiment 44. The cell culture medium of any one of further embodiments 38 to 43. wherein the monoamine oxidase inhibitor is tranylcypromine (2- PCPA) HC1.

[0392] Further embodiment 45. The cell culture medium of any one of further embodiments 38 to 44, wherein the activator of eukary otic adenylyl cyclase is forskolin. [0393] Further embodiment 46. The cell culture medium of any one of further embodiments 38 to 45. wherein the retinoid is Ch 55.

[0394] Further embodiment 47. The cell culture medium of any one of further embodiments 38 to 46, wherein the DOT1L inhibitor is EPZ004777.

[0395] Further embodiment 48. The cell culture medium of any one of further embodiments 38 to 40. wherein the culture medium is supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HCL forskolin. Ch 55, and EPZ004777. [0396] Further embodiment 49. A method of differentiating the induced pluripotent elephant cell of any one of further embodiments 34 to 37 into endoderm, mesoderm, or ectoderm. [0397] Further embodiment 50. A method of forming an embryoid body from the induced pluripotent elephant cell of any one of further embodiments 34 to 37.

[0398] Further embodiment 51. The cell of any one of further embodiments 36 to 38, wherein the agent is shRNA that targets TP53.

[0399] Further embodiment 52. The cell of any one of further embodiments 36 to 38, wherein the agent is shRNA that targets TP53 retrogenes.

[0400] Further embodiment 53. The method of any one of further embodiments 1 to 32, wherein the elephant cell is an Elephas maximus cell.

[0401] Further embodiment 54. The induced pluripotent elephant stem cell of any one of further embodiment 35 to 38, wherein the elephant cell is an Elephas maximus cell.

Claims

CLAIMS What is claimed is:

1. A method of generating induced pluripotent Afrotheria species stem cells comprising:

(a) chemically reprogramming Afrotheria species cells by culturing primary Afrotheria species cells in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF- inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a D0T1L inhibitor;

(b) transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF- inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor to generate induced pluripotent Afrotheria species stem cells; and optionally

(c) selecting for induced pluripotent Afrotheria species stem cells.

2. The method of claim 1, wherein the method comprises culturing the primary Afrotheria species cells in a culture medium 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.

3. The method of claims 1 or 2, wherein the method comprises transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6. and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in a culture medium 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.

4. The method of any one of claims 1 to 3, wherein the transfecting comprises changing the culture medium every two days.

5. The method of any one of claims 1 to 3, wherein the transfecting comprises introducing a selection marker.

6. The method of claim 5, wherein the selection marker is antibiotic resistance.

7. The method of any one of claims 1 to 6, wherein the selecting for induced pluripotent Afrotheria species stem cells comprises treatment with doxycycline and antibiotic selection and wherein the transfected chemically reprogrammed Afrotheria species cells are resistant to the antibiotic used for selection.

8. The method of claim 7, wherein the antibiotic selection comprises treatment with hygromycin or puromycin.

9. The method of claim 8, wherein the antibiotic selection comprises treatment with hygromycin, wherein the cells are treated every⁷ two days with hygromycin, and wherein the treatment lasts ten days.

10. The method of claim 8, wherein the antibiotic selection comprises daily treatment with puromycin for five days.

11. The method of any one of claims 1 to 10, wherein the selecting for induced pluripotent Afrotheria species stem cells further comprises culturing the cells in a medium supplemented with an HD AC inhibitor, a GSK-3 inhibitor, a TGF-fl inhibitor, a monoamine oxidase inhibitor, an activator of eukaryotic adenylyl cyclase, a retinoid, and a DOT1L inhibitor.

12. The method of claim 11, wherein 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.

13. The method of claims 11 or 12, wherein the method comprises changing the medium every two days and culturing for at least 150 days.

14. The method of any one of claims 7 to 13, wherein the selecting further comprises culturing the cells on a feeder layer in a 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 DOTIL inhibitor.

15. The method of claim 14, wherein 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.

16. The method of claims 14 or 15, wherein the feeder layer is an mouse embry onic fibroblast (MEF) feeder layer.

17. The method of any one of claims 14 to 16, wherein the selecting further comprises culturing the cells on an antibiotic resistant feeder layer after culturing the cells on the feeder layer with antibiotic selection.

18. The method of claim 17, wherein the antibiotic selection comprises treatment with hygromycin or puromycin.

19. The method of claim 18, 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.

20. The method of claim 18, wherein the antibiotic selection comprises daily treatment with puromycin for five days.

21. The method of any one of claims 1 to 20, wherein the HD AC inhibitor is valproic acid.

22. The method of any one of claims 1 to 21, wherein the GSK-3 inhibitor is CHIR- 99021.

23. The method of any one of claims 1 to 22, wherein the TGF-P inhibitor is RepSox.

24. The method of any one of claims 1 to 23 wherein the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1.

25. The method of any one of claims 1 to 24, wherein the activator of eukaryotic adenylyl cyclase is forskolin.

26. The method of any one of claims 1 to 25, wherein the retinoid is Ch 55.

27. The method of any one of claims 1 to 26, wherein the DOT IL inhibitor is EPZ004777.

28. The method of any one of claims 1 to 20, wherein the method comprises culturing Afrotheria species cells in a culture medium supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, forskolin. Ch 55, and EPZ004777.

29. The method of any one of claims 1 to 20 or 28, wherein the method comprises transfecting the chemically reprogrammed Afrotheria species cells with at least C4, C5, C6 and SV40LT in a culture medium supplemented with valproic acid. CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1. forskolin, Ch 55. and EPZ004777.

30. The method of any one of clams 1 to 20, 28 or 29, wherein the selecting for induced pluripotent Afrotheria species stem cells comprises culturing the cells in a medium supplemented with valproic acid, CHIR-99021, RepSox, tranylcypromine (2-PCPA) HC1, Ch 55, and EPZ004777.

31. The method of any one of claims 1 to 30, wherein the method comprises transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (hi) C6, and (iv) SV40LT.

32. The method of any one of claims 1 to 30, wherein the method comprises transfecting the chemically reprogrammed Afrotheria species cells with at least (i) C4, (ii) C5, (iii) C6, and (iv) an agent targeting TP53 and/or TP53 retrogenes.

33. The method of claim 32, wherein the agent is shRNA that targets TP53.

34. The method of claim 32, wherein the agent is shRNA that targets TP53 retrogenes.

35. The method of any one of claims 1 to 34, wherein the Afrotheria species is a cell of a species in the clade Paenungulata.

36. The method of claim 35, wherein the cell is an elephant cell or a rock hyrax cell.

37. The method of claim 36. wherein the elephant cell is an Elephas maximus cell.

38. An induced pluripotent Afrotheria species stem cell produced by the method of any one of claims 1 to 37.

39. An induced pluripotent elephant stem cell expressing at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes, wherein the stem cell is generated by chemically reprogramming elephant cells and transfecting the reprogrammed cells to express at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes.

40. An induced pluripotent elephant stem cell expressing at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes, wherein the stem cell is generated by chemically reprogramming elephant cells.

41. The induced pluripotent elephant stem cell of claim 40, wherein the expression of (i) C4, (ii) C5, (iii) C6. and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes is greater than the expression of (i) C4. (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes in elephant cells that are not chemically reprogrammed.

42. The induced pluripotent elephant stem cell of any one of claims 39 to 41, wherein the agent is shRNA that targets TP53.

43. The induced pluripotent elephant stem cell of any one of claims 39 to 41, wherein the agent is shRNA that targets TP53 retrogenes.

44. The induced pluripotent elephant stem cell of any one of claims 39 to 43, wherein the elephant cell is an Elephas maximus cell.

45. A method of differentiating the induced pluripotent elephant cell of any one of claims 39 to 44 into endoderm, mesoderm, or ectoderm.

46. A method of forming an embryoid body from the induced pluripotent elephant cell of any one of claims 39 to 44.

47. A cell culture medium for reprogramming cells comprising a 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.

48. The cell culture medium of claim 47 wherein the medium comprises a basal 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 DOT IL inhibitor.

49. The cell culture medium of claims 47 or 48, wherein 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.

50. The cell culture medium of any one of claims 47 to 49 wherein the HD AC inhibitor is valproic acid.

51. The cell culture medium of any one of claims 47 to 50, wherein the GSK-3 inhibitor is CHIR-99021.

52. The cell culture medium of any one of claims 47 to 51, wherein the TGF- inhibitor is RepSox.

53. The cell culture medium of any one of claims 47 to 52, wherein the monoamine oxidase inhibitor is tranylcypromine (2-PCPA) HC1.

54. The cell culture medium of any one of claims 47 to 53, wherein the activator of eukaryotic adenylyl cyclase is forskolin.

55. The cell culture medium of any one of claims 47 to 54, wherein the retinoid is Ch

55.

56. The cell culture medium of any one of claims 47 to 55, wherein the DOT1L inhibitor is EPZ004777.

57. The cell culture medium of any one of claims 47 to 49, wherein the culture medium is supplemented with valproic acid, CHIR-99021 , RepSox, tranylcypromine (2-PCPA) HC1, forskolin, Ch 55, and EPZ004777.

58. A kit comprising the cell culture medium of any one of claims 47 to 57 and one or more vectors comprising at least (i) C4, (ii) C5, (iii) C6, and (iv) SV40LT and/or an agent targeting TP53 and/or TP53 retrogenes.