Methods of preparing induced pluripotent stem cells from mesenchymal stem cells and methods of using the same

Abstract

Disclosed are methods of preparing induced pluripotent stem cells (iPSCs) from mesenchymal stem cells (MSCs) using compounds, e.g., small molecules and growth factors, and without the use of genetic manipulation of the cells. Further disclosed are pharmaceutical compositions comprising the iPSCs, methods of using the pharmaceutical compositions to treat diseases and disorders, and kits.

Description

METHODS OF PREPARING INDUCED PLURIPOTENT STEM CELLS FROM MESENCHYMAL STEM CELLS AND METHODS OF USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority' to U.S. Provisional Patent Application No. 63 / 631,842 that was filed April 9, 2024, the entire contents of which are hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.BACKGROUND

[0003] Current methods of preparing induced pluripotent stem cells (iPSCs) require viral reprogramming, which introduces genetic material through vectors with potential for insertional mutagenesis and other unwanted outcomes. Therefore, there is a need in the art for novel methods to prepare iPSCs that do not require viral reprogramming.SUMMARY

[0004] In an aspect of the current disclosure, methods of preparing induced pluripotent stem cells from mesenchymal stem cells are provided. In some embodiments, the methods comprise: (a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growlh factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated. coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time; (b) contacting the cells of step (a) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming grow th factor beta (TGF- b) ty pe I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time; (c) contacting the cells of step (b) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HD AC) inhibitor, an inhibitor ofS-adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N-lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and (d) contacting the cells of step (c) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacetylase (HD AC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming grow th factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HDAC) inhibitor, an inhibitor of S- adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N-lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacetylase (HDAC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods further comprise contacting the cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a MEK / ERK pathway inhibitor, an inhibitor of Wntproduction, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, heregulin- beta 1, and basic fibroblast growth factor. In some embodiments, the sufficient amount of time of step (a) is about 1 to about 10 days or, optionally, about 4 to about 10 days. In some embodiments, the sufficient amount of time of step (b) is about 1 to about 20 days or, optionally, about 16 to about 20 days. In some embodiments, the sufficient amount of time of step (c) is about 1 to about 12 days or, optionally, about 8 to about 12 days. In some embodiments, the sufficient amount of time of step (d) is about 1 to about 10 days or, optionally, about 7 to about 10 days. In some embodiments, the cells are cultured in hypoxic conditions. In some embodiments, the hypoxic conditions comprise about 1% oxygen to about 10% oxygen, optionally, about 5% oxygen. In some embodiments, the wingless (Wnt) activator comprises or consists of CHIR99021. In some embodiments, the transforming growth factor beta (TGF-b) type I receptor inhibitor comprises or consists of 616452. In some embodiments, the retinoic acid analogue comprises or consists of TTNPB. In some embodiments, the agonist of smoothened (SMO) comprises or consists of SAG. In some embodiments, the tyrosine kinase inhibitor comprises or consists of ABT-869. In some embodiments, the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor comprises or consists of Y-27632 or Tzv. In some embodiments, the inhibitor of JNK1, JNK2 and JNK3 comprises or consists of JNKIN8. In some embodiments, the monoamine oxidase inhibitor (MAOI) comprises or consists of tranylcypromine. In some embodiments, the DNA methylation inhibitor comprises or consists of 5-azacytidine. In some embodiments, the histone methyltransferase G9a inhibitor comprises or consists of UNC0224. In some embodiments, the inhibitor of JNK1 and JNK2 comprises or consists of ruxolitinib. In some embodiments, the CBP / p300 bromodomain (BRD) inhibitor comprises or consists of SGC-CBP30. In some embodiments, the histone deacetylase (HD AC) inhibitor comprises or consists of valproic acid (VPA). In some embodiments, the inhibitor of S-adenosylmethionine-dependent methyltransferase comprises or consists of DZNep. In some embodiments, the DOT1L inhibitor comprises or consists of EPZ004777. In some embodiments, the N-lysine methyltransferase SETD8 inhibitor comprises or consists of UNC0379. In some embodiments, the MEK / ERK pathway inhibitor comprises or consists of PD0325901. In some embodiments, the B-Raf inhibitor comprises or consists of SB590885. In some embodiments, the inhibitor of Wnt production comprises or consists of IWP-2. In some embodiments, the methods comprise (a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days; (b) contacting the cells of step (a) with one or more compound selected from the groupconsisting CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5 -azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) contacting the cells of step (b) with one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) contacting the cells of step (c) with one or more compound selected from the group consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise: (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT- 869, and Y-27632 for about 8 to about 10 days; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA). DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise: (a) contacting mesenchymal stem cells with one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days; (b) contacting the cells of step (a) with one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine. 5- azacytidine, UNC0224. ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) contacting the cells of step (b) with one or more compound consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) contacting the cells of step (c) with one or more compound consisting of CHIR99021, Y-27632, PD0325901. IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise: (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days; (b) culturing the cells of step(a) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the mesenchymal stem cells are derived from umbilical cord tissue, optionally, wherein the umbilical cord tissue comprises umbilical cord blood. In some embodiments, the prepared induced pluripotent stem cells express one or more factors selected from the group consisting of NANOG, OCT4, and SOX2. In some embodiments, the methods further comprise differentiating the prepared induced pluripotent stem cells.

[0005] In an aspect of the current disclosure, induced pluripotent stem cells (iPSCs) are provided. In some embodiments, the iPSCs are produced by a method comprising: (a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming grow th factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time; (b) contacting the cells of step (a) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase- 1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time; (c) contacting the cells of step (b) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho-associated. coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HDAC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N-lysinemethyltransferase SETD8 inhibitor for a sufficient amount of time; and (d) contacting the cells of step (c) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacetylase (HD AC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI). a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho-associated. coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HDAC) inhibitor, an inhibitor of S- adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N-lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacetylase (HDAC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods further comprise contacting the cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, heregulin-beta 1, and basic fibroblast growth factor. In some embodiments, the sufficient amount of time of step (a) is about 1 to about 10 days or, optionally, about 4 to about 10 days. In some embodiments, the sufficient amount of time of step (b) is about 1 to about 20 days or, optionally, about 16 to about 20 days. In some embodiments, the sufficient amount of time of step (c) is about 1 to about 12 days or, optionally, about 8 to about 12 days. In some embodiments, the sufficient amount of time of step (d) is about 1 to about 10 days or, optionally, about 7 to about 10 days. In some embodiments, the cells are cultured in hypoxic conditions. In some embodiments, the hypoxic conditions comprise about 1% oxygen to about 10% oxygen, optionally, about 5% oxygen. In some embodiments, the wingless (Wnt) activator comprises or consists of CHIR99021. In some embodiments, the transforming growth factor beta (TGF-b) type I receptor inhibitor comprises or consists of 616452. In some embodiments, the retinoic acid analogue comprises or consists of TTNPB. In some embodiments, the agonist of smoothened (SMO) comprises or consists of SAG. In some embodiments, the tyrosine kinase inhibitor comprises or consists of ABT-869. In some embodiments, the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor comprises or consists of Y-27632 or Tzv. In some embodiments, the inhibitor of JNK1, JNK2 and JNK3 comprises or consists of JNKIN8. In some embodiments, the monoamine oxidase inhibitor (MAOI) comprises or consists of tranylcypromine. In some embodiments, the DNA methylation inhibitor comprises or consists of 5-azacytidine. In some embodiments, the histone methyltransferase G9a inhibitor comprises or consists of UNC0224. In some embodiments, the inhibitor of JNK1 and JNK2 comprises or consists of ruxolitinib. In some embodiments, the CBP / p300 bromodomain (BRD) inhibitor comprises or consists of SGC-CBP30. In some embodiments, the histone deacetylase (HD AC) inhibitor comprises or consists of valproic acid (VPA). In some embodiments, the inhibitor of S-adenosylmethionine-dependent methyltransferase comprises or consists of DZNep. In some embodiments, the DOT1L inhibitor comprises or consists of EPZ004777. In some embodiments, the N-lysine methyltransferase SETD8 inhibitor comprises or consists of UNC0379. In some embodiments, the MEK / ERK pathway inhibitor comprises or consists of PD0325901. In some embodiments, the B-Raf inhibitor comprises or consists of SB590885. In some embodiments, the inhibitor of Wnt production comprises or consists of IWP-2. In some embodiments, the methods comprise (a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days; (b) contacting the cells of step (a) with one or more compound selected from the group consisting CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine,5 -azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) contacting the cells of step (b) with one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA). DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) contacting the cells of step (c) with one or more compound selected from the group consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise: (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT- 869, and Y-27632 for about 8 to about 10 days; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG. ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise: (a) contacting mesenchymal stem cells with one or more compound consisting of CHIR99021, 616452. TTNPB. SAG, ABT-869, and Y-27632 for about 8 to about 10 days; (b) contacting the cells of step (a) with one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5- azacytidine, UNC0224, ruxolitinib. and SGC-CBP30 for about 16 to about 20 days; (c) contacting the cells of step (b) with one or more compound consisting of CHIR99021. 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) contacting the cells of step (c) with one or more compound consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise: (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452,TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the mesenchymal stem cells are derived from umbilical cord tissue, optionally, wherein the umbilical cord tissue comprises umbilical cord blood. In some embodiments, the prepared induced pluripotent stem cells express one or more factors selected from the group consisting of NANOG, OCT4, and SOX2. In some embodiments, the methods further comprise differentiating the prepared induced pluripotent stem cells.

[0006] In an aspect of the current disclosure, induced pluripotent stem cells (iPSCs) derived from a mesenchymal stem cell are provided. In some embodiments, the iPSCs do not comprise an exogenous polynucleotide.

[0007] In an aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise an iPSC produced by a method comprising: (a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time; (b) contacting the cells of step (a) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming grow th factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor ofC-Jun N-terminal kinase-1 (JNK1). JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time; (c) contacting the cells of step (b) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathwayinhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HD AC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N- lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and (d) contacting the cells of step (c) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacetylase (HD AC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HDAC) inhibitor, an inhibitor of S- adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N-lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacetylase (HDAC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods further comprise contacting the cells with one or more compound selected from the groupconsisting of a wingless (Wnt) activator, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, heregulin- beta 1, and basic fibroblast growth factor. In some embodiments, the sufficient amount of time of step (a) is about 1 to about 10 days or, optionally, about 4 to about 10 days. In some embodiments, the sufficient amount of time of step (b) is about 1 to about 20 days or, optionally, about 16 to about 20 days. In some embodiments, the sufficient amount of time of step (c) is about 1 to about 12 days or, optionally, about 8 to about 12 days. In some embodiments, the sufficient amount of time of step (d) is about 1 to about 10 days or, optionally, about 7 to about 10 days. In some embodiments, the cells are cultured in hypoxic conditions. In some embodiments, the hypoxic conditions comprise about 1% oxygen to about 10% oxygen, optionally, about 5% oxygen. In some embodiments, the wingless (Wnt) activator comprises or consists of CHIR99021. In some embodiments, the transforming growth factor beta (TGF-b) type I receptor inhibitor comprises or consists of 616452. In some embodiments, the retinoic acid analogue comprises or consists of TTNPB. In some embodiments, the agonist of smoothened (SMO) comprises or consists of SAG. In some embodiments, the tyrosine kinase inhibitor comprises or consists of ABT-869. In some embodiments, the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor comprises or consists of Y-27632 or Tzv. In some embodiments, the inhibitor of JNK1, JNK2 and JNK3 comprises or consists of JNKIN8. In some embodiments, the monoamine oxidase inhibitor (MAOI) comprises or consists of tranylcypromine. In some embodiments, the DNA methylation inhibitor comprises or consists of 5-azacytidine. In some embodiments, the histone methyltransferase G9a inhibitor comprises or consists of UNC0224. In some embodiments, the inhibitor of JNK1 and JNK2 comprises or consists of ruxolitinib. In some embodiments, the CBP / p300 bromodomain (BRD) inhibitor comprises or consists of SGC-CBP30. In some embodiments, the histone deacetylase (HD AC) inhibitor comprises or consists of valproic acid (VPA). In some embodiments, the inhibitor of S-adenosylmethionine-dependent methyltransferase comprises or consists of DZNep. In some embodiments, the DOT1L inhibitor comprises or consists of EPZ004777. In some embodiments, the N-lysine methyltransferase SETD8 inhibitor comprises or consists of UNC0379. In some embodiments, the MEK / ERK pathway inhibitor comprises or consists of PD0325901. In some embodiments, the B-Raf inhibitor comprises or consists of SB590885. In some embodiments, the inhibitor of Wnt production comprises or consists of IWP-2. In some embodiments, the methods comprise (a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about10 days; (b) contacting the cells of step (a) with one or more compound selected from the group consisting CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5 -azacytidine, UNC0224, ruxolitinib. and SGC-CBP30 for about 16 to about 20 days; (c) contacting the cells of step (b) with one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) contacting the cells of step (c) with one or more compound selected from the group consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885. and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise: (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT- 869, and Y-27632 for about 8 to about 10 days; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021. Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise: (a) contacting mesenchymal stem cells with one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days; (b) contacting the cells of step (a) with one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5- azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) contacting the cells of step (b) with one or more compound consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) contacting the cells of step (c) with one or more compound consisting of CHIR99021. Y-27632. PD0325901. IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise: (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound consisting of CI4IR99021, 616452,TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632. JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VP A), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound consisting of CHIR99021, Y-27632, PD0325901, IWP-2. SB590885, and valproic acid (VP A) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the mesenchymal stem cells are derived from umbilical cord tissue, optionally, wherein the umbilical cord tissue comprises umbilical cord blood. In some embodiments, the prepared induced pluripotent stem cells express one or more factors selected from the group consisting of NANOG, OCT4, and SOX2. In some embodiments, the methods further comprise differentiating the prepared induced pluripotent stem cells.

[0008] In some embodiments, the pharmaceutical compositions comprise iPSCs derived from mesenchymal stem cells that do not comprise an exogenous polynucleotide.

[0009] In an aspect of the current disclosure, methods of treating a disease or disorder in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an iPSC produced by a method comprising: (a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time; (b) contacting the cells of step (a) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a ty rosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase- 1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI). a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase- 1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time; (c) contacting the cells of step (b) with one or more compound selected from the groupconsisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho-associated. coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HD AC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N-lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and (d) contacting the cells of step (c) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacetylase (HDAC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells, to the subject to treat the disease or disorder. In some embodiments, the methods comprise (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF- b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HDAC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N-lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathw ay inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacetylase (HDAC) inhibitor for a sufficient amount of time toprepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods further comprise contacting the cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, heregulin-beta 1, and basic fibroblast growth factor. In some embodiments, the sufficient amount of time of step (a) is about 1 to about 10 days or, optionally, about 4 to about 10 days. In some embodiments, the sufficient amount of time of step (b) is about 1 to about 20 days or, optionally, about 16 to about 20 days. In some embodiments, the sufficient amount of time of step (c) is about 1 to about 12 days or, optionally, about 8 to about 12 days. In some embodiments, the sufficient amount of time of step (d) is about 1 to about 10 days or, optionally, about 7 to about 10 days. In some embodiments, the cells are cultured in hypoxic conditions. In some embodiments, the hypoxic conditions comprise about 1% oxygen to about 10% oxygen, optionally, about 5% oxygen. In some embodiments, the wingless (Wnt) activator comprises or consists of CHIR99021. In some embodiments, the transforming growth factor beta (TGF-b) type I receptor inhibitor comprises or consists of 616452. In some embodiments, the retinoic acid analogue comprises or consists of TTNPB. In some embodiments, the agonist of smoothened (SMO) comprises or consists of SAG. In some embodiments, the tyrosine kinase inhibitor comprises or consists of ABT-869. In some embodiments, the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor comprises or consists of Y-27632 or Tzv. In some embodiments, the inhibitor of JNK1. JNK2 and JNK3 comprises or consists of JNKIN8. In some embodiments, the monoamine oxidase inhibitor (MAOI) comprises or consists of tranylcypromine. In some embodiments, the DNA methylation inhibitor comprises or consists of 5-azacytidine. In some embodiments, the histone methyltransferase G9a inhibitor comprises or consists of UNC0224. In some embodiments, the inhibitor of JNK1 and JNK2 comprises or consists of ruxolitinib. In some embodiments, the CBP / p300 bromodomain (BRD) inhibitor comprises or consists of SGC- CBP30. In some embodiments, the histone deacetylase (HDAC) inhibitor comprises or consists of valproic acid (VP A). In some embodiments, the inhibitor of S-adenosylmethionine-dependent methyltransferase comprises or consists of DZNep. In some embodiments, the DOT1L inhibitor comprises or consists of EPZ004777. In some embodiments, the N-lysine methyltransferase SETD8 inhibitor comprises or consists of UNC0379. In some embodiments, the MEK / ERK pathway inhibitor comprises or consists of PD0325901. In some embodiments, the B-Raf inhibitor comprises or consists of SB590885. In some embodiments, the inhibitor of Wnt production comprises or consists of IWP-2. In some embodiments, the methods comprise (a)contacting mesenchymal stem cells with one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days; (b) contacting the cells of step (a) with one or more compound selected from the group consisting CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5 -azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) contacting the cells of step (b) with one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA). DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) contacting the cells of step (c) with one or more compound selected from the group consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise: (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT- 869, and Y-27632 for about 8 to about 10 days; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG. ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the methods comprise: (a) contacting mesenchymal stem cells with one or more compound consisting of CHIR99021, 616452. TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days; (b) contacting the cells of step (a) with one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5- azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) contacting the cells of step (b) with one or more compound consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) contacting the cells of step (c) with one or more compound consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymalstem cells. In some embodiments, the methods comprise: (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. In some embodiments, the mesenchymal stem cells are derived from umbilical cord tissue, optionally, wherein the umbilical cord tissue comprises umbilical cord blood. In some embodiments, the prepared induced pluripotent stem cells express one or more factors selected from the group consisting of NANOG, OCT4, and SOX2. In some embodiments, the methods further comprise differentiating the prepared induced pluripotent stem cells. In some embodiments, the disease or disorder is selected from the group consisting of a cardiovascular disease or disorder, a neurological disease or disorder, a neurodegenerative disease or disorder, a musculoskeletal disease or disorder, or other disease or disorder.

[0010] In an aspect of the current disclosure, kits are provided. In some embodiments, the kits comprise one or more of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase- 1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor, a MEK / ERK pathway inhibitor, a histone deacetylase (HD AC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and anN-lysine methyltransferase SETD8 inhibitor, and an inhibitor of Wnt production. In some embodiments, the wingless (Wnt) activator comprises or consists of CHIR99021. In some embodiments, the transforming growth factor beta (TGF-b) type I receptor inhibitor comprises or consists of 616452. In some embodiments, the retinoic acid analogue comprises or consists ofTTNPB. In some embodiments, the agonist of smoothened (SMO) comprises or consists of SAG. In some embodiments, the tyrosine kinase inhibitor comprises or consists of ABT-869. In some embodiments, the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor comprises or consists of Y-27632 or Tzv. In some embodiments, the inhibitor of JNK1, JNK2 and JNK3 comprises or consists of JNKIN8. In some embodiments, the monoamine oxidase inhibitor (MAOI) comprises or consists of tranylcypromine. In some embodiments, the DNA methylation inhibitor comprises or consists of 5-azacytidine. In some embodiments, the histone methyltransferase G9a inhibitor comprises or consists of UNC0224. In some embodiments, the inhibitor of JNK1 and JNK2 comprises or consists of ruxolitinib. In some embodiments, the CBP / p300 bromodomain (BRD) inhibitor comprises or consists of SGC-CBP30. In some embodiments, the histone deacetylase (HD AC) inhibitor comprises or consists of valproic acid (VPA). In some embodiments, the inhibitor of S-adenosylmethionine-dependent methyltransferase comprises or consists of DZNep. In some embodiments, the DOT1L inhibitor comprises or consists of EPZ004777. In some embodiments, the N-lysine methyltransferase SETD8 inhibitor comprises or consists of UNC0379. In some embodiments, the MEK / ERK pathway inhibitor comprises or consists of PD0325901. In some embodiments, the B-Raf inhibitor comprises or consists of SB590885. In some embodiments, the inhibitor of Wnt production comprises or consists of IWP-2. In some embodiments, the kits further comprise instructions for using the kit to perform any of the methods disclosed herein.BRIEF DESCRIPTION OF THE FIGURES

[0011] FIGs. 1A, IB, 1C, and ID. Derivation and Characterization of Mesenchymal Stem Cells from Umbilical Cord Tissue. A. MSC Isolation from Umbilical Cord Tissue (ucMSCs): The methodology employed in extracting MSCs from umbilical cord tissue with detailed steps from tissue processing to the establishment of cell culture conditions and subsequent cell expansion strategies. B. Morphological Assessment and Osteogenic Differentiation: The cellular morphology of ucMSCs derived from a healthy donor alongside their osteogenic differentiation capabilities on day 10, as evidenced by Alizarin-Red staining to identify calcium deposits indicative of osteogenesis. C. Proliferation Capacity of ucMSCs: The 2-hour EdU proliferation assay highlighting the ucMSCs' significant proliferative ability. D. Surface Marker Characterization of ucMSCs: ucMSCs showing the positive expression of specific MSC surface markers (CD73, CD90, and CD 105) and the absence of expression for hematopoietic markers (CD34 and CD45), underscoring their MSC identify.

[0012] FIGs. 2A, 2B, 2C, and 2D. Chemical Reprogramming of ucMSCs to iPSCs. A. Reprogramming Protocol: The specific chemical reprogramming process to transform MSCs from frozen umbilical cord tissue into hCiPSCs, highlighting the different media for each stage and the duration of treatments throughout the process. B. Morphological Transformations: A series of cell images capturing key stages of cellular morphology changes during the chemical reprogramming process. Arrows indicating the emergence of primary hCiPSC colonies. C. Expression level of Pluripotency Genes in hCiPSCs: Comparison of the expression levels of crucial pluripotency genes (Nanog. OCT4, SOX2) in hCiPSCs derived from ucMSCs using qPCR, with human iPSCs from the Allen Institute serving as a positive benchmark and ucMSCs as a negative control. D. Verification of Pluripotency Markers: Immunostaining showing the high- level expression of key pluripotency markers (OCT4, SOX2, NANOG) in hCiPSCs originating from ucMSCs, affirming their pluripotent status.DETAILED DESCRIPTION

[0013] Disclosed herein are methods of preparing induced pluripotent stem cells (iPSCs) from mesenchymal stem cells, induced pluripotent stem cells produced by the methods, methods of further culturing the iPSCs to differentiate the iPSCs, and kits. The inventors discovered that culturing stem cells, e.g., mesenchymal stem cells, in the disclosed media, e.g., see Table 1, according to the disclosed methods generated iPSCs using only small molecules and growth factors and not performing any genetic manipulation of the cells, e.g., viral transduction, transfection, etc. The disclosed methods improve the art of preparing iPSCs. especially for downstream clinical applications because the cells do not comprise genetic modifications that may lead to deleterious effects, e.g., transformation of the cells into cancerous cells.Methods of preparing induced pluripotent stem cells from mesenchymal stem cells

[0014] In an aspect of the current disclosure, methods of preparing induced pluripotent stem cells (iPSCs) from mesenchymal stem cells are provided. In some embodiments, the methods comprise (a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time; (b) contacting the cells of step (a) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF- b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor,an inhibitor of C-Jun N-terminal kinase-1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time; (c) contacting the cells of step (b) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming grow th factor beta (TGF-b) type I receptor inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (EID AC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N-lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and (d) contacting the cells of step (c) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacetylase (HD AC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells.

[0015] The methods may comprise (a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a ty rosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time; (b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF- b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time; (c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacety lase (HD AC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOT1Linhibitor, and anN-lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and (d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacetylase (EID AC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells.

[0016] Mesenchymal stem cells (MSCs) for use in the disclosed methods, may be prepared or isolated using known methods, e.g., isolated or generated from umbilical cord tissue or cord blood.

[0017] As used herein, “contacting” refers to contacting a sample directly or indirectly in vitro, ex vivo, or in vivo ( / .e., within a subject as defined herein). Contacting a sample may include addition of a compound to a sample, or administration to a subject. Contacting encompasses addition to a solution, cell, administration to a tissue, mammal, subject, patient, or human. Further, contacting a cell includes adding a compound (or compounds) to a cell culture.

[0018] As used herein, “culturing” refers to growing or maintaining cells in culture. Suitable media and methods for performing cell culture are known in the art.

[0019] Each of the steps mentioned above, i.e., (a), (b). (c). and (d), may also be referred to as stage I, stage II, stage III, and stage IV, respectively. For example, below in Table 1, are exemplary compounds and / or combinations of compounds and media that may be used in each “stage” or “step” according to the disclosed methods. Further, the media listed in Table 1 may comprise one or more of the compounds or consist of each of the compounds indicated for each stage.Table 1. Exemplary compounds and media for chemical reprogramming according to the disclosed methods.

[0020] Table 1. above, provides exemplary compounds that belong to a particular class of compounds (“description,” right column). The compounds are intended to be exemplary and not limiting. Thus, the skilled artisan will understand that suitable alternatives to the exemplary7compounds may be used in the disclosed methods. Selection of the alternatives which belong to the same class of compounds is routine for the skilled person. Exemplary' compounds in each class are provided below.Table 2 - Compounds and alternatives

[0021] The disclosed methods may further comprise contacting the cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a MEK / ERKpathway inhibitor, an inhibitor of Wnt production, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, heregulin-beta 1, and basic fibroblast growth factor.

[0022] Regarding the contacting time or culture time for each of the steps of the disclosed methods, a sufficient amount of time for step (a) may be about 1 to about 10 days or, optionally, about 4 to about 10 days, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 days. A sufficient amount of time for step (b) may be about 1 to about 20 days or, optionally, about 16 to about 20 days, e.g., about 1, about 2. about 3, about 4, about 5, about 6, about 7. about 8, about 9, about 10, about 11, about 12. about 13, about 14, about 15. about 16, about 17, about 18, about 19, or about 20 days. A sufficient amount of time for step (c) may be about 1 to about 12 days or, optionally, about 8 to about 12 days, e.g., about 1, about 2, about 3, about 4, about 5. about 6, about 7, about 8, about 9, about 10, about 11 days, or about 12 days. A sufficient amount of time for step (d) may be about 1 to about 10 days or, optionally, about 7 to about 10 days, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 days.

[0023] The cells may be cultured in hypoxic conditions for one or more or each of steps (a), (b), (c), and (d). The hypoxic conditions may comprise culturing the cells in about 1% oxygen to about 10% oxygen, optionally, about 5% oxygen, e.g., about about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% oxygen.

[0024] Suitable media for culturing cells to support cell growth and health, e.g., mesenchymal stem cells or iPSCs, are known in the art and may include a basal DMEM formulation, e.g., KnockOut™ DMEM (Gibco, 10829018). The medium may be supplemented with factors to improve cell growth and health but which do not significantly influence cell differentiation, e g., one or more of or each of: 10% KnockOut™ Serum Replacement (KSR) (Gibco, 10828028), 10% FBS, 1% GlutaMax, 1% NEAA, 0.055 mM 2-mercaptoethanol. Small molecules: 50 pg / ml L- ascorbic acid 2-phosphate (Vc2P) (Sigma- Aldrich. A8960), 5 mM LiCl (Sigma- Aldrich, L4408), 1 mM nicotinamide (NAM) (Sigma-Aldrich, 72340), 2 mg / ml AlbuMax-II (Gibco, 11021045), which are known in the art.

[0025] For example, the following exemplary media may be used in the disclosed methods:

[0026] Stage I Induction Medium (step (a))

[0027] Basal DMEM formulation, e.g., KnockOut™ DMEM (Gibco, 10829018), supplemented with: 10% KnockOut™ Serum Replacement (KSR) (Gibco, 10828028), 10% FBS, 1% GlutaMax, 1% NEAA, 0.055 mM 2-mercaptoethanol. Small molecules: 50 pg / ml L-ascorbicacid 2-phosphate (Vc2P) (Sigma- Aldrich, A8960), 5 mM LiCl (Sigma- Aldrich, L4408), 1 mM nicotinamide (NAM) (Sigma- Aldrich, 72340), 2 mg / ml AlbuMax-II (Gibco, 11021045), Small molecules: CHIR999021 (10 pM). 616452 (10 pM), TTNPB (2 pM), SAG (0.5 pM), ABT-869 (1 pM), Rock inhibitor (Y-27632 (2 pM) or Tzv (2 pM)).

[0028] Stage II Induction Medium (step (b))

[0029] Basal DMEM formulation, e.g., KnockOut™ DMEM, supplemented with: 10% KSR, 10% FBS, 1% GlutaMax, 1% NEAA, 0.055 mM 2-mercaptoethanol, 50 pg / ml Vc2p, 5 mM LiCl, 1 mM NAM. 40 ng / ml bFGF (Ongene, TP750002). Small molecules: CH1R99021 (10-12 pM). 616452 (10 pM), TTNPB (2 pM), SAG (0.5 pM), ABT-869 (1 pM), Y27632 (10 pM), JNKIN8 (1 pM), tranylcypromine (10 pM), 5-azacytidine (10 pM), UNC0224 (1 pM), ruxolitinib (1 pM), SGC-CBP30 (2 pM).

[0030] Stage III Induction Medium (step (c))

[0031] Basal DMEM formulation, e.g., KnockOut™ DMEM, supplemented with: 1% N2 supplement (Gibco, 17502-048), 2% B27 supplement (Gibco, 17504-044), 1% GlutaMax, 1% NEAA, 0.055 mM 2-mercaptoethanol, 50 pg / ml Vc2p. 5 mg / ml AlbuMax-II, 20 ng / ml recombinant human heregulin (3-1 (HRG) (PeproTech. 100-03). Small molecules: CHIR99021 (1 pM), 616452 (10 pM), Y-27632 (10 pM), PD0325901 (1 pM), tranylcypromine (10 pM), VPA (500 pM), DZNep (0.2 pM), EPZ004777 (5 pM), UNC0379 (1 pM).

[0032] Stage IV Induction Medium (step (d))

[0033] Basal DMEM formulation, e g., KnockOut™ DMEM, supplemented with: 1% N2 supplement, 2% B27 supplement, 1% GlutaMax, 1% NEAA. 0.055 mM 2-mercaptoethanol, 50 pg / ml Vc2p, 20 ng / ml HRG. Small molecules: CHIR99021 (1 pM), Y-27632 (10 pM), PD0325901 (1 pM), IWP-2 (2 pM), SB590885 (0.5 pM), with VPA (500 pM) included for the first 4 days.

[0034] The cells may be further cultured or differentiated after step (d). e.g., following an 8- 12 day treatment in Stage IV conditions, cells may be dissociated using Accutase (Millipore) and replated onto Laminin coated plate at 1 : 12 ratio, with a modified Stage IV medium. This medium may comprise or consist of a basal DMEM medium, e.g., KnockOut™ DMEM, enhanced with 1% N2 and 2% B27 supplements, 1% GlutaMax, 1% NEAA, 1% penicillin-streptomycin, 0.055 mM 2-mercaptoethanol, 50 pg / ml Vc2p, 2 mg / ml AlbuMax-11, along with small molecules to support differentiation including CHIR99021 (1 pM), PD0325901 (0.5 pM), IWP-2 (2 pM), Y- 27632 (10 pM), HRG (20 ng / ml), and bFGF (100 ng / ml from Peprotech). Culture medium may be changed daily. Within 7 days. hiPSC cell colonies should begin to form. After 4-6 daysgrowing, these colonies may be mechanically cut into smaller clusters, and transferred to plates coated with Matrigel (Coming, 354248) in StemMACS™ iPS-Brew XF medium (Miltenyi Biotec, 130-104-368) supplemented with Y-27632 (10 pM). After 24 hours cells may be switched into iPS-Brew XF medium without Y-27632.Induced pluripotent stem cells

[0035] In an aspect of the current disclosure, induced pluripotent stem cells (iPSCs) are provided. In some embodiments, the iPSCs are generated by the disclosed methods, e.g., from mesenchymal stem cells.

[0036] In another aspect, iPSCs derived from mesenchymal stem cells are provided which do not comprise an exogenous polynucleotide.Pharmaceutical compositions

[0037] In an aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise the disclosed iPSCs generated by the disclosed methods.Methods of treatment

[0038] The disclosed iPSCs and pharmaceutical compositions may be used to treat a variety of diseases or disorders. For example, methods of treating a disease or disorder in a subject in need thereof are provided and may comprise administering a therapeutically effective amount of the disclosed iPSCs or pharmaceutical compositions to the subject to treat the disease or disorder. Similarly, use of the disclosed iPSCs in the manufacture of a medicament for the treatment of a disease or disorder are provided as well as the use of the disclosed pharmaceutical compositions or iPSCs for the treatment of diseases or disorders.

[0039] As used herein, “therapeutically effective amount’’ or “effective amount” refer to the amount of the pharmaceutical composition necessary to improve one sign or symptom of a subject’s disease or disorder. A therapeutically effective amount may comprise about IxlO6recombinant iPSCs to about IxlO14iPSCs, or more, e.g., about IxlO6, about IxlO7, about IxlO8, about IxlO9, about IxlO10, about IxlO11, about IxlO12, about IxlO13, or about IxlO14, or any value or range therein including the endpoints.

[0040] The pharmaceutical compositions may be administered by any appropriate route, e.g., parenterally, e.g., intravenously, illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The particular dose and administration route can be determined by a physician.

[0041] A subject in need thereof, as used herein, refers to a subject in need of treatment for a disease or disorder, e.g., a cardiovascular disease or disorder, a neurological disease or disorder, a neurodegenerative disease or disorder, a musculoskeletal disease or disorder, or other disease or disorder.Kits

[0042] In an aspect of the current disclosure, kits are provided. The kits may comprise one or more of the disclosed compounds or growth factors, e.g., a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase- 1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI). a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase- 1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor, a MEK / ERK pathway inhibitor, a histone deacetylase (HD AC) inhibitor, an inhibitor of S- adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N-lysine methyltransferase SETD8 inhibitor, and an inhibitor of Wnt production and may, optionally, comprise instructions for using the kit to perform the disclosed methods.Definitions

[0043] The present invention is described herein using several definitions, as set forth below and throughout the application.

[0044] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0045] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0046] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0047] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0048] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0049] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone. A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or B or “A and B.”

[0050] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0051] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to usea specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.” Exemplary embodiments1. A method of preparing induced pluripotent stem cells from mesenchymal stem cells, the method comprising:(a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-0) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time;(b) contacting the cells of step (a) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-P) ty pe I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase- 1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C- Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time;(c) contacting the cells of step (b) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho-associated. coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HD AC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N- lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and(d) contacting the cells of step (c) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacety lase (HD AC)inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells.2. A method of preparing induced pluripotent stem cells from mesenchymal stem cells, the method comprising:(a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-P) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time;(b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming grow th factor beta (TGF-P) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase- 1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time;(c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) t pe I receptor inhibitor, a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HD AC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N-lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and(d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histonedeacetylase (HDAC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells.3. The method of any one of the preceding embodiments, further comprising contacting the cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, heregulin-beta 1, and basic fibroblast growth factor.4. The method of any one of the preceding embodiments, wherein the sufficient amount of time of step (a) is about 1 to about 10 days or, optionally, about 4 to about 10 days.5. The method of any one of the preceding embodiments, wherein the sufficient amount of time of step (b) is about 1 to about 20 days or. optionally, about 16 to about 20 days.6. The method of any one of the preceding embodiments, wherein the sufficient amount of time of step (c) is about 1 to about 12 days or, optionally, about 8 to about 12 days.7. The method of any one of the preceding embodiments, wherein the sufficient amount of time of step (d) is about 1 to about 10 days or, optionally, about 7 to about 10 days.8. The method of any one of the preceding embodiments, wherein the cells are cultured in hypoxic conditions.9. The method of embodiment 6, wherein the hypoxic conditions comprise about 1% oxygen to about 10% oxygen, optionally, about 5% oxygen.10. The method of any one of the preceding embodiments, wherein the wingless (Wnt) activator comprises or consists of CHIR99021.11. The method of any one of the preceding embodiments, wherein the transforming growth factor beta (TGF-b) type I receptor inhibitor comprises or consists of 616452.12. The method of any one of the preceding embodiments, wherein the retinoic acid analogue comprises or consists of TTNPB13. The method of any one of the preceding embodiments, wherein the agonist of smoothened (SMO) comprises or consists of SAG.14. The method of any one of the preceding embodiments, wherein the tyrosine kinase inhibitor comprises or consists of ABT-869.15. The method of any one of the preceding embodiments, wherein the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor comprises or consists of Y-27632 or Tzv.16. The method of any one of the preceding embodiments, wherein the inhibitor of JNK1, JNK2 and JNK3 comprises or consists of JNKIN8.17. The method of any one of the preceding embodiments, wherein the monoamine oxidase inhibitor (MAOI) comprises or consists of tranylcypromine.18. The method of any one of the preceding embodiments, wherein the DNA methylation inhibitor comprises or consists of 5-azacytidine.19. The method of any one of the preceding embodiments, wherein the histone methyltransferase G9a inhibitor comprises or consists of UNC0224.20. The method of any one of the preceding embodiments, wherein the inhibitor of JNK1 and JNK2 comprises or consists of ruxolitinib.21. The method of any one of the preceding embodiments, wherein the CBP / p300 bromodomain (BRD) inhibitor comprises or consists of SGC-CBP30.22. The method of any one of the preceding embodiments, wherein the histone deacetylase (HD AC) inhibitor comprises or consists of valproic acid (VPA).23. The method of any one of the preceding embodiments, wherein the inhibitor of S- adenosylmethionine-dependent methyltransferase comprises or consists of DZNep.24. The method of any one of the preceding embodiments, wherein the DOT 1 L inhibitor comprises or consists of EPZ004777.25. The method of any one of the preceding embodiments, wherein the N-lysine methyltransferase SETD8 inhibitor comprises or consists of UNC0379.26. The method of any one of the preceding embodiments, wherein the MEK / ERK pathway inhibitor comprises or consists of PD0325901.27. The method of any one of the preceding embodiments, wherein the B-Raf inhibitor comprises or consists of SB590885.28. The method of any one of the preceding embodiments, wherein the inhibitor of Wnt production comprises or consists of IWP-2.29. A method comprising:(a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days;(b) contacting the cells of step (a) with one or more compound selected from the group consisting CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5 -azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days;(c) contacting the cells of step (b) with one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VP A), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and(d) contacting the cells of step (c) with one or more compound selected from the group consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VP A) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. A method comprising:(a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days;(b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days;(c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VP A), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and(d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VP A) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells. A method comprising:(a) contacting mesenchymal stem cells with one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days;(b) contacting the cells of step (a) with one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days;(c) contacting the cells of step (b) with one or more compound consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid(VP A), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and(d) contacting the cells of step (c) with one or more compound consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VP A) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells.32. A method comprising:(a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days;(b) culturing the cells of step (a) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days;(c) culturing the cells of step (b) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, Y-27632. PD0325901, tranylcypromine, valproic acid (VP A), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and(d) culturing the cells of step (c) in a suitable medium comprising one or more compound consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VP A) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells.33. The method of any one of the preceding embodiments, wherein the mesenchymal stem cells are derived from umbilical cord tissue, optionally, wherein the umbilical cord tissue comprises umbilical cord blood.34. The method of any one of the preceding embodiments, wherein the prepared induced pluripotent stem cells express one or more factors selected from the group consisting of NANOG, OCT4, and SOX235. The method of any one of the preceding embodiments, further comprising differentiating the prepared induced pluripotent stem cells.36. An induced pluripotent stem cell (iPSC) produced by the method of any one of the preceding embodiments.37. An induced pluripotent stem cell derived from a mesenchymal stem cell that does not comprise an exogenous polynucleotide.38. A pharmaceutical composition comprising the iPSC of embodiment 36 or 37, optionally, further comprising a pharmaceutically acceptable carrier or excipient.39. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of embodiment 38 to the subject to treat the disease or disorder.40. The method of embodiment 39, wherein the disease or disorder is selected from the group consisting of a cardiovascular disease or disorder, a neurological disease or disorder, a neurodegenerative disease or disorder, a musculoskeletal disease or disorder, or other disease or disorder.41. A kit comprising one or more of a wingless (Wnt) activator, a transforming growth factor beta (TGF- ) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase- 1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor, a MEK / ERK pathway inhibitor, a histone deacety lase (HD AC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOTI L inhibitor, and an N-lysine methyltransferase SETD8 inhibitor, and an inhibitor of Wnt production.42. The kit of embodiment 41, wherein the wingless (Wnt) activator comprises or consists of CHIR99021.43. The kit of embodiment 41 or 42, wherein the transforming growth factor beta (TGF- b) type I receptor inhibitor comprises or consists of 616452.44. The kit of any one of embodiments 41-43, wherein the retinoic acid analogue comprises or consists of TTNPB45. The kit of any one of embodiments 41-44, wherein the agonist of smoothened (SMO) comprises or consists of SAG.46. The kit of any one of embodiments 41-45, wherein the tyrosine kinase inhibitor comprises or consists of ABT-869.47. The kit of any one of embodiments 41-46, wherein the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor comprises or consists of Y-27632 or Tzv.48. The kit of any one of embodiments 41-47, wherein the inhibitor of JNK1, JNK2 and JNK3 comprises or consists of JNKIN8.49. The kit of any one of embodiments 41-48, wherein the monoamine oxidase inhibitor (MAOI) comprises or consists of tranylcypromine.50. The kit of any one of embodiments 41-49, wherein the DNA methylation inhibitor comprises or consists of 5 -azacytidine.51. The kit of any one of embodiments 41-50, wherein the histone methyltransferase G9a inhibitor comprises or consists of UNC0224.52. The kit of any one of embodiments 41-51, wherein the inhibitor of JNK1 and JNK2 comprises or consists of ruxolitinib.53. The kit of any one of embodiments 41-52, wherein the CBP / p300 bromodomain (BRD) inhibitor comprises or consists of SGC-CBP30.54. The kit of any one of embodiments 41-53, wherein the histone deacety lase (HDAC) inhibitor comprises or consists of valproic acid (VP A).55. The kit of any one of embodiments 41-54. wherein the inhibitor of S- adenosylmethionine-dependent methyltransferase comprises or consists of DZNep.56. The kit of any one of embodiments 41-55, wherein the DOT1L inhibitor comprises or consists of EPZ004777.57. The kit of any one of embodiments 41-56. wherein the N-lysine methyltransferase SETD8 inhibitor comprises or consists of UNC0379.58. The kit of any one of embodiments 41-57, wherein the MEK / ERK pathway inhibitor comprises or consists of PD0325901.59. The kit of any one of embodiments 41-58, wherein the B-Raf inhibitor comprises or consists of SB590885.The kit of any one of embodiments 41-59, wherein the inhibitor of Wnt production comprises or consists of IWP-2.61. The kit of any one of embodiments 41-60, further comprising instructions for using the kit to perform the method of any one of embodiments 1-35.EXAMPLES

[0052] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0053] Reference is made to Nansubuga, C., Donna K. Mahnke, Siqi Li, Abigail Multerer, Jake Minx, Bradley Miller, Mengcheng Shen, Andreas Beyer, Lu Han, Joy Lincoln, Chun Liu "Efficient Generation of Human Pluripotent Stem Cells from Frozen Cord Tissue via Chemical Reprogramming ’ doi.org / 10. 1101 / 2024.04. 10.588154, a manuscript by the inventors, which is incorporated by reference herein in its entirety.Example 1 - Efficient Generation of Human Pluripotent Stem Cells from Frozen Cord Tissue via Chemical Reprogramming

[0054] ABSTRACT

[0055] Chemical reprogramming presents an innovative approach for generating induced pluripotent stem cells (iPSCs), bypassing the genetic instability and safe concern associated with viral vector approach. We describe a novel, efficient chemical method for reprogramming human umbilical cord tissue-derived mesenchymal stem cells (MSCs) into induced pluripotent stem cells (iPSCs). Compared to previous sources like adipose tissue and skin, frozen umbilical cord tissue offers an abundant, non-invasive, long-term storable, and ethically sound cell source. Our findings not only showcase the feasibility and safety of utilizing chemical reprogramming on cells from frozen umbilical cords but also underscore its potential in regenerative medicine, especially for developing safer and more effective therapies for cardiovascular diseases.

[0056] Chemical reprogramming offers a compelling alternative to viral methods for generating induced pluripotent stem cells (iPSCs), a cornerstone in regenerative medicine and biomedical research1. Unlike viral reprogramming, which introduces genetic material through vectors with potential for insertional mutagenesis, chemical reprogramming employs small molecules to overpass the genetic modifications, thus minimizing risks of genetic instability and enhancing safety profilest2. This approach not only mitigates concerns related to genomic alterations but also offers a more controllable and reversible mechanism, critical for clinical applications. Furthermore, the scalability and cost-effectiveness of chemical methods surpass those of viral techniques, presenting a more feasible pathway for widespread therapeutic use and personalized medicine3,4. By addressing the limitations associated with viral vectors, chemicalreprogramming emerges as a promising avenue, potentially revolutionizing the generation of iPSCs for the study and treatment of cardiovascular diseases and beyond.

[0057] Current human chemical reprogramming method was primarily conducted on human mesenchymal stem cells (MSCs) from adipose tissue or dermis tissues1,3. However, umbilical cord tissue emerges as a superior alternative for several reasons: it is an abundant source of MSCs5; its frozen form ensures both sustainability and easy accessibility of cells without necessitating invasive procurement methods like blood PBMCs6; MSCs from umbilical cord tissue exhibit a higher proliferation rate, potentially increasing the efficiency of reprogramming processes7. Furthermore, utilizing young cells from umbilical cords, as opposed to cells from older blood donors, leverages the advantage of younger donor cells, which have been reported to reprogram more effectively than those from older donors8.

[0058] Here for the first time, we successfully employ chemical reprogramming on MSCs isolated from frozen umbilical cord tissue. It not only enriches the toolkit of regenerative medicine but also paves the way for innovative therapeutic strategies that are safer, more efficient, and ethically sound.

[0059] Isolate MSCs from frozen cord tissue

[0060] Umbilical cord tissues were initially processed and cryogenically stored at the Cord Blood Registry in Tucson, AZ. The process began with cutting the tissue into 1 cm segments within a clean petti dish. These segments were further cut into halves and placed into a 50 mL tube filled with PBS for rinsing. Subsequently, the halves were transferred to another sterile dish, and a 4 mm biopsy punch was used to obtain tissue pieces with uniform size. These samples were then allocated into 5 mL cryotubes, each filled with 2 mL cryopreservation solution and containing approximately 2-2.5 g of tissue. Initially, the samples were frozen at -80°C, and after 18-24 hours, they were transferred to liquid nitrogen (LN2) storage for long-term preservation. The frozen tissues were warmed in a 37°C water bath to quickly thaw on site (FIG. 1A). Tissue segments were then placed on a 5 x 5 grid on a 10 cm culture dish coated with a MSC attachmentpromoting substrate. To ensure firmly attached, tissues were left to dry for 10 mins before gradually adding 15 mL of complete MSC media. After 7-8 days incubation, fibroblast-like cells were observed. Then original tissue segments were carefully removed and replaced with fresh MSC media. Between day 14 to day 16, confluent MSCs were detached and passaged for expansion. Additionally, a cryovial from the original culture dish was preserved. The MSCs from frozen umbilical cord tissues are referred as ucMSCs.

[0061] Characterization of ucMSC from frozen cord tissue

[0062] These MSCs, referred to as ucMSCs, were subjected to an osteogenic differentiation medium, under which they were differentiated to mature osteoblasts. The mineralized extracellular calcium deposits were validated by Alizarin-Red staining (FIG. IB). The staining results confirmed the presence of calcium deposits, underscoring the differentiation of ucMSCs into osteoblasts. In addition to their differentiation capacity, ucMSCs exhibited a significantly higher proliferative capacity, showing 32.1% EdU incorporation (FIG. 1C). This high proliferative capability suggests that ucMSCs possess a high degree of viability and activity, making them highly suitable for reprogramming where cell proliferation is desirable. To further characterize the isolated ucMSCs, a comprehensive analysis of their surface markers was conducted using fluorescence- activated cell sorting (FACS). The FACS analysis showed that ucMSCs have ty pical MSC markers and the absence of blood cell markers, which are not present on MSCs. The FACS results confirmed that the ucMSCs strongly expressed the MSC markers CD73, CD90, and CD105, establishing they are MSCs. Conversely, the ucMSCs did not exhibit the blood cell markers CD34 and CD45 (FIG. ID). This comprehensive characterization confirms the mesenchymal stem cell identity of ucMSCs.

[0063] Chemical reprogramming of ucMSC to human chemical induced iPSC (hCiPSC)

[0064] To initiate the chemical reprogramming of ucMSCs, the cells were seeded at a density of 1-1.5 x 10A4cells per well in a 12-well plate with DMEM medium supplemented with 15% FBS (FIG. 2A). Cells were treated with stage I induction medium the next day and immediately maintained in 5% 02 hypoxia incubator. Cells were cultured in stage I induction medium under hypoxia condition for 8 days. By days 4-6 of stage I, epithelial-like cells began to emerge, achieving approximately 80% confluence. On day 8, cells were switched to stage II induction medium and under regular 02 incubator for the rest of process. The emergence of multilayered cell colonies was observed during Stage II within 8-12 days. The colonies were expanded rapidly and were cultured with stage III medium for another 12 days. VPA was included for the first four days of induction process in stage IV medium and the appearance of primary hCiPS cell colonies were observed after 6-8 days culture. Primary' colonies from ucMSCs were dissociated and replaced for expansion. Compact hCiPSC colonies were manually picked up and mechanically- cut into small clamps as typical iPSC culture (FIG. 2B).

[0065] Characterization of hCiPSC from ucMSC

[0066] To assess the pluripotent characteristics of hCiPSCs generated from ucMSCs, we examined the expression levels of key pluripotency genes, namely Nanog, OCT4, and SOX2. These genes, NANOG, OCT4, and SOX2, are recognized as critical markers for pluripotency.For comparison, we employed hiPSCs from the Allen Institute as a positive control and ucMSCs as the baseline negative control. Quantitative real-time PCR (qPCR) analysis showed a comparable high expression level of NANOG, OCT4, and SOX2 in our hCiPSCs when compared to the iPSCs from Allen Institute, while they were absent in ucMSCs (Figure 2C). Furthermore, Immunofluorescence staining highlighted the robust protein level expression of these pluripotency markers in the hCiPSCs, reinforcing their pluripotent status (FIG. 2D).

[0067] DISCUSSION

[0068] In this study, we used small molecule chemical reprogramming to generate human pluripotent stem cells from frozen umbilical cord tissue. Human somatic cells are resistant to chemical stimuli by exhibiting a stable epigenome and low plasticity, therefore, the restricted human epigenetic landscape is unlocked into a plastic state by chemically induced dedifferentiation of small molecules, allowing the release of small molecules into human cells and reprogramming of somatic cells into induced pluripotent stem cells1.

[0069] Currently the most commonly used method is Sendai virus reprogramming, which is well established for reprogramming somatic cells to generate iPSCs by introducing the reprogramming factors OCT4, SOX2, KLF4, and CMYC (OKSM)2,9. However, Sendai virus is the persistent expression of exogenous factors of OKSM genes in the cells, and the continued expression of Sendai virus carrying exogenous Yamanaka factors over multiple passages poses significant risks for clinical use10. In specific, c-Myc is one of the most frequently mutated genes in human cancer, and previous studies have shown that chimeric mice generated from iPSC by inducing retrovirus- mediated transfection of four reprogramming factors frequently develop tumorsn.

[0070] In contrast, chemical reprogramming methods offer a safer and more controlled approach by eliminating the need for exogenous factors like viral vectors or genetic modifications. Chemical reprogramming involves the use of small molecules to induce cellular dedifferentiation and pluripotency, without permanently altering the cell's genome or introducing foreign elements. Small molecules can be readily synthesized, optimized, and standardized, allowing for reproducible and cost-effective protocols that can be easily adapted for various cell types and applications. This scalability is crucial for the widespread implementation of cell-based therapies and regenerative medicine approaches. Previous studies have found that mouse somatic cells can be generated into iPSC via small molecule compounds12. The recent success of chemical reprogramming from human adipose derived mesenchymal stem cells is a paradigm shift in reprogramming technology.

[0071] Various types of somatic cells have been shown to be reprogrammed to generate induced pluripotent stem cells. Cell proliferation has been shown to affect reprogramming efficiency, decreasing programming efficiency with increased donor age, and aged cells retain their DNA mutations during reprogramming8. Umbilical cord cells are the youngest cells that stored in public cord blood banks capture researchers interests to create iPSC using them13. Umbilical cord tissue MSCs can be easily isolated, have the same advantages as umbilical cord blood cells, and are simple to operate, and can serve as a source of young donor cells14. In this study, we found that MSCs can be isolated from frozen umbilical cord tissue and retained all MSCs cell surface markers. They are highly proliferative and can be passaged long term to yield millions of cells during the initial plating as others reported15. Furthermore, because they are young cells from birth, these cells are absent of environment or aging caused somatic mutations, serving as a perfect source for studying familial mutation associated disease, such as congenital heart disease.

[0072] Taking the advantage of umbilical cord MSCs and chemical reprogramming method, we have successfully generated human iPSC from MSCs isolated from frozen umbilical cord tissue using chemical reprogramming. This approach provides a promising avenue for expanding the range of iPSC sources and advancing regenerative medicine applications, particularly in cardiovascular diseases.

[0073] MATERIALS AND METHODS

[0074] ucMSC isolation and culture

[0075] ucMSCs were isolated from frozen umbilical cord tissue as described above following a published protocol16. Complete MSC medium were prepared with MEM Alpha (IX) + GlutaMAX™ -1 (GIBCO, 32561037), PLTMax GMP Clinical Grade Supplement (Mill Creek Life Sciences, PLTMax27GMP), and Heparin Solution (0.2%) (STEMCELL Technologies, 07980). CELLstart™ Humanized Substrate for Cell Culture (GIBCO, A10142-01) was used for tissue segment attachment and ucMSC expansion.

[0076] Chemical reprogramming medium Stage I Induction Medium

[0077] KnockOut™ DMEM (Gibco, 10829018) supplemented with: 10% KnockOut™ Serum Replacement (KSR) (Gibco. 10828028), 10% FBS, 1% GlutaMax, 1% NEAA, 0.055 mM 2- mercaptoethanol. Small molecules: 50 pg / ml L-ascorbic acid 2-phosphate (Vc2P) (Sigma- Aldrich, A8960), 5 mM LiCl (Sigma-Aldrich, L4408), 1 mM nicotinamide (NAM) (Sigma- Aldrich, 72340), 2 mg / ml AlbuMax-II (Gibco, 11021045), Small molecules: CHIR999021 (10pM), 616452 (10 pM), TTNPB (2 pM), SAG (0.5 pM), ABT-869 (1 pM), Rock inhibitor (Y- 27632 (2 pM) or Tzv (2 pM)).

[0078] Stage II Induction Medium

[0079] KnockOut™ DMEM supplemented with: 10% KSR, 10% FBS, 1% GlutaMax, 1% NEAA, 0.055 mM 2-mercaptoethanol, 50 pg / ml Vc2p, 5 mM LiCl, 1 mM NAM, 40 ng / ml bFGF (Origene, TP750002). Small molecules: CHIR99021 (10-12 pM), 616452 (10 pM), TTNPB (2 pM), SAG (0.5 pM), ABT-10080] 869 (1 pM), Y27632 (10 pM). JNKIN8 (1 pM). tranylcypromine (10 pM). 5- azacytidine (10 pM),

[0081] UNC0224 (1 pM), ruxolitinib (1 pM), SGC-CBP30 (2 pM).

[0082] Stage III Induction Medium

[0083] KnockOut™ DMEM supplemented with: 1% N2 supplement (Gibco, 17502-048), 2% B27 supplement (Gibco, 17504-044), 1% GlutaMax, 1% NEAA, 0.055 mM 2-mercaptoethanol, 50 pg / ml Vc2p, 5 mg / ml AlbuMax-II, 20 ng / ml recombinant human heregulin P-1 (HRG) (PeproTech, 100-03). Small molecules: CHIR99021 (1 pM), 616452 (10 pM), Y-27632 (10 pM), PD0325901 (1 pM), tranylcypromine (10 pM), VPA (500 pM), DZNep (0.2 pM). EPZ004777 (5 pM), UNC0379 (1 pM).

[0084] Stage IV Induction Medium

[0085] KnockOut™ DMEM supplemented with: 1 % N2 supplement, 2% B27 supplement, 1 % GlutaMax, 1% NEAA, 0.055 mM 2-mercaptoethanol, 50 pg / ml Vc2p, 20 ng / ml HRG. Small molecules: CHIR99021 (1 pM), Y-27632 (10 pM), PD0325901 (1 pM), IWP-2 (2 pM). SB590885 (0.5 pM), with VPA (500 pM) included for the first 4 days.

[0086] Derivation and culture of hCiPSCs

[0087] Following an 8-12 day treatment in Stage IV conditions, cells were dissociated using Accutase (Millipore) and replated onto Laminin coated plate at 1 : 12 ratio, with a modified Stage IV medium. This medium consisted of KnockOut™ DMEM enhanced with 1% N2 and 2% B27 supplements, 1% GlutaMax, 1% NEAA, 1% penicillin-streptomycin, 0.055 mM 2- mercaptoethanol, 50 pg / ml Vc2p, 2 mg / ml AlbuMax-II, along with small molecules including CHIR99021 (1 pM), PD0325901 (0.5 pM), IWP-2 (2 pM), Y-27632 (10 pM), HRG (20 ng / ml), and bFGF (100 ng / ml from Peprotech). Culture medium was changed daily. Within 7 days, hCiPS cell colonies started to form. After 4-6 days growing, these colonies were mechanically cut into smaller clusters, and transferred to plates coated with Matrigel (Coming, 354248) inStemMACS™ iPS-Brew XF medium (Miltenyi Biotec, 130-104-368) supplemented with Y- 27632 (10 pM). After 24 hours cells were switch into iPS-Brew XF medium without Y-27632.

[0088] MSC Osteogenic Differentiation

[0089] ucMSCs were cultured in Mesenchymal Stem Cell Osteogenic Differentiation Medium (PromoCell, C-28013) for osteogenic differentiation. After 10 days of differentiation, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 30 minutes. Cells were then stained by adding sufficient Alizarin-Red Staining Solution (Sigma-Aldrich, TMS-008-C) following manufacturer's instruction.

[0090] Fluorescence-activated Cell Sorting (FACS) analysis for Cell Surface Staining and MSC markers

[0091] Proliferation assay: EdU (10 pM) was added to the ucMSC culture medium 2hr before harvest. The cells were then trypsinized and fixed in 4% formaldehyde. EdU incorporation was determined with the Click-iT™ EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (Invitrogen, Cl 0419) according to the manufacturer's instructions.

[0092] MSC markers: A total of 5*1O5cells were resuspended and incubated with fluorescence- conjugated antibody mixture (CD34-PE; CD45-APC; CD73-FITC; CD90-Pacific Blue; CD105-PE- Cy7) for 20 min at room temperature. Next, cells were incubated with biotinylated secondary antibodies for 30 min on ice followed FACS buffer wash for 3 times. The fluorescence intensity of the cells was measured using a flow cytometer.

[0093] Quantitative RT-PCR analysis

[0094] Total RNA was prepared from the hCiPSC using the RNeasy Plus Mini Kit (Qiagen). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Quantitative RT-PCR was carried out using CFX96 real-time PCR detection system (Bio-Rad) with TaqMan Universal PCR Master Mix (Applied Biosystems). The relative mRNA expression of the targeted genes was normalized to the GADPH as endogenous control. Gene expression was calculated using 2v tmethod. Human iPSC line (AICS-0060-027iPSC from Stem cell) purchased from Allen Institute was used as positive control.

[0095] Immunofluorescence staining

[0096] Immunofluorescence staining was performed to analyze OCT4, SOX2, NANOG protein expression. Cells fixed using 4% paraformaldehyde for 2d and washed with 0.1% Triton X-100 (Sigma-Aldrich). Blocking was done using 2.5% donkey serum (Sigma- Aldrich) and then incubated with the primary antibodies (Cell signaling Technology, StemLight™ Pluripotency Antibody Kit #9656) in 2.5% donkey serum-PBS for overnight at 4°C. On the next day. afterthree washes with PBS, the cells were further incubated with secondary antibodies (Thermo Fisher, Alexa Fluro 488 and 594) for 1 hour at room temperature. Next, cells were w ashed with PBS and mounted with Slow Fade Gold Antifade Reagent with 4,6-diamidino-2-phenylindole (DAPI; Life Technologies) before imaging. All immunofluorescent images were captured using a Nikon Eclipse 80i fluorescence microscope.

[0097] References1. Guan J, Wang G, Wang J, Zhang Z, Fu Y, Cheng L, Meng G, Lyu Y, Zhu J, Li Y, et al. Chemical reprogramming of human somatic cells to pluripotent stem cells. Nature. 2022;605:325-331. doi: 10.1038 / s41586-022-04593-52. Kunitomi A, Hirohata R, Arreola V, Osawa M, Kato TM, Nomura M, Kawaguchi J, HaraH. Kusano K, Takashima Y, et al. Improved Sendai viral system for reprogramming to naive pluripotency. Cell Rep Methods. 2022:2: 100317. doi: 10.1016 / j.crmeth.2022.1003173. Liuyang S, Wang G, Wang Y,He H, Lyu Y, Cheng L, Yang Z, Guan J, Fu Y,Zhu J, et al. Highly efficient and rapid generation of human pluripotent stem cells by chemical reprogramming. Cell Stem Cell. 2023;30:450-459 e459. doi: 10.1016 / j.stem.2023.02.0084. Lange L, Esteban MA, Schambach A. Back to pluripotency: fully chemically induced reboot of human somatic cells. Signal Transduct Target Ther. 2022;7:244. doi: 10.1038 / s41392- 022-01109-55. Nagamura-Inoue T, Mukai T. Umbilical Cord is a Rich Source of Mesenchymal Stromal Cells for Cell Therapy. Curr Stem Cell Res Ther. 2016; 1 1 :634-642. doi: 10.2174 / 1574888x106661510261150176. Beeravolu N, McKee C, Alamri A, Mikhael S, Brown C, Perez-Cruet M, Chaudhry GR. Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta. J Vis Exp. 2017. doi: 10.3791 / 552247. Wang Q, Wang Y, Chang C, Ma F, Peng D, Yang S, An Y, Deng Q, Wang Q, Gao F, et al. Comparative analysis of mesenchymal stem / stromal cells derived from human induced pluripotent stem cells and the cognate umbilical cord mesenchymal stem / stromal cells. Heliyon. 2023;9:el2683. doi: 10.1016 / j.heliyon.2022.el26838. Trokovic R, Weltner J, Noisa P, Raivio T, Otonkoski T. Combined negative effectof donor age and time in culture on the reprogramming efficiency into induced pluripotent stem cells. Stem Cell Res. 2015;15:254-262. doi: 10.1016 / j.scr.2015.06.001 Takahashi K, Tanabe K, Ohnuki M, Narita M, IchisakaT,TomodaK, YamanakaS. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861-872. doi: 10. 1016 / j. cell.2007. 11.019 Angeles ADL, Hug CB, Gladyshev VN, Church GM. Velychko S. Sendai virus persistence questions the transient naive reprogramming method for iPSC generation. bioRxiv. 2024:2024.2003.2007.583804. doi: 10.1101 / 2024.03.07.583804 Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 2007;448:313-317. doi: 10.1038 / nature05934 Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, Zhao T, Ye J, Yang W, Liu K, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 2013;341 :651-654. doi: 10.1126 / science.1239278 Zhou H. Rao MS. Can cord blood banks transform into induced pluripotent stem cell banks?Cytotherapy. 2015;17:756-764. doi: 10. 1016 / j.jcyt.2015.02.008 Mohamed A, Chow T, Whiteley J, Fantin A, Sorra K, Hicks R, Rogers IM. Umbilical Cord Tissue as a Source of Young Cells for the Derivation of Induced Pluripotent Stem Cells Using Non-Integrating Episomal Vectors and Feeder- Free Conditions. Cells. 2020;10. doi: 10.3390 / cellsl0010049 Raileanu VN, Whiteley J, Chow T, Kollara A, Mohamed A, Keating A, Rogers IM. Banking Mesenchymal Stromal Cells from Umbilical Cord Tissue: Large Sample Size Analysis Reveals Consistency Between Donors. Stem Cells Transl Med. 2019;8: 1041-1054. doi: 10. 1002 / sctm. 19-0022 Skiles ML, Marzan AJ, Brown KS, Shamonki JM. Comparison of umbilical cord tissue- derived mesenchymal stromal cells isolated from ciyopreserved material and extracted by explantation and digestion methods utilizing a split manufacturing model. Cytotherapy. 2020:22:581 -591 , doi: 10.1016 / j.jcyt.2020.06.002

[0098] In the foregoing description, it will be readily apparent to one skilled in the art that vary ing substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0099] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

Claims

CLAIMS1. A method of preparing induced pluripotent stem cells from mesenchymal stem cells, the method comprising:(a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-P) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time;(b) contacting the cells of step (a) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-P) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase- 1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C- Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time;(c) contacting the cells of step (b) with one or more compound selected from the group consisting of a w ingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HD AC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N- lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and(d) contacting the cells of step (c) with one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacetylase (HD AC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells.

2. A method of preparing induced pluripotent stem cells from mesenchymal stem cells, the method comprising:(a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming grow th factor beta (TGF-P) t pe I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor for a sufficient amount of time;(b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-P) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO), a tyrosine kinase inhibitor, and a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor for a sufficient amount of time;(c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a transforming growth factor beta (TGF-b) type I receptor inhibitor, a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathway inhibitor, a monoamine oxidase inhibitor (MAOI), a histone deacetylase (HD AC) inhibitor, an inhibitor of S-adenosylmethionine-dependent methyltransferase, a DOT1L inhibitor, and an N-lysine methyltransferase SETD8 inhibitor for a sufficient amount of time; and(d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of a wingless (Wnt) activator, a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor, a MEK / ERK pathw ay inhibitor, an inhibitor of Wnt production, a B-Raf inhibitor, and a histone deacety lase (HD AC) inhibitor for a sufficient amount of time to prepare induced pluripotent stem cells from mesenchymal stem cells.

3. The method of claim 1 or 2, further comprising contacting the cells with one or more compound selected from the group consisting of a wingless (Wnt) activator, a MEK / ERK pathway inhibitor, an inhibitor of Wnt production, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, heregulin-beta 1, and basic fibroblast growth factor.

4. The method of claim 1 or 2, wherein the sufficient amount of time of step (a) is about 1 to about 10 days or, optionally, about 4 to about 10 days.

5. The method of claim 1 or 2, wherein the sufficient amount of time of step (b) is about 1 to about 20 days or, optionally, about 16 to about 20 days.

6. The method of claim 1 or 2. wherein the sufficient amount of time of step (c) is about 1 to about 12 days or, optionally, about 8 to about 12 days.

7. The method of claim 1 or 2, wherein the sufficient amount of time of step (d) is about 1 to about 10 days or, optionally, about 7 to about 10 days.

8. The method of claim 1 or 2, wherein the cells are cultured in hypoxic conditions.

9. The method of claim 6, wherein the hypoxic conditions comprise about 1% oxygen to about 10% oxygen, optionally, about 5% oxygen.

10. The method of claim 1 or 2, wherein the wingless (Wnt) activator comprises or consists of CHIR99021.

11. The method of claim 1 or 2, wherein the transforming growth factor beta (TGF-b) type I receptor inhibitor comprises or consists of 616452.

12. The method of claim 1 or 2, wherein the retinoic acid analogue comprises or consists of TTNPB.

13. The method of claim 1 or 2, wherein the agonist of smoothened (SMO) comprises or consists of SAG.

14. The method of claim 1 or 2, wherein the tyrosine kinase inhibitor comprises or consists of ABT-869.

15. The method of claim 1 or 2, wherein the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor comprises or consists of Y-27632 or Tzv.

16. The method of claim 1 or 2, wherein the inhibitor of JNK1, JNK2 and JNK3 comprises or consists of JNKIN8.

17. The method of claim 1 or 2, wherein the monoamine oxidase inhibitor (MAOI) comprises or consists of tranylcypromine.

18. The method of claim 1 or 2, wherein the DNA methylation inhibitor comprises or consists of 5-azacytidine.

19. The method of claim 1 or 2, wherein the histone methyltransferase G9a inhibitor comprises or consists of UNC0224.

20. The method of claim 1 or 2, wherein the inhibitor of JNK1 and JNK2 comprises or consists of ruxolitinib.

21. The method of claim 1 or 2, wherein the CBP / p300 bromodomain (BRD) inhibitor comprises or consists of SGC-CBP30.

22. The method of claim 1 or 2, wherein the histone deacetylase (HDAC) inhibitor comprises or consists of valproic acid (VP A).

23. The method of claim 1 or 2, wherein the inhibitor of S-adenosylmethionine- dependent methyltransferase comprises or consists of DZNep.

24. The method of claim 1 or 2, wherein the DOT1L inhibitor comprises or consists of EPZ004777.

25. The method of claim 1 or 2, wherein the N-lysine methyltransferase SETD8 inhibitor comprises or consists of UNC0379.

26. The method of claim 1 or 2, wherein the MEK / ERK pathway inhibitor comprises or consists of PD0325901.

27. The method of claim 1 or 2, wherein the B-Raf inhibitor comprises or consists of SB590885.

28. The method of claim 1 or 2, wherein the inhibitor of Wnt production comprises or consists of IWP-2.

29. A method comprising:(a) contacting mesenchymal stem cells with one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days;(b) contacting the cells of step (a) with one or more compound selected from the group consisting CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5 -azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days;(c) contacting the cells of step (b) with one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VP A), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and(d) contacting the cells of step (c) with one or more compound selected from the group consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VP A) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells.

30. A method comprising:(a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days;(b) culturing the cells of step (a) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, TTNPB. SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days;(c) culturing the cells of step (b) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VPA), DZNep, EPZ004777. and UNC0379 for about 8 days to about 12 days; and(d) culturing the cells of step (c) in a suitable medium comprising one or more compound selected from the group consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VPA) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells.

31. A method comprising:(a) contacting mesenchymal stem cells with one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days;(b) contacting the cells of step (a) with one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days;(c) contacting the cells of step (b) with one or more compound consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid(VP A), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and(d) contacting the cells of step (c) with one or more compound consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VP A) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells.

32. A method comprising:(a) culturing mesenchymal stem cells in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, and Y-27632 for about 8 to about 10 days;(b) culturing the cells of step (a) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, TTNPB, SAG, ABT-869, Y-27632, JNKIN8, tranylcypromine, 5-azacytidine, UNC0224, ruxolitinib, and SGC-CBP30 for about 16 to about 20 days;(c) culturing the cells of step (b) in a suitable medium comprising one or more compound consisting of CHIR99021, 616452, Y-27632, PD0325901, tranylcypromine, valproic acid (VP A), DZNep, EPZ004777, and UNC0379 for about 8 days to about 12 days; and(d) culturing the cells of step (c) in a suitable medium comprising one or more compound consisting of CHIR99021, Y-27632, PD0325901, IWP-2, SB590885, and valproic acid (VP A) for about 7 days to about 10 days to prepare induced pluripotent stem cells from mesenchymal stem cells.

33. The method of any one of claims 1, 2, or 29-32, wherein the mesenchymal stem cells are derived from umbilical cord tissue, optionally, wherein the umbilical cord tissue comprises umbilical cord blood.

34. The method of any one of claims 1, 2. or 29-32, wherein the prepared induced pluripotent stem cells express one or more factors selected from the group consisting of NANOG, OCT4, and SOX2.

35. The method of any one of claims 1, 2, or 29-32, further comprising differentiating the prepared induced pluripotent stem cells.

36. An induced pluripotent stem cell (iPSC) produced by the method of any one of claims 1, 2, or 29-32.

37. An induced pluripotent stem cell derived from a mesenchymal stem cell that does not comprise an exogenous polynucleotide.

38. A pharmaceutical composition comprising the iPSC of claim 36 or 37, optionally, further comprising a pharmaceutically acceptable carrier or excipient.

39. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 38 to the subject to treat the disease or disorder.

40. The method of claim 39, wherein the disease or disorder is selected from the group consisting of a cardiovascular disease or disorder, a neurological disease or disorder, a neurodegenerative disease or disorder, a musculoskeletal disease or disorder, or other disease or disorder.

41. A kit comprising one or more of a wingless (Wnt) activator, a transforming growth factor beta (TGF-P) type I receptor inhibitor, a retinoic acid analogue, an agonist of smoothened (SMO). a ty rosine kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, an inhibitor of C-Jun N-terminal kinase- 1 (JNK1) and JNK2; a monoamine oxidase inhibitor (MAOI), a DNA methylation inhibitor, a histone methyltransferase G9a inhibitor, an inhibitor of C-Jun N-terminal kinase-1 (JNK1), JNK2, and JNK3; an inhibitor of JNK1 and JNK2; and a CBP / p300 bromodomain (BRD) inhibitor, a MEK / ERK pathway inhibitor, a histone deacetylase (HD AC) inhibitor, aninhibitor of S-adenosylmethionine-dependent methyltransferase, aDOTIL inhibitor, and an N-lysine methyltransferase SETD8 inhibitor, and an inhibitor of Wnt production.

42. The kit of claim 41, wherein the wingless (Wnt) activator comprises or consists of CHIR99021.

43. The kit of claim 41 or 42, wherein the transforming growth factor beta (TGF-b) type I receptor inhibitor comprises or consists of 616452.

44. The kit of claim 41, wherein the retinoic acid analogue comprises or consists of TTNPB45. The kit of claim 41, wherein the agonist of smoothened (SMO) comprises or consists of SAG.

46. The kit of claim 41, wherein the tyrosine kinase inhibitor comprises or consists of ABT-869.

47. The kit of claim 41, wherein the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor comprises or consists of Y-27632 or Tzv.

48. The kit of claim 41, wherein the inhibitor of JNK1, JNK2 and JNK3 comprises or consists of JNKIN8.

49. The kit of claim 41, wherein the monoamine oxidase inhibitor (MAOI) comprises or consists of tranylcypromine.

50. The kit of claim 41, wherein the DNA methylation inhibitor comprises or consists of 5-azacytidine.

51. The kit of claim 41 , wherein the histone methyltransferase G9a inhibitor comprises or consists of UNC0224.

52. The kit of claim 41. wherein the inhibitor of JNK1 and JNK2 comprises or consists of ruxolitinib.

53. The kit of claim 41 , wherein the CBP / p300 bromodomain (BRD) inhibitor comprises or consists of SGC-CBP30.

54. The kit of claim 41 , wherein the histone deacetylase (HD AC) inhibitor comprises or consists of valproic acid (VP A).

55. The kit of claim 41, wherein the inhibitor of S-adenosylmethionine-dependent methyltransferase comprises or consists of DZNep.

56. The kit of claim 41, wherein the DOT IL inhibitor comprises or consists of EPZ004777.

57. The kit of claim 41, wherein the N-lysine methyltransferase SETD8 inhibitor comprises or consists of UNC0379.

58. The kit of claim 41, wherein the MEK / ERK pathway inhibitor comprises or consists of PD0325901.

59. The kit of claim 41. wherein the B-Raf inhibitor comprises or consists of SB590885.

60. The kit of claim 41, wherein the inhibitor of Wnt production comprises or consists of IWP-2.

61. The kit of any one of claims 41-60, further comprising instructions for using the kit to perform the method of any one of claims 1-35.

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