Method of producing multipotent stem cells
The present invention provides a method of producing a multipotent stem cell, said method comprising culturing at least one fibroblast cell in the presence of an effective amount of at least one small molecule reprogramming factor(s) that induces the cell to de-differentiate into a multipotent stem cell, wherein the method excludes the use of reprogramming factor(s) that are not small molecules. The small molecule reprogramming factor(s) may include a G9a HMTase inhibitor(s) and/or a MEK inhibitor(s) optionally in combination with other small molecule reprogramming factor(s). The invention also includes methods of differentiating the multipotent stem cells, cells produced by the methods, assays using the cells and kits for use in the methods.
1. A method of producing a multipotent neural stem cell that expresses ALP, Sox2, and SSEA1, said method comprising culturing at least one fibroblast cell in the presence of an effective amount of at least one small molecule reprogramming factor(s) that induces the cell to de-differentiate into a multipotent neural stem cell that expresses ALP, Sox2 and SSEA1, wherein the small molecule reprogramming factor(s) is selected from the group consisting of G9a HMTase inhibitors and MEK inhibitors, and, wherein the method excludes the use of exogenous reprogramming factor(s) that are not small molecules that induce the cell to de-differentiate into a multipotent neural stem cell.
2. The method of claim 1 , wherein the small molecule reprogramming factor(s) is a G9a HMTase inhibitor(s).
3. The method of claim 2 , wherein the G9a HMTase inhibitor(s) is in combination with an effective amount of at least one further small molecule reprogramming factor(s) selected from the group consisting of a histone deacetylase (HDAC) inhibitor(s), a MEK inhibitor(s), a DNA methyltransferase inhibitor(s), a glycogen synthase kinase 3 (GSK3) inhibitor(s), Vitamin C, and a Activin receptor-like kinase (ALK) receptor inhibitor(s).
4. The method of claim 2 , wherein the G9a HMTase inhibitor(s) is in combination with a DNA methyltransferase inhibitor(s).
5. The method of claim 2 , wherein the G9a HMTase inhibitor(s) is in combination with a DNA methyltransferase inhibitor(s) and a MEK inhibitor(s).
6. The method of claim 1 , wherein the small molecule reprogramming factor(s) is a MEK inhibitor(s).
7. The method of claim 1 , wherein the MEK inhibitor(s) is in combination with an effective amount of at least one further small molecule reprogramming factor(s) selected from the group consisting of a HDAC inhibitor(s), a G9a HMTase inhibitor(s), a DNA methyltransferase inhibitor(s) a GSK3 inhibitor(s), Vitamin C, and a ALK receptor inhibitor(s).
8. The method of claim 1 , wherein the G9a HMTase inhibitor(s) is 2-(hexahydro-4-methyl-1H-1,4-diazepin-1-yl)-6,7-dimethoxy-N-[1-(phenylmethyl)-4-piperidinyl]-4-quinazolinamine trihydrochloride hydrate (BIX01294).
9. The method of claim 3 , wherein the DNA methyltransferase inhibitor(s) is 1H-Indole-3-propanoic acid, α-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)-, (αS)— (RG108).
10. The method of claim 1 , wherein the MEK inhibitor(s) is N-(2,3-dihydroxy-propoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide (PD325901).
11. The method of claim 1 , wherein the multipotent neural stem cell is a human cell.
12. The method of claim 1 , wherein the culturing comprises:
(a) performing an at least one induction cycle comprising culturing for approximately one day the at least one fibroblast cell in the presence of an effective amount of at least one small molecule reprogramming factor(s) that induces the cell to de-differentiate into a multipotent neural stem cell, and then culturing for approximately two days the at least one cell in the absence of said effective amount of at least one small molecule reprogramming factor(s), and optionally
(b) culturing the at least one cell of step (a) in media adapted to support multipotent neural stem cell growth for a suitable period.