Method for inducing oligodendrocyte precursor cells from oct4-induced human somatic cells through direct reprogramming

Abstract

Provided is a method of inducing oligodendrocyte precursor cells (OPCS) through direct reprogramming from human somatic cells into which a nucleic acid molecule encoding an Oct4 protein or Oct4 protein-treated human somatic cells. The method of inducing OPCs by treating Oct4-overexpressing human somatic cells with a low molecular weight substance may establish OPCs with high efficiency in a short period of time through direct reprogramming without via neural stem cells, and thus the OPCs are useful as a cell therapeutic agent for an intractable demyelinating disease.

1 . A method of inducing oligodendrocyte precursor cells (OPCs) from human somatic cells through direct reprogramming, comprising:
 culturing human somatic cells, into which a nucleic acid molecule encoding an Oct4 protein is introduced, in a medium containing (i) a TGF-β type I receptor inhibitor; (ii) an inhibitor of Rho-associated kinase (ROCK inhibitor); (iii) a histone deacetylase inhibitor; and (iv) a sonic hedgehog agonist (Shh agonist).   
     
     
         2 . The method of  claim 1 , wherein the medium further comprises a calcium channel agonist. 
     
     
         3 . A method of inducing oligodendrocyte precursor cells (OPCs) from human somatic cells through direct reprogramming, comprising:
 culturing human somatic cells in a medium containing (i) a TGF-β type I receptor inhibitor; (ii) an inhibitor of Rho-associated kinase (ROCK inhibitor); (iii) a histone deacetylase inhibitor; and (iv) a sonic hedgehog agonist (Shh agonist),   wherein the human somatic cells are treated with an Oct4 protein before, during or after the culture.   
     
     
         4 . The method of  claim 3 , wherein the medium further comprises a calcium channel agonist. 
     
     
         5 . The method of  claim 1 , wherein the medium further comprises any one selected from the group consisting of RG108, BIX01294, SP600125, lysophosphatidic acid, Bayk8644, forskolin, dexamethasone, EX527 and rolipram. 
     
     
         6 . The method of  claim 1 , wherein the TGF-β type I receptor inhibitor is A83-01, the ROCK inhibitor is thiazovivin, the histone deacetylase inhibitor is valproic acid, and the Shh agonist is purmorphamine. 
     
     
         7 . The method of  claim 2 , wherein the calcium channel agonist is forskolin. 
     
     
         8 . The method of  claim 1 , wherein the medium is DMEM containing N2, B27, penicillin/streptomycin, non-essential amino acids, bFGF, PDGF and ascorbic acid. 
     
     
         9 . The method of  claim 1 , wherein the human somatic cells are selected from the group consisting of foreskin fibroblasts, hair-follicle dermal papillae, IMR90 lung fibroblasts and dermal fibroblasts. 
     
     
         10 . The method of  claim 1 , wherein the OPCs express any one or more markers selected from the group consisting of PDGFRα, A2B5, Olig2, Sox10, S100b and ZFP536. 
     
     
         11 . The method of  claim 1 , wherein the OPCs do not express Sox1, Sox2 and Pax6 markers. 
     
     
         12 . A composition for inducing oligodendrocyte precursor cells (OPCs) through direct reprogramming from human somatic cells into which a nucleic acid molecule encoding an Oct4 protein is introduced or which are treated with Oct4 protein, comprising:
 (i) a TGF-β type I receptor inhibitor; (ii) an inhibitor of Rho-associated kinase (ROCK inhibitor); (iii) a histone deacetylase inhibitor; and (iv) a sonic hedgehog agonist (Shh agonist) as active ingredients.   
     
     
         13 . The composition of  claim 12 , further comprising:
 a calcium channel agonist.   
     
     
         14 . The composition of  claim 12 , further comprising:
 any one selected from the group consisting of RG108, BIX01294, SP600125, lysophosphatidic acid, Bayk8644, forskolin, dexamethasone, EX527 and rolipram.   
     
     
         15 . The composition of  claim 12 , wherein the TGF-β type I receptor inhibitor is A83-01, the ROCK inhibitor is thiazovivin, the histone deacetylase inhibitor is valproic acid, and the Shh agonist is purmorphamine. 
     
     
         16 . The composition of  claim 13 , wherein the calcium channel agonist is forskolin. 
     
     
         17 . The composition of  claim 12 , wherein the OPCs do not express Sox1, Sox2 and Pax6 markers. 
     
     
         18 . A method of differentiating oligodendrocyte precursor cells (OPCs) into oligodendrocytes, comprising:
 culturing the OPCs prepared by the method of  claim 1  in a medium containing an inhibitor of Rho-associated kinase (ROCK inhibitor), a calcium channel agonist and a leukemia inhibitory factor (LIF).   
     
     
         19 . The method of  claim 18 , wherein the medium is DMEM containing N2, B27, penicillin/streptomycin, non-essential amino acids, ascorbic acid and triiodo-1-thyronine (T3).