Generation of neural stem cells and motor neurons

Abstract

A method of generating a population of cells useful for treating a brain disorder in a subject is disclosed. The method comprises contacting mesenchymal stem cells (MSCs) with at least one exogenous miRNA having a nucleic acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 15-19 and 27-35, thereby generating a population of cells and/or generating neurotrophic factors that may provide important signals to damaged tissues or locally residing stem cells. MSCs differentiated by miRs may also secrete miRs and deliver them to adjacent cells and therefore provide important signals to neighboring endogenous normal or malignant cells.

1 . A method of promoting mesenchymal stem cell (MSC) differentiation toward a neuronal stem cell, the method comprising:
 (i) culturing a MSC in a medium supporting neuronal stem cell growth and differentiation;   (ii) contacting said MSC with an exogenous agent that down-regulates an amount, activity or both of Related to testis-specific, vespid and pathogenesis protein 1 (RTVP-1); and   (iii) confirming increased expression at least one neuronal marker selected from the group consisting of nestin and Sox2 by detecting expression of said marker on said MSC, thereby promoting differentiation of the MSC into the neuronal stem cell.   
     
     
         2 . The method of  claim 1 , wherein said contacting comprises introducing into said MSC a microRNA (miR) or siRNA that down-regulates RTVP-1. 
     
     
         3 . The method of  claim 2 , wherein said miR is miR-137. 
     
     
         4 . The method of  claim 2 , wherein said siRNA comprises the nucleotide sequence provided in SEQ ID NO: 481. 
     
     
         5 . The method of  claim 2 , wherein said introducing comprises any one of:
 (i) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes a pre-miRNA of said miR;   (ii) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes said miR;   (iii) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes an shRNA comprising said siRNA; and   (iv) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes said siRNA.   
     
     
         6 . The method of  claim 1 , further comprising introducing into said MSC an exogenous molecule selected from miR-218, miR-504, miR-9, miR-125 and a miR-31 antagomir before said confirming. 
     
     
         7 . The method of  claim 6 , comprising introducing into said MSC exogenous miR-218. 
     
     
         8 . The method of  claim 6 , wherein said introducing comprises any one of:
 (i) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes a pre-miRNA of said miR; and   (ii) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes said miR.   
     
     
         9 . A method of promoting mesenchymal stem cell (MSC) differentiation toward a neuronal stem cell, the method comprising:
 (i) culturing a MSC in a medium supporting neuronal stem cell growth and differentiation;   (ii) contacting said MSC with an exogenous agent that down-regulates an amount, activity or both of Related to testis-specific, vespid and pathogenesis protein 1 (RTVP-1); and   (iii) confirming expression of at least one neuronal stem cell marker selected from the group consisting of nestin and Sox2, wherein said expressing results in at least 50% of the MSCs expressing said neuronal stem cell marker, thereby promoting differentiation of the MSC into the neuronal stem cell.   
     
     
         10 . The method of  claim 9 , wherein said contacting comprises introducing into said MSC a microRNA (miR) or siRNA that down-regulates RTVP-1. 
     
     
         11 . The method of  claim 10 , wherein said miR is miR-137. 
     
     
         12 . The method of  claim 10 , wherein said siRNA comprises the nucleotide sequence provided in SEQ ID NO: 481. 
     
     
         13 . The method of  claim 10 , wherein said introducing comprises any one of:
 (i) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes a pre-miRNA of said miR;   (ii) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes said miR;   (iii) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes an shRNA comprising said siRNA; and   (iv) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes said siRNA.   
     
     
         14 . The method of  claim 9 , further comprising introducing into said MSC an exogenous molecule selected from miR-218, miR-504, miR-9, miR-125 and a miR-31 antagomir before said confirming. 
     
     
         15 . The method of  claim 14 , comprising introducing into said MSC exogenous miR-218. 
     
     
         16 . The method of  claim 14 , wherein said introducing comprises any one of:
 (i) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes a pre-miRNA of said miR; and   (ii) transfecting said MSCs with an expression vector which comprises a polynucleotide sequence which encodes said miR.   
     
     
         17 . A method of treating a subject suffering from a brain cancer, the method comprising administering to said subject an MSC differentiated toward a neuronal stem cell by the method of  claim 1 , thereby treating a subject suffering from a brain cancer. 
     
     
         18 . The method of  claim 17 , wherein said brain cancer is a glioma. 
     
     
         19 . A method of treating a subject suffering from a brain cancer, the method comprising administering to said subject an MSC differentiated toward a neuronal stem cell by the method of  claim 9 , thereby treating a subject suffering from a brain cancer. 
     
     
         20 . The method of  claim 17 , wherein said brain cancer is a glioma.