Method for highly efficient conversion of human stem cells to lineage-specific neurons

Abstract

The present invention relates to the field of stem cells. More specifically, the present invention provides methods and compositions useful for the highly efficient conversion of human stem cells to lineage-specific neurons. In a specific embodiment, a method of inducing differentiation of human stem cells into dopaminergic (DA) neurons comprises the steps of (a) transfecting human stem cells with a lentiviral vector encoding Atoh1, wherein the vector is Dox inducible; and (b) growing the transfected cells in culture in the presence of Dox, Sonic Hedgehog (SHH) and FGF-8b until DA neurons are induced.

1 . A method for inducing differentiation of neuronal cells from human stem cells comprising the steps of (a) transfecting human stem cells with an expression vector encoding Atoh1; and (b) growing the transfected cells in culture until the stem cells are differentiated. 
     
     
         2 . The method of  claim 1 , wherein the human stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs) or neural stem cells (NSCs). 
     
     
         3 . The method of  claim 2 , wherein the neural stem cells are fetal or adult neural stem cells. 
     
     
         4 . The method of  claim 1 , wherein the expression vector is a viral vector. 
     
     
         5 . The method of  claim 4 , wherein the viral vector is from a lentivirus, adeno-associated virus, herpes simplex virus, Senai virus or baculovirus. 
     
     
         6 . The method of  claim 5 , wherein the viral vector is from a lentivirus. 
     
     
         7 . The method of  claim 1 , wherein the expression vector is non-viral. 
     
     
         8 . The method of  claim 1 , wherein the expression vector encoding Atoh1 is inducible. 
     
     
         9 . The method of  claim 6 , wherein the lentiviral expression vector is doxycycline (Dox) inducible. 
     
     
         10 . The method of  claim 9 , wherein the cells are grown in the presence of a sufficient concentration of Dox in order to complete differentiation into neuronal cells. 
     
     
         11 . The method of  claim 9 , wherein step (b) further comprises the steps outlined in Table 1. 
     
     
         12 . The method of  claim 1 , wherein prior to step (b), the method further comprises the step of transfecting the human stem cells with an expression vector encoding NeuroD1. 
     
     
         13 . The method of  claim 12 , wherein prior to step (b), the method further comprises the step of transfecting the human stem cells with an expression vector encoding Neurogenin 2. 
     
     
         14 . The method of  claim 1 , wherein the expression vector also encodes NeuroD1 and/or Neurogenin 2. 
     
     
         15 . The method of  claim 1 , further comprising exposing the cells to sufficient concentrations of additional growth factors. 
     
     
         16 . A method of inducing differentiation of human stem cells into dopaminergic (DA) neurons comprising the steps of:
 a. transfecting human stem cells with a lentiviral vector encoding Atoh1, wherein the vector is Dox inducible; and   b. growing the transfected cells in culture in the presence of Dox, Sonic Hedgehog (SHH) and FGF-8b until DA neurons are induced.   
     
     
         17 . The method of  claim 16 , wherein the human stem cells are iPSCs. 
     
     
         18 . The method of  claim 16 , wherein the human stem cells are ESCs. 
     
     
         19 . The method of  claim 17 , wherein step (b) further comprises the steps outlined in Table 2. 
     
     
         20 . The method of  claim 17 , wherein the human stem cells are NSCs. 
     
     
         21 . The method of  claim 20 , wherein step (b) further comprises the steps outlined in Table 3. 
     
     
         22 . The method of  claim 16 , wherein prior to step (b), the method further comprises the step of transfecting the human stem cells with an expression vector encoding NeuroD1. 
     
     
         23 . The method of  claim 22 , wherein prior to step (b), the method further comprises the step of transfecting the human stem cells with an expression vector encoding Neurogenin 2. 
     
     
         24 . The method of  claim 16 , wherein the lentiviral vector also encodes NeuroD1 and/or Neurogenin 2. 
     
     
         25 . A method for treating a patient suffering from a neurodegenerative disease comprising the steps of:
 a. obtaining stem cells from the patient;   b. initiating differentiation of the stem cells into a population of differentiated cells using the methods of  claim 16 ;   c. analyzing the development of differentiated neurons in culture; and   d. transplanting the differentiated cells into the patient's brain.   
     
     
         26 . The method of  claim 25 , wherein the neurodegenerative disease is Parkinson's disease and the differentiated cells are DA neurons. 
     
     
         27 . A population of neuronal cells prepared using the method of  claim 16 .