Methods of differentiation to neuronal cells and kits therefor

Abstract

Embodiments herein provide methods of differentiating neural stem cells to neuronal cells while concomitantly retarding neural stem cell proliferation. Resultant cultures demonstrate reduced clumping of cells, increased purity of neuronal cells and accelerated electrophysiology as compared to control methods.

1 - 11 . (canceled) 
     
     
         12 . A method of reducing cell clumping during differentiation of neural stem cells to neuronal cells, comprising:
 culturing the neural stem cells in a differentiation medium for a time and under conditions to form neuronal cells,   wherein the differentiation medium comprises a serum-free neural stem cell culture medium, and a serum-free supplement comprising at least one gamma secretase inhibitor,   wherein cell clumping is reduced at least 50% when compared to culturing the neural stem cells in the differentiation medium lacking the at least one gamma secretase inhibitor.   
     
     
         13 . The method of  claim 12 , wherein the serum-free supplement of the differentiation medium comprises a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027, LY411575, MK-0752, a salt thereof, and a combination thereof. 
     
     
         14 . The method of  claim 13 , wherein the gamma secretase inhibitor is present in the differentiation medium at a concentration of 0.2 micromolar to 2.0 micromolar. 
     
     
         15 . The method of  claim 12  wherein cell clumping is reduced at least 75% when compared to culturing the neural stem cells in the differentiation medium lacking the at least one gamma secretase inhibitor. 
     
     
         16 - 20 . (canceled) 
     
     
         21 . The method of  claim 12 , wherein the neural stem cells are derived from an induced pluripotent stem cells. 
     
     
         22 . The method of  claim 12 , wherein the neural stem cells are derived from an embryonic stem cell. 
     
     
         23 . The method of  claim 12 , wherein the neural stem cells comprise SOX1 positive neural stem cells. 
     
     
         24 . The method of  claim 12 , wherein the neuronal cells comprise MAP2 positive neuronal cells. 
     
     
         25 . The method of  claim 12 , wherein the gamma secretase inhibitor is present in the differentiation medium at a concentration of 0.1 micromolar to 80 micromolar. 
     
     
         26 . The method of  claim 12 , wherein the gamma secretase inhibitor is present in the differentiation medium at a concentration of up to 2.0 micromolar. 
     
     
         27 . The method of  claim 13 , wherein the gamma secretase inhibitor is present in the differentiation medium at a concentration of 0.1 micromolar to 40 micromolar. 
     
     
         28 . The method of  claim 13 , wherein the serum-free supplement of the differentiation medium comprises a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027, LY411575, a salt thereof, and a combination thereof. 
     
     
         29 . The method of  claim 24 , wherein the gamma secretase inhibitor is present in the differentiation medium at a concentration of 0.2 micromolar to 10 micromolar. 
     
     
         30 . The method of  claim 24 , wherein the gamma secretase inhibitor is present in the differentiation medium at a concentration of 0.2 micromolar to 2.0 micromolar.