Methods and compositions for spinal cord cells

Abstract

Described here are systems and methods for deriving both spinal motor neurons and brain microvascular endothelial cells from induced pluripotent stem cells using distinct methods and combining them in a chip format. Neurons cultured alone in chip microvolume displayed increased calcium transient function and chip-specific gene expression. When seeded with endothelial cells, interaction further enhanced neural function, elicited vascular-neural interaction, niche gene expression with enhanced in vivo-like signatures arising from the chip co-cultures. Development of novel media formulations further allow for improved readout of differentiation process, by eliminating additives that otherwise confound differentiation processes and resulting phenotypes.

1 . A method of generating spinal neural progenitor cells (spNPCs) comprising:
 providing induced pluripotent stem cells (iPSCs);   differentiating iPSCs into neural ectodermal cells by culturing in neural induction media; and   dissociating neural ectodermal cells, replating on a cell culture substrate and further culturing the replated neural ectodermal cells in differentiation media to generate spNPCs.   
     
     
         2 . The method of  claim 1 , wherein the iPSCs are cultured in neural induction media for a period of about 5-7 days. 
     
     
         3 . The method of  claim 1 , wherein the replated neural ectodermal cells are cultured in differentiation media for a period of about 5-7 days. 
     
     
         4 . The method of  claim 1 , wherein the neural induction media comprises one or more of: LDN193189, SB431542, and CHIR99021. 
     
     
         5 . The method of  claim 1 , wherein the cell culture substrate is matrigel. 
     
     
         6 . The method of  claim 1 , wherein the differentiation media comprises one or more of: ascorbic acid, retinoic acid, SAG. 
     
     
         7 . The method of  claim 1 , further comprising freezing the spNPCs. 
     
     
         8 . A cryopreserved solution of spNPCs made by the method of  claim 7 . 
     
     
         9 . A cell culture supplement, comprising:
 one or more first additives selected from the group consisting of: Albumin, Bovine Serum, Sodium Bicarbonate, L-Ascorbic Acid,   
       Putrescine, D(+)-Galactose, Holo-transferrin, Catalase, L-Carnitine, Glutathione (reduced), Sodium Selenite, Ethanolamine, and T3 (triiodo-L-thyronine); and
 one or more second additives selected from the group consisting of: Corticosterone, Linoleic Acid, Linolenic Acid, Lipoic Acid (thioctic acid), Progesterone, Retinol Acetate, a-Tocopherol (vitamin E), and a-Tocopherol acetate. 
 
     
     
         10 . A cell culture media comprising:
 the cell culture supplement of  claim 9 , and a base media.   
     
     
         11 . The cell culture media of  claim 10 , wherein the base media comprises DMEM/F12. 
     
     
         12 . A method of generating spinal neural progenitor cells (spNPCs) comprising:
 providing induced pluripotent stem cells (iPSCs);   differentiating iPSCs into neural ectodermal cells by culturing in neural induction media for about 6 days; and   dissociating neural ectodermal cells, replating on a cell culture substrate and further culturing the replated neural ectodermal cells in differentiation media for about 6 days to generate spNPCs.   
     
     
         13 . The method of  claim 12 , wherein the neural induction media, differentiation media, or both, comprise a supplement comprising:
 one or more first additives selected from the group consisting of: Albumin, Bovine Serum, Sodium Bicarbonate, L-Ascorbic Acid,   
       Putrescine, D(+)-Galactose, Holo-transferrin, Catalase, L-Carnitine, Glutathione (reduced), Sodium Selenite, Ethanolamine, and T3 (triiodo-L-thyronine); and
 one or more second additives selected from the group consisting of: Corticosterone, Linoleic Acid, Linolenic Acid, Lipoic Acid (thioctic acid), Progesterone, Retinol Acetate, a-Tocopherol (vitamin E), and a-Tocopherol acetate. 
 
     
     
         14 . The method of  claim 12 , wherein the neural induction media comprises one or more of: LDN193189, SB431542, and CHIR99021. 
     
     
         15 . The method of  claim 12 , wherein the cell culture substrate is matrigel. 
     
     
         16 . The method of  claim 12 , wherein the differentiation media comprises one or more of: ascorbic acid, retinoic acid, SAG. 
     
     
         17 . A quantity of spNPCs made by the method of  claim 12 . 
     
     
         18 . The method of  claim 12 , further comprising freezing the spNPCs. 
     
     
         19 . A cryopreserved solution of spNPCs made by the method of  claim 18 .