Differentiation of human pluripotent stem cells to multipotent neural crest cells

Abstract

The present invention relates to the differentiation of human pluripotent cells, including human pluripotent stems cells to produce a self-renewing multipotent neural crest cell population in a single step method without the requirement of isolation of intermediate cells and without appreciable contamination (in certain preferred instances, virtually none) with Pax6+ neural progenitor cells in the population of p75+Hnk1+Ap2+ multipotent neural crest-like cells. The multipotent neural crest cell population obtained can be clonally amplified and maintained for >25 passages (>100 days) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells and mesenchymal precursor cells.

1 - 50 . (canceled) 
     
     
         51 . A method of producing p75+Hnk1+Ap2+ multipotent neural crest-like cells from pluripotent stem cells comprising differentiating said pluripotent stem cells in a differentiation medium consisting essentially of an effective amount of a Wingless (Wnt) signaling agonist in combination with an effective amount of an Activin A/Smad pathway inhibitor, and optionally, an effective amount of a bone morphogenic protein inhibitor. 
     
     
         52 . The method according to  claim 51  wherein said pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells. 
     
     
         53 . The method according to  claim 51  wherein said neural crest-like cells comprise at least 70% or 80% or, preferably 90% of a population of said neural crest-like stem cells. 
     
     
         54 . The method according to  claim 51  wherein said neural crest-like cells comprise at least 95% of a population of said neural crest-like cells. 
     
     
         55 . The method according to  claim 51  wherein said neural crest-like cells comprise at least 99% of a population of said neural crest-like cells. 
     
     
         56 . The method according to  claim 51  wherein said Wnt signaling promoter is a GSK3 inhibitor. 
     
     
         57 . The method according to  claim 56  wherein said GSK3 inhibitor is selected from the group consisting of GSK3 inhibitor IX (BIO), CHIR 99021, TD2D8, and A1070722. 
     
     
         58 . The method according to  claim 51  wherein said Wnt signaling promoter is a Wnt protein. 
     
     
         59 . The method according to  claim 58  wherein said Wnt protein is Wnt3a. 
     
     
         60 . The method according to  claim 51  wherein said Activin A/Smad pathway inhibitor is SB-431542, A83-01, R268712, or GW788288. 
     
     
         61 . The method according to a  claim 51  wherein said pluripotent stem cells are human pluripotent stem cells. 
     
     
         62 . The method according to  claim 51  wherein said pluripotent stem cells are human embryonic stem cells or human induced pluripotent cells. 
     
     
         63 . The method according to  claim 51  wherein a GSK inhibitor, or a Wnt protein and an Activin A/Smad pathway inhibitor. 
     
     
         64 . The method according to  claim 63  wherein said pluripotent cells are differentiated in a differentiation medium consisting essentially of a GSK inhibitor or a Wnt protein and an Activin A/Smad pathway inhibitor. 
     
     
         65 . The method according to  claim 64  wherein a GSK3 inhibitor or a said Wnt protein is used with an Activin A/Smad pathway inhibitor. 
     
     
         66 . The method according to  claim 51  wherein said differentiation medium may further include an effective amount of a BMP signaling inhibitor. 
     
     
         67 . The method according to  claim 66  wherein said BMP inhibitor is selected from a group including ALK2/3 inhibitors such as LDN 193189, TP 0184, Noggin and Compound C (dorsomorphorin). 
     
     
         68 . A method of producing p75+Hnk1+ multipotent neural crest-like cells from pluripotent stem cells, comprising differentiating said pluripotent stem cells in a differentiation medium consisting essentially of an effective amount of a Wnt signaling agonist in combination with an effective amount of an Activin A/Smad pathway inhibitor, optionally in combination with an effective amount of a BMP inhibitor for a period of ranging from about 4 to about 40 days. 
     
     
         69 . The method according to  claim 68  wherein said pluripotent cells are differentiated to neural crest-like cells for a period ranging from between about 6 and 20 days. 
     
     
         70 . The method according to  claim 68  wherein said neural crest-like cells comprise at least 70% of a population of said neural crest-like cells and said neural progenitor cells. 
     
     
         71 . The method according to  claim 68  wherein said neural crest-like cells comprise at least 80% of a population of said neural crest-like cells and said neural progenitor cells. 
     
     
         72 . The method according to  claim 68  wherein said neural crest-like cells comprise at least 90% of a population of said neural crest-like cells and said neural progenitor cells. 
     
     
         73 . The method according to  claim 68  wherein said neural crest-like cells comprise at least 95% of a population of said neural crest-like cells and said neural progenitor cells. 
     
     
         74 . The method according to  claim 68  wherein said neural crest-like cells comprise at least 99% of a population of said neural crest-like cells and said neural progenitor cells.