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 . 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 Wnt signaling promoter in combination with an effective amount of an Activin A/Smad pathway inhibitor, and optionally, an effective amount of a bone morphogenic protein inhibitor, the resulting neural crest-like stem cells being appreciably free of PAX6+ neural progenitor cells. 
     
     
         2 . The method according to  claim 1  wherein said pluripotent stem cells are human pluripotent stem cells. 
     
     
         3 . The method according to  claim 2  wherein said neural crest-like cells are produced without separation from said neural progenitor cells. 
     
     
         4 . The method according to  claim 1  wherein said neural crest-like cells comprise at least 90% of a population of said neural crest-like stem cells and said neural progenitor cells. 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . The method according to  claim 1  wherein said Wnt signaling promoter is a GSK inhibitor. 
     
     
         8 . The method according to  claim 7  wherein said GSK inhibitor is BIO. 
     
     
         9 . The method according to  claim 1  wherein said Wnt signaling promoter is a Wnt protein. 
     
     
         10 . The method according to  claim 9  wherein said Wnt protein is Wnt3a. 
     
     
         11 . The method according to  claim 1  wherein said Activin A/Smad pathway inhibitor is SB431542. 
     
     
         12 . (canceled) 
     
     
         13 . The method according to  claim 1  wherein said pluripotent stem cells are human embryonic stem cells or human induced pluripotent cells. 
     
     
         14 . The method according to  claim 1  wherein said GSK inhibitor is BIO, said Wnt protein is Wnt3a and said Activin A/Smad pathway inhibitor is SB431542. 
     
     
         15 . The method according to  claim 13  wherein said pluripotent cells are differentiated in a differentiation medium consisting essentially of a GSK inhibitor, a Wnt protein and an Activin A/Smad pathway inhibitor. 
     
     
         16 . (canceled) 
     
     
         17 . The method according to  claim 1  wherein said differentiation medium further includes an effective amount of a BMP inhibitor. 
     
     
         18 . The method according to  claim 17  wherein said BMP inhibitor is selected from the group consisting of noggin, chordin, follistatin, sclerostin, gremlin, dorsomorphorin, connective tissue growth factor (CTGF). 
     
     
         19 . The method according to  claim 17  wherein said BMP inhibitor is noggin. 
     
     
         20 . A method of producing p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from human pluripotent stem cells, in particular human embryonic stem cells, comprising differentiating said pluripotent stem cells in a differentiation medium consisting essentially of an effective amount of a Wnt signaling promoter 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 7 to about 20 days, the resulting neural crest-like cells being appreciably free of Pax6+ neural progenitor cells. 
     
     
         21 . The method according to  claim 20  wherein said pluripotent cells are differentiated to neural crest-like cells for a period ranging from between about 10 and 15 days. 
     
     
         22 . The method according to  claim 20  wherein said neural crest-like cells are produced without separation from said neural progenitor cells. 
     
     
         23 . The method according to  claim 20  wherein said neural crest-like cells comprise at least 90% of a population of said neural crest-like cells and said neural progenitor cells. 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . The method according to  claim 20  wherein said Wnt signaling promoter is a GSK inhibitor. 
     
     
         27 . The method according to  claim 26  wherein said GSK inhibitor is BIO. 
     
     
         28 . The method according to  claim 20  wherein said Wnt signaling promoter is a Wnt protein. 
     
     
         29 . The method according to  claim 28  wherein said Wnt protein is Wnt3a. 
     
     
         30 . The method according to  claim 20  wherein said Activin A/Smad pathway inhibitor is SB431542. 
     
     
         31 . (canceled) 
     
     
         32 . The method according to  claim 20  wherein said pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells. 
     
     
         33 . The method according to  claim 20  wherein said GSK inhibitor is BIO, said Wnt protein is Wnt3a and said Activin A/Smad pathway inhibitor is SB431542. 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . The method according to  claim 20  wherein said differentiation medium includes a BMP inhibitor. 
     
     
         37 . The method according to  claim 36  wherein said BMP inhibitor is selected from the group consisting of noggin, chordin, follistatin, sclerostin, gremlin, dorsomorphin, connective tissue growth factor (CTGF) and mixtures thereof. 
     
     
         38 . The method according to  claim 37  wherein said BMP inhibitor is noggin. 
     
     
         39 . The method according to  claim 2  wherein said p75+ Hnk1+ Ap2+ neural crest-like cells are further differentiated into peripheral neurons or mesenchymal progenitor cells. 
     
     
         40 . The method according to  claim 39  wherein said mesenchymal progenitor cells are further differentiated into smooth muscle cells, adipocytes, osteocytes and/or chondrocytes. 
     
     
         41 . A composition comprising a population of p75+ Hnk1+ Ap2+ multipotent neural crest-like cells in a differentiation medium consisting essentially of at least one Wnt signaling promoter in combination with an Activin A/Smad pathway inhibitor and optionally a BMP inhibitor, said composition being appreciably free of PAX6+ neural progenitor cells. 
     
     
         42 . The composition according to  claim 40  wherein said Wnt signaling promoter is BIO. 
     
     
         43 . The composition according to  claim 40  wherein said Wnt signaling promoter is Wnt3a. 
     
     
         44 . The composition according to  claim 40  wherein said Activin A/Smad pathway inhibitor is SB431542. 
     
     
         45 . The composition according to  claim 40  wherein said Wnt signaling promoter is a combination of BIO and Wnt3a and said Activin A/Smad pathway inhibitor is SB431542. 
     
     
         46 . The composition according to  claim 40  which includes a BMP inhibitor. 
     
     
         47 . The composition according to  claim 45  wherein said BMP inhibitor is noggin, chordin, follistatin, sclerostin, gremlin, dorsomorphin, connective tissue growth factor (CTGF) or a mixture thereof. 
     
     
         48 . The composition according to  claim 46  wherein said BMP inhibitor is noggin. 
     
     
         49 . A composition consisting essentially of a population of p75+ Hnk1+ Ap2+ multipotent neural crest-like stem cells in a differentiation medium produced by exposing human pluripotent stem cells to a combination of effective amounts of BIO, Wnt3a and SB431542, and optionally noggin in a differentiation medium for a period ranging from about 10 to about 14 days, said composition being appreciably free of PAX6+ neural progenitor cells. 
     
     
         50 . The composition according to  claim 49  wherein said pluripotent stem cells are exposed to BIO, Wnt3a and SB431542, and optionally noggin in a differentiation medium for a period ranging from about 10 days to about 12 days.