Methods for differentiating pluripotent stem cells in dynamic suspension culture

A method for inducing differentiation of human pluripotent stem cells into paired box 6 (PAX6) positive neuroectoderm cells, the method comprising:

• a) obtaining a suspension culture of non-embryoid body (non-EB) aggregates of undifferentiated human pluripotent stem cells, wherein the human pluripotent stem cells remain in an undifferentiated state;
• b) culturing the non-EB aggregates from a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta (TGFβ)/Activin/Nodal signaling selected from an inhibitor of activin receptor-like kinase 5 (ALK5), SB431542, LY2157299, GW788388, A-77-01, A-83-01 or SB505124 and at least one inhibitor of bone morphogenetic protein (BMP) signaling selected from an activin receptor-like kinase 2 (ALK2), Dorsomorphin, DMH-1, K02288, ML3467, LDN193189 or Noggin protein for a first time period, thereby inducing differentiation to neuroectoderm lineage cells, wherein the first time period is three to four days;
• c) culturing the neuroectoderm lineage cells from b) in dynamic suspension in the presence of retinoic acid and at least one agonist of Smoothened receptor for a second time period, wherein the second time period is three to four days;
• until the cells have matured into paired box 6 (PAX6) positive neuroectoderm cells.

A method for obtaining a population of cells comprising glial progenitor cells from undifferentiated human pluripotent stem cells, the method comprising:

• a) culturing undifferentiated human pluripotent stem cells that have been disaggregated and form a single-cell suspension in dynamic suspension to obtain non-embryoid body (non-EB) aggregates, wherein the human pluripotent stem cells in the non-EB aggregates remain in an undifferentiated state;
• b) culturing the non-EB aggregates from a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta (TGFf3)/Activin/Nodal signaling selected from an inhibitor of activin receptor-like kinase 5 (ALK5), SB431542, LY2157299, GW788388, A-77-01, A-83-01 or SB505124 and at least one inhibitor of bone morphogenetic protein (BMP) signaling selected from an activin receptor-like kinase 2 (ALK2), Dorsomorphin, DMH-1, K02288, ML3467, LDN193189 or Noggin protein for a first time period, thereby inducing differentiation to neuroectoderm lineage cells, wherein the first time time period is three to four days;
• c) culturing the neuroectoderm lineage cells from b) in dynamic suspension in the presence of retinoic acid and at least one agonist of Smoothened receptor for a second time period, wherein the second time period is three to four days;
• and
• d) culturing the neuroectoderm lineage cells from c) in dynamic suspension in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for a further time period, until the cells have matured into glial progenitor cells, wherein steps a) through d) are performed over a period of 21 days.