Methods for differentiating pluripotent stem cells in dynamic suspension culture

A method of obtaining a differentiated cell population comprising oligodendrocyte progenitor cells (OPCs) comprising:

• a) obtaining a suspension culture of non-embryoid body aggregates of undifferentiated human pluripotent stem cells, wherein the human pluripotent stem cells remain in an undifferentiated state, and wherein obtaining the suspension culture of non-embryoid body comprises culturing a single-cell suspension of human pluripotent stem cells in a dynamic suspension culture;
• b) culturing the non-embryoid body aggregates from a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta/Activin/Nodal signaling and at least one inhibitor of bone morphogenetic protein signaling for a first time period, thereby inducing differentiation to neuroectoderm, wherein the first period is one to four days;
• c) culturing the non-embryoid body aggregates 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 period is one to four days;
• d) culturing the aggregates from c) in dynamic suspension in the presence of basic fibroblast growth factor, epidermal growth factor for a further time period, until the cells have matured into glial progenitor cells;
• and
• e) harvesting the cells from d) and plating them onto a substrate, and culturing the cells adherently in the presence of epidermal growth factor and platelet-derived growth factor AA for a further time period, until the cells have matured into oligodendrocyte progenitor cells, wherein the adherent culturing is performed for a period of 21 days.