Remyelination is the goal of potential cell transplantation therapies for demyelinating diseases and other central nervous system injuries. Transplantation of oligodendrocyte precursor cells (OPCs) can result in remyelination in the central nervous system, and induced pluripotent stem cells (iPSCs) are envisioned to be an autograft cell source of transplantation therapy for many cell types. However, it remains time-consuming and difficult to generate OPCs from iPSCs. Clonal sphere preparations are reliable cell culture methods for purifying select populations of proliferating cells. To make clonal neurospheres from human embryonic stem cell (ESC)/iPSC colonies, we have found that a monolayer differentiation phase helps to increase the numbers of neural precursor cells. Indeed, we have compared a direct isolation of neural stem cells from human ESC/iPSC colonies (protocol 1) with monolayer neural differentiation, followed by clonal neural stem cell sphere preparations (protocol 2). The two-step method combining monolayer neuralization, followed by clonal sphere preparations, is more useful than direct sphere preparations in generating mature human oligodendrocytes. The initial monolayer culture stage appears to bias cells toward the oligodendrocyte lineage. This method of deriving oligodendrocyte lineage spheres from iPSCs represents a novel strategy for generating OPCs.
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