?A major challenge of the current hESC/iPSC differentiation paradigm is the inability to effectively capture and long-term, stably expand lineage-specific stem cells that retain broad differentiation potential and, more importantly, developmental stage-specific differentiation propensity. To overcome this challenge, the combination of a GSK3 inhibitor (CHIR99021, 3 μM) and a TGFβ receptor inhibitor (SB431542, 2 μM), pNSC-CSB, was developed to be used alone or further combined with a Notch pathway inhibitor to rapidly and efficiently convert hESCs/iPSCs into a homogenous population of primitive neural stem cells? (pNSCs) within 7 days under chemically defined conditions. Most importantly, these pNSCs were shown to stably self-renew in the presence of leukemia inhibitory factor/LIF and the small molecule combination of CHIR99021 and SB431542 (pNSC-CSB). Under this condition, long-term expanded pNSCs retain high neurogenic potential and responsiveness to instructive patterning cues toward midbrain and hindbrain neuronal subtypes, and exhibit in vivo integration.
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