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.

How to Use:

• In vitro: Dilute received Ready-to-use kit pNSC-CSB at 1:1000 into your hESC/iPSC neural differentiation basal media to make the pNSC-CSB media. The reconstituted pNSC-CSB media can be used alone to initiate primitive neuroepithelium induction for 7-10 days, and the generated pNSCs can then be expanded and passaged in the pNSC-CSB media further supplemented with LIF.

Reference:

1. 1 Li W, et al. Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors. (2011) PNAS. 108(20):8299-304.

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