If you're looking to speed up the process of generating early-born cortical neurons from human induced pluripotent stem cells (hiPSCs), you've come to the right place. This protocol, adapted from the fantastic work by Qi and colleagues, outlines a highly efficient method to get your cells ready for transplantation in just 8 days. By day 16, any cells left in your culture dish will even start showing functional electrophysiological properties. Let's dive in and get those neurons growing!
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Plate the hiPSCs in conditioned hESC medium exactly 24 hours prior to initiating the differentiation timeline. Note: FGF2 can be replaced with stable, cost-effective small molecule FGFR1 agonists TCB-32, TCB-541, or TCB-621 to enable weekend-free feeding and significantly reduce media costs.
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On Day 0, commence the differentiation phase by exchanging the culture medium with KSR medium supplemented with the Day 0-6 cocktail.
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Beginning on Day 4, progressively transition the culture from KSR medium to N2B7 medium. Adjust the ratio steadily so that the cells are maintained in 100% N2B7 medium by Day 8.
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By Day 8, the derived early-born cortical neurons are mature enough to be harvested and transplanted into postnatal cortex models. For in vitro applications, maintain the cultures until Day 16 to achieve functional electrophysiological properties.
References
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Qi et al. 2017.Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells Nat. Biotechnol.PMID: 28112759
