Human pluripotent stem cell culture and neural differentiation

Human stm cell maintain and culture:

1. hPSCs  were maintained under feeder free conditions by coating with vitronectin (Life technology) or matrigel (BD Biosciences). 

2. Change the medium every day for 3-4 days of maintenance in E8 medium (Life technology).

3. hPSCs were dissociated by using EDTA (Lonza, 1 mL /well) for 1-2 minutes in 37 degrees, and wash with DMEMF12.

4. reseed the cells at the density of 1 × 10⁵ cells per well of a 6-well plate.

Neural differentiation:

1. 70% hPSCs were detached by dispase(Life technology) to form embryoid bodies (EBs),cultured in neural induction medium

2. cultured the EBs in neural induction medium(NIM) as previously described: 500 ml of NIM contains 5 ml of N2 supplement, 5 ml of NEAA, and 490 ml of DMEM/F12.

3. After floating for 7 days, EBs were attached on 6-well plate.

4. Rosette structures can be observed at d10–16. keep culturing in neural induction medium.For ventral differentiation, treat the cells with 500nM SAG from d10 to d25.

5. At d16, rosette colonies were detached by a 1-ml pipette manually. Non-neuroepithelialcolonies can be removed at this stage.

6. Neurospheres were continuous floated in NIM, and then dissociated byTrypLE (Life technology) and plated on vitronectin (Life technology) and poly-l-ornithine (Sigma) pre-coated coverslips for further neuronal differentiation.

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