Objective: Our objective was to establish and evaluate a protocol for deriving proliferative neural cultures from induced pluripotent stem cells (iPSC).

Background: For generating patient specific neurons, iPSC can be neurally induced and differentiated. Keeping neural stem cells (NSC) under proliferative conditions, followed by terminal differentiation when needed can greatly enhance the numbers of neurons generated. To our knowledge, the protocol utilizing dual SMAD inhibition (Compton et al. (2009)) has not yet been studied regarding generating cultures of proliferating NSC, especially in comparison to an embryoid body (EB) based protocol.

Methods: Neural induction of human iPSC started by deprivation of fibroblast growth factor (FGF) and dual SMAD inhibition by the small molecules LDN-193189 and SB-431542 under adherent culture conditions as well as derived from EBs. After 10 days, we transferred cells to low-attachment culture plates, where they formed neurospheres (NSPH). The proliferating NSPH were passaged, RNA collected, transcribed to cDNA and analysed by qPCR. Furthermore, NSPH were plated down at regular intervals for immunofluorescent stainings and either analysed directly or differentiated first by addition of growth factors, dibutyl cAMP and the notch inhibitor DAPT.

Results: Both protocols showed culture failure by arrested proliferation after a certain number of passages. The EB based protocol yielded higher amounts of cells expressing the neural precursor marker nestin than the adherent protocol. In contrast, fewer cells expressed the neuronal marker ß-III tubulin, with no difference between the protocols. QPCR showed increasing nestin expression and a loss of regional identity during proliferation. Likewise, the differentiation with the EB based protocol resulted in a higher yield of MAP2+ neurons. Cortical neurons (BRN2+), GABAergic and dopaminergic (TH+) neurons were found. In contrast to the adherent protocol, cells from the EB protocol showed a higher expression of the transcription factor sonic hedgehog (SHH), known to promote proliferation of NSC.

Conclusions: Although improvements in the area of culture consistency and reliability are desirable, the EB based protocol seems to be preferable to the adherent protocol for neural induction and subsequent culturing of neural precursors.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/comparison-of-two-protocols-for-neural-induction-and-cultivation-of-ipscs-derived-neural-stem-cells/