Obtain hPSC-Derived NPCs using an EB-Based Protocol with the STEMdiff™ Neural Induction System

STEMdiff™ Neural Induction Medium (Catalog #05835/05839) and the STEMdiff™ SMADi Neural Induction Kit (Catalog #08581/08582) are defined, serum-free media for the efficient and reproducible derivation of neural progenitor cells (NPCs) from human pluripotent stem cells (hPSCs). This media are compatible with both embryoid body (EB)-based and monolayer culture-based neural induction protocols.

EB-Based Protocol:

The STEMdiff™ Neural Induction Medium and the STEMdiff™ SMADi Neural Induction Kit allow for the highly efficient differentiation of hPSCs to neural rosettes, and STEMdiff™ Neural Rosette Selection Reagent (Catalog # 05832) selectively detaches neural rosettes for subsequent isolation of NPCs at high purity. Optimizing the differentiation procedure from beginning to end gives superior results. Here are some important tips for successful neural induction using our EB-based protocol:

  • If starting with hPSCs grown on feeders, it may be best to switch to a feeder-independent system using mTeSR™1 (Catalog #85850) plus Corning® Matrigel® hESC-Qualified Matrix (Corning Catalog # 354277) or Vitronectin™-XF (Catalog #07180) first, before attempting the neural induction protocol.
  • It is important to start with a high quality hPSC culture. This means that no more than 10% of the colonies in the starting population should display morphological differentiation, and cells should be in exponential growth phase.
  • Use a ‘control’ cell line the first time you use this protocol. The following cell lines have been tested and found to work using the protocol provided in our Technical Manual: H1, H7, H9, WLS-1C and WLS-4D1.
  • Use the AggreWell™800 (Catalog #34811) plate, as larger aggregates are more efficient in terms of NPC isolation later on.
  • To avoid co-selection of flat cells (neural crest cells), do not over-incubate with STEMdiff™ Neural Rosette Selection Reagent or excessively wash the plate with DMEM/F-12 when removing the neural rosette clusters.
  • Pre-warm the medium before use; do not add cold medium from the fridge directly to your cells.
    • PAX6 Antibody (early neural progenitor marker)
    • SOX-1 Antibody (neural progenitor marker)
    • Nestin Antibody (pan-neural marker)
    • OCT4/OCT3 Antibody (undifferentiated hPSC marker)
  • Other useful markers to confirm neural rosette formation include:
    • ZO-1 (marker for tight junctions, expressed in the lumen of neural rosettes)
    • SOX-10 (expressed by flat, neural crest cells, at the periphery of neural rosettes)

    For further information on how to characterize hPSC-derived NPCs, please refer to this Technical Tip.

NPCs derived using STEMdiff™ Neural Induction Medium are shown to be functional and capable of differentiation to mature neuronal and glial cells. For more information on the downstream applications for NPCs derived using this method, please refer to the Brochure.

Monolayer Culture-Based Protocol:

For tips on using STEMdiff™ Neural Induction Medium and the STEMdiff™ SMADi Neural Induction Kit in a monolayer-culture based neural induction protocol, please refer to this Technical Tip.

While the monolayer-based protocol is fast and simple (generating PAX6+ NPCs in 6-9 days), assessment of marker expression is necessary to confirm neural induction (cell morphology is not always a reliable indicator). The EB-based protocol is robust, with a strong publication record, and allows for visual confirmation of neural rosette formation. It also efficiently enriches for CNS-type NPCs by selecting for neural rosettes and removing ‘flat’ neural crest cells from mixed NPC cultures.

For further assistance and information, please contact techsupport@stemcell.com.

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