Neural Induction for Human Pluripotent Stem Cells (hPSCs) using Monolayer Culture

Although neural induction of hPSCs using embryoid body (EB) formation allows for visual assessment of induction success via the formation of neural rosettes, the whole process can take anywhere between 16 and 19 days to get single-cell neural progenitor cells (NPCs). As a result, monolayer culture-based neural induction methods have recently gained popularity, since they enable single-cell NPCs to be obtained in as few as six days. STEMdiff™ Neural Induction Media (NIM) and the STEMdiff™ SMADi Neural Induction Kit are versatile serum-free, defined media that can be used for both methods. For the EB-based protocol, please refer to the STEMdiff™ Neural Induction Technical Manual. For the complete monolayer procedure, please refer to this Technical Bulletin.

Below is a list of tips for using STEMdiff™ NIM for monolayer culture–based neural induction:

  1. Cell density is critical for achieving success. For initial plating, seed hPSCs between 200,000 and 250,000 cells/cm2. If seeding at too low a density, the efficiency of neural induction will be reduced.
  2. Ensure that 10 μM Y-27632 ROCK inhibitor is added to STEMdiff™ NIM for the first day after passage. This will ensure better survival of single cells.
  3. Use cultureware coated with poly-L-ornithine/laminin (PLO/L) or Matrigel™ (Corning Matrigel® hESC-Qualified Matrix).
  4. Cells are seeded at high density in the monolayer culture protocol and generally form a nearly confluent monolayer 3 days after the initial plating, however additional days in culture are required to promote neural induction.
  5. Visual inspection of cultures is not a reliable method of confirming neural induction, because morphology is highly variable in this monolayer culture system. For initial experiments, prepare extra wells to perform immunocytochemistry, or other phenotypic characterization, at various time points. We recommend using PAX6 (a marker for neural induction) and OCT4 (a marker of pluripotency) to verify that neural induction is successful, before passaging the cells.
  6. The first passage is typically performed 6-9 days after plating, or when all cells are PAX6+ and OCT4-, using ACCUTASE™ (5-10 min at 37°C).
  7. After this first passage, NPCs should be passaged once they reach 70-80% confluency, and seeded in the next passage at a density between 125,000 and 200,000 cells/ cm2.
  8. Maintain NPCs in STEMdiff™ Neural Induction Medium until passage 3, and in STEMdiff™ Neural Progenitor Medium (NPM) thereafter. Switching to STEMdiff™ NPM at an earlier stage may enhance the proliferation of unwanted cells, such as OCT4 positive cells.
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