Summary
A literature-derived dual-SMAD neural induction protocol for human pluripotent stem cells. Scientific review is still required: a concentration discrepancy and incomplete medium specifications remain unresolved.
Reagent checklist
| Reagent | Concentration | Supplier | Catalogue number |
|---|---|---|---|
| Matrigel | Coating dilution not reported | BD Biosciences | 354234 |
| Accutase | Ready-to-use; volume not reported | Not reported | Not reported |
| FGF2 | 10 ng/mL | Not reported | Not reported |
| Noggin | 500 ng/mL | Not reported | Not reported |
| SB431542 | 10 nM as archived; disputed | Tocris Bioscience* | 1614* |
| Y-27632 | 10 µM; later preparation method | Not reported | Not reported |
| KnockOut Serum Replacement | 15% in later SRM formulation | Invitrogen | 10828-028 |
| N2 medium | See historical formulation | Prepared in-house | No single catalogue number |
Preparation
1. Dissociate and deplete feeders Accutase: 20 minutes; gelatin preplate: one hour, 37°C, ROCK inhibition. Retain nonadherent cells.
2. Plate and expand Matrigel: 18,000 cells/cm². MEF-conditioned medium, FGF2, ROCK inhibition; withdraw ROCK inhibitor and expand three days.
HCMX review checklist Before approving this record, document cell-line provenance, passage, mycoplasma status, coating lot, seeding viability and the chosen medium formulation.
Protocol
Day 0 KSR medium plus both inhibitors; concentration unresolved.
Day 5 Withdraw SB431542; 25% N2, retain Noggin.
Days 7 & 9 50%, then 75% N2; retain Noggin.
Day 11 Assess neural identity.
Exact feed volumes and unreported intervals need author/laboratory confirmation; this draft does not invent them.
Validation
Check PAX6/OCT4; strong PAX6 is reported by day 7.
Suggested HCMX reporting fields Cell line · differentiation batch · analysis day · marker-positive fraction · assay method · images · deviations. Community ratings assess the record’s usefulness, not experimental success.
References
Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology 27, 275–280 (2009). https://doi.org/10.1038/nbt.1529
Tomishima M. Neural induction – Dual SMAD inhibition. StemBook (2012). https://www.ncbi.nlm.nih.gov/books/NBK133262/?report=reader
Corrigendum record: https://www.nature.com/articles/nbt0509-485a
Source note: the archived 2009 method lists SB431542 at 10 nM, while the later Tomishima protocol uses 10 µM. The discrepancy has not been silently corrected; verify the intended concentration and current product formulation before use.
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