Directed Differentiation of Human ESCs and iPSCs Toward Retinal Lineage
Source Meyer et al. · <UNKNOWN> · 10.1073/pnas.0905245106
Abstract
This protocol describes the directed differentiation of human embryonic stem cells (H9) and induced pluripotent stem cells (IMR90 clone 4) toward retinal lineage fate, generating neuroepithelial rosettes that differentiate into either neural retinal progenitors or retinal pigment epithelium cells. The method employs feeder-free suspension culture with chemically defined media and specific growth factor supplementation to achieve eye field specification and subsequent retinal differentiation.
Protocol overview
14 steps across 4 phases
- 1 Expand hESCs and iPSCs on feeder layer
- 2 Prepare embryonic stem cell medium (ESCM)
- 3 Passage cells every 5-6 days
- 1 Enzymatically lift cells from MEF feeder layer
- 2 Culture cells as aggregates in suspension
- 1 Switch to chemically defined neural induction medium (NIM)
- 2 Allow aggregates to attach to culture dish with laminin
- 3 Allow formation of neuroepithelial rosettes
- 4 Optional: Add BMP4 and Wnt3A antagonists (days 0-10 variant)
- 5 Optional: Add FGF inhibitor SU5402 (variant experiment)
- 1 Switch to retinal differentiation medium (RDM)
- 2 Mechanically isolate rosettes for neural retinal progenitor pathway
- 3 Culture isolated rosettes in suspension for neurosphere formation
- 4 Maintain rosettes on substrate for RPE differentiation pathway
Full SOP
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Attribution
This SOP was authored by Organthis based on the published method in Meyer et al.. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.
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