hESC cultures were disaggregated using accutase for 20 minutes, washed using hESC media and pre-plated on gelatin for 1 hour at 37°C in the presence of ROCK inhibitor to remove MEFs. The nonadherent hESC were washed and plated on matrigel at a density of 10,000–25,000 cells/cm2 on matrigel (BD) coated dishes in MEF conditioned hESC media (CM) spiked with 10 ng/mL of FGF-2 and ROCK-inhibitor. Ideal cell density was found to be 18,000 cells/cm2. The ROCK inhibitor was withdrawn, and hESC were allowed to expand in CM for 3 days or until they were nearly confluent. The initial differentiation media conditions included knock out serum replacement (KSR) media with 10 nM TGF-b inhibitor (SB431542, Tocris) and 500 ng/mL of Noggin (R&D). Upon day 5 of differentiation, the TGF-b inhibitor was withdrawn and increasing amounts of N2 media (25%, 50%, 75%) was added to the KSR media every two days while maintaining 500 ng/mL of Noggin. For MS5 induction, established methods previously reported were used22 (link).
Protocol detail
hESC Differentiation to MS5 Cells
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Corresponding Organization : Kettering University
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Variable analysis
independent variables
- Disaggregation of hESC cultures using accutase for 20 minutes
- Presence of ROCK inhibitor during pre-plating on gelatin
- Plating density of hESC on matrigel-coated dishes (10,000–25,000 cells/cm^2)
- Concentration of FGF-2 in MEF conditioned hESC media (10 ng/mL)
- Presence of ROCK inhibitor in MEF conditioned hESC media
- Withdrawal of ROCK inhibitor after 3 days of expansion
- Composition of initial differentiation media (KSR media with 10 nM TGF-b inhibitor and 500 ng/mL of Noggin)
- Gradual increase of N2 media (25%, 50%, 75%) in KSR media every two days
- Maintenance of 500 ng/mL of Noggin during differentiation
- Expansion and proliferation of hESC in culture
- Induction of MS5 cell lineage
- Washing of hESC using hESC media after disaggregation
- Pre-plating on gelatin for 1 hour at 37°C to remove MEFs
- Coating of dishes with matrigel
- Use of MEF conditioned hESC media
- Withdrawal of TGF-b inhibitor on day 5 of differentiation
- Maintenance of 500 ng/mL of Noggin during differentiation
- Positive control: Established methods for MS5 induction as previously reported
- Negative control: Not explicitly mentioned
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