STEMdiff™ APEL™2 Medium

Protocol tips

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	Differentiation is enhanced by co-culturing with endothelial cells

Protocol tips
Differentiation is enhanced by co-culturing with endothelial cells

Publication protocol

Kidney differentiation in microwell plates: hPSCs were dissociated with Accutase (Stem Cell Technologies) and plated onto microwell plates pre-coated with GelTrex in mTeSR1 supplemented with 10 µM Rho-kinase inhibitor Y27632 (StemGent). The media was replaced with mTeSR1 + 1.5 % GelTrex at 16 hours, 12 µM CHIR99021 in Advanced RPMI + Glutamax (Life Technologies) at 60 hours, and RB (Advanced RPMI + Glutamax + B27 Supplement, from Life Technologies) at 96 hours. Volumes used are as follows: 500 uL for 24-well plates, 100 uL for 96-well plates, and 50 uL for 384-well plates. RB was changed two days later and every three days thereafter. For experiments involving modulation of ECs media was supplemented with VEGF165 (Peprotech, 12.5 to 200 ng/ml). Alternatively (Protocol B, Figure S1A), the protocol described by Takasato et al. was adapted for adherent culture: undifferentiated hPSCs were plated overnight and treated the following morning with 8 µM CHIR99021 in APEL media (StemCell Technologies) for 48–72 hr, 30 ng/ml FGF9 (Peprotech) + 1µg/ml heparin (StemCellTechnologies) in APEL for 96 hr, and cultured thereafter in APEL, replaced every three days. Alternatively, to generate endothelial cells without kidney organoids, 100,000 hPSCs/cm2 were plated in mTeSR1 + 10 µM Y27632 + 1 µM CHIR99021, replaced with RPMI + B27 minus insulin + 1.5% Geltrex + 50 ng/mL Activin A (R&D) at 24h, RPMI + B27 minus insulin + 40 ng/mL BMP4 (Peprotech) + 1 µL CHIR99021 at 61 h, and StemPro 34 (Thermo Fisher Scientific) + 2 mM Glutamax + 50 µg/mL ascorbic acid (Sigma) + 10 ng/mL BMP4 + 5 ng/mL bFGF (Peprotech) + 300 ng/mL VEGF165 at 85 h for a 72-hour incubation. Robotic instrumentation consisted of a BioMek el406 plate washer with microplate stacker from Beckman-Coulter Matrix Technologies, WellMate Dispenser and Stacker and a CyBio CyBi-Well Vario Workstation which allows dispensing of small amounts of reagents, cells, and compounds. Manual instrumentation consisted of Integra Voyager and Viaflo II electronic multichannel pipets.

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Manufacturer protocol

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