The differentiation protocol was adapted from Takasato et al34 (link) and described earlier.35 (link) In brief, human iPSCs at a density of 20 000-25 000 per cm2 were cultured in a monolayer in STEMdiff APEL2 medium (APEL2) (STEMCELL Technologies, Vancouver, Canada) supplemented with 8 µM CHIR99021 (R&D Systems, Minneapolis, USA), 5% protein-free hybridoma medium II (PFHMII, Thermo Fisher Scientific) and 1% antibiotic-antimycotic (Thermo Fisher Scientific) for 3-4 days. Recombinant human FGF9 (200 ng/mL; R&D Systems) and heparin (1 μg/mL; Sigma-Aldrich) were added after 3-4 days. After 7 days of monolayer culture, the cells were pulsed for 1 h with 5 μM CHIR99021, pelleted, and transferred onto a Transwell membrane (0.4 μm pore polyester membrane, Corning, New York, USA). For 5 days, the organoids were stimulated with 200 ng/mL Recombinant human FGF9 and 1 μg/mL heparin after which growth factors were removed.
Stemdiff apel2 medium apel2
Manufactured by STEMCELL
Sourced in Canada
STEMdiff APEL2 medium (APEL2) is a serum-free, xeno-free culture medium optimized for the maintenance and differentiation of human pluripotent stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Heparin, by Merck Group (2 mentions)
Chir99021, by Corning (2 mentions)
Recombinant human fgf9, by R&D Systems (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Chir99021, by R&D Systems (2 mentions)
Transwell membrane, by Corning (1 mentions)
Pfhm 2, by Thermo Fisher Scientific (1 mentions)
Protein free hybridoma medium 2, by Thermo Fisher Scientific (1 mentions)
2 protocols using stemdiff apel2 medium apel2
1
Differentiation of Human iPSCs into Kidney Organoids
2
Kidney Organoid Generation from iPSCs
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As adapted from Takasato et al. [16 (link)] and our earlier publication [14 (link)], iPSC were plated at a density of 20,000–25,000 cells/cm2 and cultured in STEMdiff APEL2 medium (APEL2) (STEMCELL Technologies, Vancouver, Canada) supplemented with 8 µM CHIR99021 (R&D Systems, Minneapolis, United States), 5% Protein Free Hybridoma Medium II and 1% Antibiotic-Antimycotic (both Thermo Fisher Scientific) for 3-4 days. Hereafter, 200 ng/mL recombinant human FGF9 (R&D Systems) and 1 μg/mL heparin (Sigma Aldrich, Zwijndrecht, Netherlands) were added until day 7. 500,000 cells were transferred as a pellet onto a 0.4 μm pore polyester transwell membrane (Corning, New York, United States) at an air–liquid interphase and treated for 1 h with 5 μM CHIR99021. Organoids were stimulated with 200 ng/mL recombinant human FGF9 and 1 μg/mL heparin for 5 days, followed by 13 days culture without growth factors. Medium was refreshed every other day.
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