Mesodermal progenitors were induced from iPSCs as described [19 (link)] with certain optimizations. Briefly,
iPSCs were cultured on Vitronectin coated played in Essential 8 complete medium.
At the time of induction, cells are 60-70% confluent and are dissociated from
the well using Tryp1E at 37C for 10 minutes. Disassociated cells are then washed
and resuspended in X-Vivo 15 medium (Lonza) supplemented with rhActivin A
(R&D Systems), rhBMP4 (R&D Systems), rhVEGF (R&D Systems), rhFGF
(R&D Systems) at 10ng/mL and ROCK Inhibitor at 10μM (Tocris). After
counting, 2.5x10^6 cells are seeded into a single Vitronectin coated well
in a 6-well dish in 2mL X-Vivo 15 media supplemented with the same factors.
Medium was then changed each day with the same growth factors as above without
ROCK Inhibitor. On day 5, cells are incubated with 1X Accutase (Stem Cell,
Vancouver, Canada) for 10 minutes at 37C and washed twice with PBS before
staining with EPCAM and CD56 antibodies to prepare for fluorescence activated
cell sorting. Stained cells were sorted on a FACS ARIA instrument (BD
Biosciences, San Jose, CA) for CD56+ EPCAM-cells.
iPSCs were cultured on Vitronectin coated played in Essential 8 complete medium.
At the time of induction, cells are 60-70% confluent and are dissociated from
the well using Tryp1E at 37C for 10 minutes. Disassociated cells are then washed
and resuspended in X-Vivo 15 medium (Lonza) supplemented with rhActivin A
(R&D Systems), rhBMP4 (R&D Systems), rhVEGF (R&D Systems), rhFGF
(R&D Systems) at 10ng/mL and ROCK Inhibitor at 10μM (Tocris). After
counting, 2.5x10^6 cells are seeded into a single Vitronectin coated well
in a 6-well dish in 2mL X-Vivo 15 media supplemented with the same factors.
Medium was then changed each day with the same growth factors as above without
ROCK Inhibitor. On day 5, cells are incubated with 1X Accutase (Stem Cell,
Vancouver, Canada) for 10 minutes at 37C and washed twice with PBS before
staining with EPCAM and CD56 antibodies to prepare for fluorescence activated
cell sorting. Stained cells were sorted on a FACS ARIA instrument (BD
Biosciences, San Jose, CA) for CD56+ EPCAM-cells.
