Generating Vascular Organoids
In Brief
Wimmer et al. describe a protocol to generate human blood vessel organoids from H9 ES cells or iPS cells.
Human H9 ES cells or iPS cells were disaggregated then resuspended in differentiation medium 1 containing Y-27632 (50 μM) and plated into one well of an ultra-low attachment six-well plate for cell aggregation. On day 3 cell aggregates were treated with 12 μM CHIR 99021, then on days 5, 7 and 9 BMP4, VEGF-A and FGF-2 were added.
On day 11, differentiation media 2 containing VEGF-A, FGF-2 and SB 431542 (10 μM) was added to increase the yield of endothelial cells and suppress pericyte formation.
On day 13 cell aggregates were embedded in Matrigel:collagen I (1:1) gels and differentiation media 3 was added. Media was changed every second to third day. Approximately day 18, vascular networks were established, extracted from the gels and further cultured in 96-well low-attachment plates. These vascular networks self-assembled into vascular organoids and could be cultured for up to 3 months.
Cocktails
| Differentiation Media 1 | Differentiation Media 2 | Differentiation Media 3 | |||
|---|---|---|---|---|---|
| DMEM:F12 medium | DMEM:F12 medium | DMEM:F12 medium | |||
| 20% KOSR | 20% KOSR | 20% KOSR | |||
| Glutamax (equivalent to GlutaminePlus (B90210)) | Glutamax | Glutamax | |||
| NEAA | NEAA | NEAA | |||
| Y-27632 (Cat.No. 1254) | 50 μM | VEGF-A (293-VE) | 30 ng ml-1 | 15% FBS | |
| CHIR 99021 (added day 3) (Cat.No. 4423) | 20 μM | FGF (233-FB) | 30 ng ml-1 | VEGF-A (293-VE) | 100 ng ml-1 |
| BMP-4 (314-BP) (added days 5, 7 and 9) | 30 ng ml-1 | SB 431542 (Cat.No. 1614) | 10 μM | FGF (233-FB) | 100 ng ml-1 |
| VEGF-A (293-VE) (added days 5, 7 and 9) | 30 ng ml-1 | ||||
| FGF (233-FB) (added days 5, 7 and 9) | 30 ng ml-1 | ||||
Reference
Wimmer et al. (2019) Human blood vessel organoids as a model of diabetic vasculopathy. Nature 565 505 PMID: 30651639