| 生长方案: |
For monolayer-based differentiation of cardiomyocytes (CMs), hPSCs were cultured between P20-P40 and seeded at a density of 3x105 cells/well of a 6-well plate and grown for 48 hr to 90% confluence in a humidified incubator with 5% CO2 at 37℃. On day 0, the medium was replaced with RPMI 1640 supplemented with B27 without insulin and 6 µM CHIR99021. On day 1, the medium was changed to RPMI 1640 supplemented with B27 without insulin for 48 hr. Day 3, medium was refreshed to RPMI 1640 supplemented with B27 without insulin and 2 µM C59 for another 48 hr. On day 5, the medium was changed back to RPMI-B27 without insulin for 48 hr, and then switched to RPMI 1640 plus normal B27 until day 12 with a medium change every 2 days. On day 12, the medium was transiently changed to RPMI 1640 without D-glucose containing ascorbic acid, human albumin and DL-Lactate for two days to allow metabolic purification of CMs. From that day on, fresh RPMI 1640-B27 was changed every two days; Macrophages were derived from hPSCs by adapting a previously reported protocol28. hPSCs were cultured between P20-P40. First, H9 or H1 cells were lifted with ReLeSR (STEMCELL Technologies) as small clusters onto Matrigel-coated 6-well plates at low density. On day 0, IF9S medium was supplemented with 50 ng/ml BMP-4, 15 ng/ml Activin A and 1.5 µm CHIR99021. On day 2, medium was refreshed with IF9S medium with 50 ng/ml VEGF, 50 ng/ml bFGF, 50 ng/ml SCF (R&D Systems) and 10 µm SB431542 (Cayman Chemical). On day 5 and da y7, medium was changed to IF9S with 50 ng/ml IL-6 (R&D Systems), 12 ng/ml IL-3 (R&D Systems), 50 ng/ml VEGF, 50 ng/ml bFGF, 50 ng/ml SCF and 50 ng/ml TPO (R&D Systems). On day 9, cells were dissociated with TrypLE (Life Technologies) and re-suspended in IF9S medium with 50 ng/ml IL-6, 12 ng/ml IL-3 and 80 ng/ml M-CSF (R&D Systems). On day 13 and day 15, medium was changed and supplemented with 50 ng/ml IL-6, 12 ng/ml IL-3 and 80 ng/ml M-CSF. All differentiation steps were cultured at 37°C, 5% CO2. |
| 处理方案: |
To analyze the gene expression of CMs co-cultured with H9-derived macrophages using RNA-seq, we used trans-well plates. CMs were seeded on the lower plate of 24 well plates and macrophages were seeded onto the upper chamber. After 24 hr, drug treatments were done to CMs and macrophages in the upper chamber were infected with SARS-CoV-2 (MOI=0.1). The trans-well chamber will allow cytokines to go through but not macrophages. 24 hpi, CMs were collected to extract total RNA. For analyzing macrophages infected with virus, macrophages were seeded onto 24 well plates and infected with SARS-CoV-2 (MOI=0.1). 24 hpi, macrophages were collected for total RNA. |
