Blood-generating heart-forming organoids recapitulate co-development of the human haematopoietic system and the embryonic heart
DOI: 10.1038/s41556-024-01526-4
Authors: Miriana Dardano; Felix Kleemiß; Maike Kosanke; Dorina Lang; Liam Wilson; Annika Franke; Jana Teske; Akshatha Shivaraj; Jeanne de la Roche; Martin Fischer; Lucas Lange; Axel Schambach; Lika Drakhlis; Robert Zweigerdt
Step 1: iPSC/ESC maintainance
day 0 ~ 1
culture “adherent” human PSC in “E8” for “1” days. For ECM, use “Geltrex” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| Y27632 | 1.0e-5 | mol/l |
| FGF | 1.0e-4 | g/l |
| insulin | 0.02 | g/l |
| Ascorbic Acid | 0.064 | g/l |
| NaHCO3 | 0.543 | g/l |
| Na2SeO3 | 1.4e-5 | g/l |
| Transferrin | 0.0107 | g/l |
| TGFB | 2.0e-6 | g/l |
Ideally, “PSC” should be obtained at the end of culture.
Step 2: Primitive streak 1
day 1 ~ 2
culture “spheroid” human PSC in “RPMI 1640” for “1” days. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| B27 | 2 | % |
| CHIR90021 | 7.5e-6 | mol/l |
| BMP4 | 1.0e-5 | g/l |
| FGF | 5.0e-6 | g/l |
Step 3: Primitive streak 2
day 2 ~ 4
culture “spheroid” human PSC in “RPMI 1640” for “2” days. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| B27 | 2 | % |
| IWP2 | 5.0e-6 | mol/l |
| VEGF | 5.0e-5 | g/l |
| FGF | 1.0e-5 | g/l |
| SCF | 1.0e-4 | g/l |
| EPO | 1.7e-5 | g/l |
| IL-6 | 1.0e-5 | g/l |
| IL-11 | 1.0e-5 | g/l |
| IGF1 | 2.5e-5 | g/l |
Step 4: mesoderm 1
day 4 ~ 6
culture “spheroid” human PSC in “E8” for “2” days. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| BMP4 | 1.0e-5 | g/l |
Step 5: mesoderm 2
day 6 ~ 8
culture “spheroid” human PSC in “RPMI 1640” for “2” days. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| B27 | 2 | % |
| VEGF | 5.0e-5 | g/l |
| FGF | 1.0e-5 | g/l |
Step 6: endothelial 1
day 8 ~ 13
culture “spheroid” human PSC in “RPMI 1640” for “5” days. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “5” days.
| concentration | unit | |
|---|---|---|
| B27 | 2 | % |
| IWP2 | 5.0e-6 | mol/l |
| VEGF | 5.0e-5 | g/l |
| FGF | 1.0e-5 | g/l |
| SCF | 1.0e-4 | g/l |
| EPO | 1.7e-5 | g/l |
| IL-6 | 1.0e-5 | g/l |
| IL-11 | 1.0e-5 | g/l |
| IGF1 | 2.5e-5 | g/l |
| TPO | 3.0e-5 | g/l |
| FLT3 | 1.0e-5 | g/l |
| IL-3 | 3.0e-5 | g/l |
| BMP4 | 1.0e-5 | g/l |
| SHH | 2.0e-5 | g/l |
| PSA | 1 | % |
Ideally, “EC” should express “PECAM1”, “CDH5”, “CD34” at the end of culture. Ideally, “VE” should express “NR2F2”, “NT5E” at the end of culture. Ideally, “AE” should express “DLL4”, “CXCR4”, “GJA4”, “SOX17”, “MECOM” at the end of culture. Ideally, “HE” should express “ALDH1A1”, “RUNX1” at the end of culture. Ideally, “ELC” should express “NKX2.5”, “MYH6”, “MYL3”, “NPPA”, “TNNC1”, “ACTC1”, “CDH5”, “PECAM1” at the end of culture.