Role of YAP in hematopoietic differentiation and erythroid lineage specification of human-induced pluripotent stem cells
DOI: 10.1186/s13287-023-03508-z
Authors: Chuti Laowtammathron; Chanchao Lorthongpanich; Nittaya Jiamvoraphong; Pimonwan Srisook; Phatchanat Klaihmon; Pakpoom Kheolamai; Sudjit Luanpitpong; Surapol Issaragrisil
Step 1: iPSC/ESC maintainance
day 0 ~ 1 (duration missing in database; assumed 1 day)
culture “adherent” human PSC and human PSC and human PSC in “Nutristem medium”. “PSC” should be “downregulated” for “YAP1”. “PSC” should be “upregulated” for “YAP1”. For ECM, use “Matrigel” at “N/A” “N/A”.
Ideally, “PSC” should express “POU5F1”, “NANOG”, “SOX2” at the end of culture.
Step 2: Primitive streak 1
day 1 ~ 2
culture “spheroid” human PSC and human PSC and human PSC in “StemPro-34 medium” for “1” days. “PSC” should be “downregulated” for “YAP1”. “PSC” should be “upregulated” for “YAP1”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| Glutamine | 0.002 | mol/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| Ascorbic Acid | 0.05 | g/l |
| Y27632 | 1.0e-5 | mol/l |
| BMP4 | 1.0e-5 | g/l |
Step 3: Primitive streak 2
day 2 ~ 4
culture “spheroid” human PSC and human PSC and human PSC in “StemPro-34 medium” for “2” days. “PSC” should be “downregulated” for “YAP1”. “PSC” should be “upregulated” for “YAP1”. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| Glutamine | 0.002 | mol/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| Ascorbic Acid | 0.05 | g/l |
| BMP4 | 1.0e-5 | g/l |
| FGF | 5.0e-6 | g/l |
| CHIR90021 | 3.0e-6 | mol/l |
Step 4: endothelial 1
day 4 ~ 7
culture “adherent” human PSC and human PSC and human PSC in “StemPro-34 medium” for “3” days. “PSC” should be “downregulated” for “YAP1”. “PSC” should be “upregulated” for “YAP1”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “3” days.
| concentration | unit | |
|---|---|---|
| Glutamine | 0.002 | mol/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| Ascorbic Acid | 0.05 | g/l |
| FGF | 5.0e-6 | g/l |
| VEGF | 1.5e-5 | g/l |
| IL-3 | 3.0e-5 | g/l |
| IL-6 | 1.0e-5 | g/l |
| IL-11 | 5.0e-6 | g/l |
| IGF1 | 2.5e-5 | g/l |
| SCF | 5.0e-5 | g/l |
| EPO | 2000 | U/l |
| TPO | 3.0e-5 | g/l |
| FLT3L | 1.0e-5 | g/l |
Ideally, “HSPC” should express “CD34”, “CD43”, “PTPRC” at the end of culture.
Step 5: endothelial 2
day 7 ~ 14
culture “adherent” human PSC and human PSC and human PSC in “StemPro-34 medium” for “7” days. “PSC” should be “downregulated” for “YAP1”. “PSC” should be “upregulated” for “YAP1”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “7” days.
| concentration | unit | |
|---|---|---|
| Glutamine | 0.002 | mol/l |
| MTG | 4.0e-4 | mol/l |
| Transferrin | 0.15 | g/l |
| Ascorbic Acid | 0.05 | g/l |
| FGF | 5.0e-6 | g/l |
| VEGF | 1.5e-5 | g/l |
| IL-3 | 3.0e-5 | g/l |
| IL-6 | 1.0e-5 | g/l |
| IL-11 | 5.0e-6 | g/l |
| IGF1 | 2.5e-5 | g/l |
| SCF | 5.0e-5 | g/l |
| EPO | 2000 | U/l |
| TPO | 3.0e-5 | g/l |
| FLT3L | 1.0e-5 | g/l |
| BMP4 | 1.0e-5 | g/l |
Ideally, “HSPC” should express “CD34”, “CD43”, “PTPRC” at the end of culture.