Hypoxia drives hematopoiesis with the enhancement of T lineage through eliciting arterial specification of hematopoietic endothelial progenitors from hESC

DOI: 10.1186/s13287-022-02967-0

Authors: Ning Wang; Chuxin Chen; Yang Cheng; Yingjie Fu; Zhiyong Zhong; Yu Yang; Ling Lv; Honglin Chen; Jian Huang; Yuyou Duan

Step 1: iPSC/ESC maintainance

day 0 ~ 1 (duration missing in database; assumed 1 day)

culture “adherent” human ESC in “DMEM/F12”. For ECM, use “Fibroblast” at “N/A” “N/A”. Medium should contain factors shown in the following table.

Ideally, “ESC” should be obtained at the end of culture.

Step 2: mesoderm 1

day 1 ~ 5

culture “spheroid” human ESC in “APEL medium” for “4” days. Medium should contain factors shown in the following table, and should be changed once in “4” days.

Ideally, “mesoderm” should express “CDX4”, “KDR”, “BMP4”, “TBXT”, “SOX17”, “CD56”, “HIF-1alpha” at the end of culture.

Step 3: mesoderm 2

day 5 ~ 8

culture “spheroid” human mesoderm in “APEL medium” for “3” days. Medium should contain factors shown in the following table, and should be changed once in “3” days.

Ideally, “HEP” should express “PECAM1”, “CD34”, “CDH5” at the end of culture.

Step 4: endothelial 1

day 8 ~ 12

culture “adherent” human mesoderm in “APEL medium” for “4” 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 “4” days.

Ideally, “HEP” should be obtained at the end of culture. Ideally, “HEP” should express “PECAM1”, “CD34”, “CDH5”, “CDH5”, “SOX17”, “DLL4”, “NOTCH1”, “EPHB4” at the end of culture.

Step 5: endothelial 2

day 12 ~ 16

culture “adherent” human HEP in “APEL medium” for “4” 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 “4” days.

Ideally, “HEP” should express “PECAM1”, “CD34”, “CDH5”, “RUNX1”, “DLL4”, “NOTCH1”, “VEGF”, “NR2F2”, “CDH5”, “PTPRC” at the end of culture. Ideally, “HPC” should express “CD34”, “PTPRC”, “HIF-1alpha”, “GATA1” at the end of culture.