Identification of Cell Fate Determining Transcription Factors for Generating Brain Endothelial Cells

DOI: 10.1007/s12015-025-10842-7

Authors: Roya Ramezankhani; Jonathan De Smedt; Burak Toprakhisar; Bernard K. van der Veer; Tine Tricot; Gert Vanmarcke; Bradley Balaton; Leo van Grunsven; Massoud Vosough; Yoke Chin Chai; Catherine Verfaillie

Step 1: iPSC/ESC maintainance

day 0 ~ 1

culture “adherent” human ESC in “mTeSR” for “1” days. “ESC” should be “upregulated” for “ETV2”. 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.

Step 2: endothelial 1

day 1 ~ 7

culture “adherent” human ESC in “NMM” for “6” days. “ESC” should be “upregulated” for “ETV2”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “6” days.

Ideally, “EC” should express “PECAM1”, “CDH5”, “ETS1”, “ETV6”, “FLI1”, “ERG”, “CLDN5” at the end of culture.

Step 3: endothelial 2

day 7 ~ 11

culture “adherent” human ESC in “NMM” for “4” days. “ESC” should be “downregulated” for “ETV2”, “upregulated” for “ZIC3”, “upregulated” for “FOXF2”, “upregulated” for “FOXQ1”, “upregulated” for “TCF7”, “upregulated” for “KLF4”, “upregulated” for “PRDM5”, “upregulated” for “TFAP2A”, “upregulated” for “TFAP2C”, “upregulated” for “SPIB”, “upregulated” for “DLX2”, “upregulated” for “PAX5”, “upregulated” for “HNF4A”. 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, “EC” should express “PECAM1”, “CDH5”, “CLDN5”, “ETS1”, “FLI1”, “ERG”, “CD34”, “OCLN”, “SLC2A1”, “SLC2A3”, “SLC3A2”, “ABCG1”, “SLC16A1”, “ZO1”, “SMAD1”, “TCF7L2”, “TCF7L1”, “CTBP2”, “CDH2”, “GNAQ”, “ITPR2”, “ARID1A” at the end of culture.