Analysis of endothelial-to-haematopoietic transition at the single cell level identifies cell cycle regulation as a driver of differentiation

DOI: 10.1186/s13059-020-02058-4

Authors: Giovanni Canu; Emmanouil Athanasiadis; Rodrigo A. Grandy; Jose Garcia-Bernardo; Paulina M. Strzelecka; Ludovic Vallier; Daniel Ortmann; Ana Cvejic

Step 1: iPSC/ESC maintainance

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

culture “adherent” human PSC in “E6 medium”. For ECM, use “Vitronectin” at “0.01” “g/l”. Medium should contain factors shown in the following table.

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

Step 2: Primitive streak 1

day 1 ~ 2

culture “spheroid” human PSC in “IMDM” for “1” days. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Step 3: Primitive streak 2

day 2 ~ 3

culture “spheroid” human PSC in “IMDM” for “1” days. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Step 4: mesoderm 1

day 3 ~ 4

culture “spheroid” human PSC in “IMDM” for “1” days. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Step 5: mesoderm 2

day 4 ~ 6

culture “spheroid” human PSC in “StemPro” for “2” days. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Step 6: endothelial 1

day 6 ~ 9

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

Ideally, “EC” should express “CDH5”, “PECAM1”, “KDR”, “THY1”, “CD34”, “CD44” at the end of culture.

Step 7: endothelial 2

day 9 ~ 11

culture “adherent” human EC in “StemPro” for “2” days. For ECM, use “Matrigel” at “0.35” “g/m2”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Ideally, “HPC” should express “RUNX1”, “CDH5”, “CD43”, “CD44” at the end of culture.