Efficient generation of human NOTCH ligand-expressing haemogenic endothelial cells as infrastructure for in vitro haematopoiesis and lymphopoiesis

DOI: 10.1038/s41467-024-51974-7

Authors: Shicheng Sun; Ali Motazedian; Jacky Y. Li; Kevin Wijanarko; Joe Jiang Zhu; Kothila Tharmarajah; Kathleen A. Strumila; Anton Shkaruta; L. Rayburn Nigos; Jacqueline V. Schiesser; Yi Yu; Paul J. Neeson; Elizabeth S. Ng; Andrew G. Elefanty; Edouard G. Stanley

Step 1: iPSC/ESC maintainance

day 0 ~ 1

culture “adherent” human PSC in “E8” for “1” days. For ECM, use “Geltrex” at “1” “%”. Medium should be changed once in “1” days.

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

Step 2: Primitive streak 1

day 1 ~ 2

culture “adherent” human PSC in “unspecified medium” for “1” days. For ECM, use “Geltrex” at “1” “%”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Ideally, “PS” should express “MIXL1” at the end of culture.

Step 3: Primitive streak 2

day 2 ~ 3

culture “adherent” human PSC in “unspecified medium” for “1” days. For ECM, use “Geltrex” at “1” “%”. 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 “adherent” human PSC in “unspecified medium” for “1” days. For ECM, use “Geltrex” at “1” “%”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Ideally, “mesoderm” should express “CD13”, “KDR” at the end of culture.

Step 5: endothelial 1

day 4 ~ 6

culture “adherent” human PSC in “unspecified medium” for “2” days. For ECM, use “Geltrex” at “1” “%”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Ideally, “EC” should express “DLL4”, “CXCR4”, “CDH5”, “CD34”, “PECAM1”, “SOX17”, “GJA4”, “GJA5” at the end of culture.

Step 6: endothelial 2

day 6 ~ 8

culture “adherent” human EC in “unspecified medium” for “2” days. For ECM, use “Geltrex” at “1” “%”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

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