Robust differentiation of human pluripotent stem cells into mural progenitor cells via transient activation of NKX3.1

DOI: 10.1038/s41467-024-52678-8

Authors: Umji Lee; Yadong Zhang; Yonglin Zhu; Allen Chilun Luo; Liyan Gong; Daniel M. Tremmel; Yunhye Kim; Victoria Sofia Villarreal; Xi Wang; Ruei-Zeng Lin; Miao Cui; Minglin Ma; Ke Yuan; Kai Wang; Kaifu Chen; Juan M. Melero-Martin

Step 1: iPSC/ESC maintainance

day 0 ~ 1

culture “adherent” human PSC in “mTeSR Plus” for “1” days. “PSC” should be “upregulated” for “NKX3.1”. 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.

Ideally, “PSC” should express “POU5F1”, “SOX2”, “NANOG” at the end of culture.

Step 2: mesoderm 1

day 1 ~ 2

culture “adherent” human PSC in “Advanced DMEM/F-12” for “1” days. “PSC” should be “upregulated” for “NKX3.1”. 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.

Ideally, “mesoderm” should express “TBXT”, “TBX6”, “MSGN1”, “MIXL1” at the end of culture.

Step 3: mesoderm 2

day 2 ~ 3

culture “adherent” human mesoderm in “Advanced DMEM/F-12” for “1” days. “mesoderm” should be “upregulated” for “NKX3.1”. 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.

Ideally, “iMPC” should express “NKX3.1”, “CSPG4”, “PDGFRB”, “DES” at the end of culture.

Step 4: endothelial 1

day 3 ~ 10

culture “adherent” human iMPC and human EC and human MSC in “EGM2” for “7” days. For ECM, use “Gelatin” at “1” “%”. Medium should be changed once in “7” days.

Ideally, “Pericyte/SMC” should express “PDGFRB”, “NT5E”, “CSPG4” at the end of culture. Ideally, “Pericyte/SMC” should express “ACTA2”, “CNN1”, “TAGLN”, “MYOCD”, “TPM1”, “MYH11”, “FN1”, “COL1A1”, “COL1A2” at the end of culture.