Targeted CRISPR activation is functional in engineered human pluripotent stem cells but undergoes silencing after differentiation into cardiomyocytes and endothelium

DOI: 10.1007/s00018-023-05101-2

Authors: Elaheh Karbassi; Ruby Padgett; Alessandro Bertero; Hans Reinecke; Jordan M. Klaiman; Xiulan Yang; Stephen D. Hauschka; Charles E. Murry

Step 1: iPSC/ESC maintainance

day 0 ~ 1

culture “adherent” human PSC in “mTeSR” for “1” 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 “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 “RPMI-1640” for “1” days. For ECM, use “Matrigel” at “1” “x”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

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

Step 3: mesoderm 1

day 2 ~ 3

culture “adherent” human PS in “RPMI-1640” for “1” days. For ECM, use “Matrigel” at “1” “x”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

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

Step 4: endothelial 1

day 3 ~ 6

culture “adherent” human mesoderm 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 be obtained at the end of culture.

Step 5: endothelial 2

day 6 ~ 7 (duration missing in database; assumed 1 day)

culture “adherent” human EC in “EGM2”. For ECM, use “Gelatin” at “N/A” “N/A”. Medium should contain factors shown in the following table.

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