Generation of an EFNB2-2A-mCherry reporter human embryonic stem cell line using CRISPR/Cas9-mediated site-specific homologous recombination

DOI: 10.1016/j.scr.2021.102241

Authors: Ying Huang; Hongchun Wu; Xinglong Han; Jie Wu; Miao Yu; Zhen-Ao Zhao; Zhenya Shen; Shijun Hu; Wei Lei

Step 1: iPSC/ESC maintainance

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

culture “adherent” human ESC in “E8”. “ESC” should be “unspecified regulation” for “EFNB2”. For ECM, use “Matrigel” at “N/A” “N/A”.

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

Step 2: Primitive streak 1

day 1 ~ 3

culture “adherent” human ESC in “CDM3” for “2” days. “ESC” should be “unspecified regulation” for “EFNB2”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Step 3: mesoderm 1

day 3 ~ 4

culture “adherent” human ESC in “CDM3” for “1” days. “ESC” should be “unspecified regulation” for “EFNB2”. 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 4: endothelial 1

day 4 ~ 7

culture “adherent” human ESC in “CDM3” for “3” days. “ESC” should be “unspecified regulation” for “EFNB2”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “3” days.

Ideally, “EC” should express “EFNB2” at the end of culture.