Mouse embryonic stem cell-derived blood–brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis

DOI: 10.1186/s12987-023-00437-0

Authors: Anna Jezierski; Jez Huang; Arsalan S. Haqqani; Julie Haukenfrers; Ziying Liu; Ewa Baumann; Caroline Sodja; Claudie Charlebois; Christie E. Delaney; Alexandra T. Star; Qing Liu; Danica B. Stanimirovic

Step 1: iPSC/ESC maintainance

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

culture “adherent” human PSC in “mTeSR”.

Step 2: Primitive streak 1

day 1 ~ 6

culture “adherent” human PSC in “KnockOut DMEM/F12” for “5” days. Medium should contain factors shown in the following table, and should be changed once in “5” days.

Step 3: mesoderm 1

day 6 ~ 7

culture “adherent” unknown species cell and mouse ESC in “DMEM” for “1” days. For ECM, use “Collagen IV” 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” at the end of culture.

Step 4: mesoderm 2

day 7 ~ 10

culture “adherent” mouse ESC in “DMEM” for “3” days. For ECM, use “Collagen IV” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “3” days.

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

Step 5: endothelial 1

day 10 ~ 19

culture “adherent” human PSC in “human serum free endothelial medium” for “9” days. Medium should contain factors shown in the following table, and should be changed once in “9” days.

Ideally, “EC” should be obtained at the end of culture. Ideally, “EC” should express “GLUT1”, “PECAM1”, “CDH5”, “Claudin 5”, “ZO1”, “Occludin”, “KDR” at the end of culture.

Step 6: endothelial 2

day 19 ~ 30

culture “device” human EC and mouse EC in “human serum free endothelial medium” for “11” days. For ECM, use “Fibronectin/Collagen IV” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “11” days.

Ideally, “EC” should be obtained at the end of culture. Ideally, “EC” should express “GLUT1”, “PECAM1”, “ZO1”, “Occludin”, “Claudin 5”, “CDH5”, “Fzd3”, “Fzd7”, “Fzd2”, “Lrp5”, “Lrp6”, “Wnt2”, “Wnt4”, “Wnt5a”, “Wnt5b”, “Wnt6”, “Wnt7a”, “Wnt7b”, “Wnt9a”, “Wnt10a”, “Wnt10b”, “Wnt11”, “Sfrp1”, “Sfrp2”, “Ddk2”, “SOX17”, “ZO3”, “Claudin 9”, “Claudin 7”, “Claudin 4”, “Claudin 6”, “Claudin 3”, “Claudin 12”, “Claudin 23”, “SLC7A5”, “ABCB1”, “ABCG2”, “ABCC1”, “ABCC4”, “ABCC5”, “TfR”, “INSR”, “IGF1R”, “LRP1”, “LRP8”, “TMEM30A” at the end of culture.