Generation of Multipotential Mesendodermal Progenitors from Mouse Embryonic Stem Cells via Sustained Wnt Pathway Activation

DOI: 10.1074/jbc.M704287200

Authors: Manjiri Manohar Bakre; Aina Hoi; Jamie Chen Yee Mong; Yvonne Yiling Koh; Kee Yew Wong; Lawrence W. Stanton

Step 1: iPSC/ESC maintainance

day 0 ~ 2

culture “adherent” mouse ESC in “DMEM” for “2” days. For ECM, use “Gelatin” at “0.1” “%”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

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

Step 2: Primitive streak 1

day 2 ~ 4

culture “adherent” mouse ESC in “DMEM” for “2” days. For ECM, use “Gelatin” at “0.1” “%”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Ideally, “PS” should express “TBXT”, “KDR”, “Gsc”, “Foxa2”, “Lefty”, “Nodal”, “Sprouty2”, “FGF8”, “Lhx1”, “AFP” at the end of culture.

Step 3: endothelial 1

day 4 ~ 14

culture “spheroid” mouse ESC in “unspecified medium” for “10” days. Medium should be changed once in “10” days.

Ideally, “EC” should express “KDR”, “Flt-1”, “PECAM1”, “CD34”, “TEK” at the end of culture.

Step 4: endothelial 2

day 14 ~ 17

culture “adherent” mouse ESC in “unspecified medium” for “3” days. For ECM, use “Collagen” 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 “KDR”, “Flt-1”, “PECAM1”, “CD34”, “TEK” at the end of culture.