RAAS-deficient organoids indicate delayed angiogenesis as a possible cause for autosomal recessive renal tubular dysgenesis

DOI: 10.1038/s41467-023-43795-x

Authors: Naomi Pode-Shakked; Megan Slack; Nambirajan Sundaram; Ruth Schreiber; Kyle W. McCracken; Benjamin Dekel; Michael Helmrath; Raphael Kopan

Step 1: iPSC/ESC maintainance

day 0 ~ 1

culture “adherent” human PSC and human PSC in “mTeSR” for “1” days. “PSC” should be “downregulated” for “ACE”. “PSC” should be “downregulated” for “AGTR1”. For ECM, use “Cultrex” 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 “ACE” at the end of culture. Ideally, “PSC” should express “AGTR1” at the end of culture.

Step 2: Primitive streak 1

day 1 ~ 2

culture “adherent” human PSC and human PSC in “Advanced RPMI 1640” for “1” days. “PSC” should be “downregulated” for “ACE”. “PSC” should be “downregulated” for “AGTR1”. 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 3: Primitive streak 2

day 2 ~ 3

culture “adherent” human PSC and human PSC in “Advanced RPMI 1640” for “1” days. “PSC” should be “downregulated” for “ACE”. “PSC” should be “downregulated” for “AGTR1”. 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: mesoderm 1

day 3 ~ 4

culture “adherent” human PSC and human PSC in “Advanced RPMI 1640” for “1” days. “PSC” should be “downregulated” for “ACE”. “PSC” should be “downregulated” for “AGTR1”. 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 5: mesoderm 2

day 4 ~ 5

culture “device” human PSC and human PSC in “Advanced RPMI 1640” for “1” days. “PSC” should be “downregulated” for “ACE”. “PSC” should be “downregulated” for “AGTR1”. Medium should contain factors shown in the following table, and should be changed once in “1” days.