Vascular progenitors generated from tankyrase inhibitor-regulated naïve diabetic human iPSC potentiate efficient revascularization of ischemic retina
DOI: 10.1038/s41467-020-14764-5
Authors: Tea Soon Park; Ludovic Zimmerlin; Rebecca Evans-Moses; Justin Thomas; Jeffrey S. Huo; Riya Kanherkar; Alice He; Nensi Ruzgar; Rhonda Grebe; Imran Bhutto; Michael Barbato; Michael A. Koldobskiy; Gerard Lutty; Elias T. Zambidis
Step 1: iPSC/ESC maintainance
day 0 ~ 1 (duration missing in database; assumed 1 day)
culture “adherent” unknown species cell and human PSC in “DMEM/F12”. For ECM, use “Fibroblast” at “N/A” “N/A”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| XAV939 | 4.0e-6 | mol/l |
| KSR | 20 | % |
| NEAA | 1.0e-4 | mol/l |
| GlutaMAX | 0.001 | mol/l |
| 2-mercaptoethanol | 1.0e-4 | mol/l |
| LIF | 2.0e-5 | g/l |
| CHIR90021 | 3.0e-6 | mol/l |
| PD0325901 | 1.0e-6 | mol/l |
| FGF | 5.0e-5 | g/l |
| TGF | 2.0e-6 | g/l |
| Ascorbic Acid | 0.064 | g/l |
| sodium selenite | 1.4e-5 | g/l |
| Transferrin | 0.0107 | g/l |
| insulin | 0.02 | g/l |
| HEPES | 0.015 | mol/l |
| sodium bicarbonate | 0.014 | mol/l |
Ideally, “PSC” should express “TRA-1-81”, “NANOG”, “NR5A2”, “DPPA3”, “E-CADHERIN”, “TFAP2C”, “SSEA4”, “P-STAT3”, “AXIN1” at the end of culture.
Step 2: Primitive streak 1
day 1 ~ 3
culture “adherent” unknown species cell in “APEL-2Li” for “2” days. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| Activin A | 2.5e-5 | g/l |
| VEGF | 5.0e-5 | g/l |
| BMP4 | 3.0e-5 | g/l |
| CHIR90021 | 1.5e-6 | mol/l |
Step 3: mesoderm 1
day 3 ~ 5
culture “adherent” unknown species cell in “APEL-Li” for “2” days. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| VEGF | 5.0e-5 | g/l |
| SB431542 | 1.0e-5 | mol/l |
Step 4: endothelial 1
day 5 ~ 7
culture “adherent” unknown species cell and human PSC in “APEL-Li” for “2” days. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| Activin A | 2.5e-5 | g/l |
| VEGF | 5.0e-5 | g/l |
| BMP4 | 3.0e-5 | g/l |
| CHIR90021 | 1.5e-6 | mol/l |
| SB431542 | 1.0e-5 | mol/l |
Ideally, “EC” should express “PECAM1”, “CD34”, “CDH5”, “KDR”, “MCAM”, “VWF”, “endothelin-1”, “ICAM2”, “CXCR4”, “CD105”, “CD140b”, “THY1”, “NG2”, “CD143”, “DLL1”, “FZD7” at the end of culture.
Step 5: endothelial 2
day 7 ~ 14
culture “adherent” unknown species cell and human EC in “EGM2” for “7” days. For ECM, use “FN1” at “0.01” “g/l”. Medium should be changed once in “7” days.