Autologous endothelialized small-caliber vascular grafts engineered from blood-derived induced pluripotent stem cells
DOI: 10.1016/j.actbio.2019.07.032
Authors: Melanie Generali; Elisa A. Casanova; Debora Kehl; Debora Wanner; Simon P. Hoerstrup; Paolo Cinelli; Benedikt Weber
Step 1: iPSC/ESC maintainance
day 0 ~ 1 (duration missing in database; assumed 1 day)
culture “adherent” human PSC in “DMEM/F12”. Medium should contain factors shown in the following table.
| concentration | unit | |
|---|---|---|
| KSR | 20 | % |
| NEAA | 0.1 | mol/l |
| 2-mercaptoethanol | 0.05 | mol/l |
| Glutamine | 0.001 | mol/l |
| PSA | 50000 | U/l |
| FGF | 1.0e-5 | g/l |
Ideally, “PSC” should express “POU5F1”, “SOX2”, “NANOG”, “REX1”, “DPPA3”, “TRA-1-81”, “TRA-1-60” at the end of culture.
Step 2: Primitive streak 1
day 1 ~ 11
culture “spheroid” human PSC in “DMEM” for “10” days. Medium should contain factors shown in the following table, and should be changed once in “10” days.
| concentration | unit | |
|---|---|---|
| FBS | 20 | % |
| Glutamine | 0.002 | mol/l |
| 2-mercaptoethanol | 1.0e-4 | mol/l |
| NEAA | 1.0e-4 | mol/l |
| Ascorbic Acid | 5.0e-5 | g/l |
| PSA | 125000 | U/l |
| streptomycin | 125 | g/l |
Step 3: endothelial 1
day 11 ~ 36
culture “adherent” human PSC in “EGM2” for “25” days. For ECM, use “FN1” at “N/A” “N/A”. Medium should be changed once in “25” days.
Ideally, “EC” should express “PECAM1”, “VWF”, “endoglin”, “NOS3”, “POU5F1”, “NANOG”, “REX1” at the end of culture.