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Comparison of two different culture conditions for derivation of early hiPSC

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Different culture-systems for derivation of induced pluripotent stem cells (iPSC) in vitro from human fibroblasts have been established. Here we compared the efficacy of two different feeder-free culture-systems; Matrigel-coated surfaces in combination with mTeSR1 medium versus Vitronectin-coated surfaces in combination with Essential 8 (E8) medium. The comparison was performed by counting the number of emerging iPSC-looking colonies of re-programmed fibroblasts. The fibroblasts were re-programmed using episomal plasmids expressing OCT3/4, SOX2, KLF4, L-MYC, LIN28 and a p53 knock down shP53. Three different fibroblast lines, K40 and K48 from healthy controls and BBS1 from a patient with Bardet-Biedl syndrome, were used in two independent setups. The BBS1 line was used in both setups in combination with K40 and K48 respectively. In all four re-programming experiments, we observed a significantly higher number of emerging colonies with the combination Matrigel/mTeSR1 as compared to the combination Vitronectin/E8. The presence of iPSC was verified by alkaline phosphatase and Tra-1-60 staining. Furthermore, a higher expression of the pluripotency-associated markers NANOG and SOX2 in cells under Matrigel/mTeSR1 conditions compared with Vitronectin/E8 supported the higher proportion of iPSC on Matrigel/mTeSR1 plates. In conclusion the combination Matrigel/mTeSR1 is more efficient for derivation of iPSC compared to the Vitronectin/E8 combination.

OriginalsprogEngelsk
TidsskriftJournal of Cell Biology
Vol/bind42
Udgave nummer11
Sider (fra-til)1467-1473
ISSN0021-9525
DOI
StatusUdgivet - 30 mar. 2018

ID: 53543977