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Comparison of two different culture conditions for derivation of early hiPSC
Author(s) -
Hey Caroline A. B.,
Saltõkova Katarina B.,
Bisgaard Hanne C.,
Møller Lisbeth B.
Publication year - 2018
Publication title -
cell biology international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.932
H-Index - 77
eISSN - 1095-8355
pISSN - 1065-6995
DOI - 10.1002/cbin.10966
Subject(s) - matrigel , lin28 , sox2 , homeobox protein nanog , induced pluripotent stem cell , vitronectin , alkaline phosphatase , chemistry , cell culture , microbiology and biotechnology , biology , biochemistry , embryonic stem cell , in vitro , extracellular matrix , genetics , fibronectin , enzyme , gene
Abstract 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.