|
Takagi Ryo
Department Research Institutes and Facilities, Research Institutes and Facilities Position Assistant Professor |
|
| Article types | Original article |
| Language | English |
| Peer review | Peer reviewed |
| Title | Laminin-511-derived recombinant fragment and Rho kinase inhibitor Y-27632 facilitate serial cultivation of keratinocytes differentiated from human embryonic stem cells. |
| Journal | Formal name：Regenerative Therapy ISSN code：2352-3204 |
| Domestic / Foregin | Foregin |
| Volume, Issue, Page | 18,pp.242-252 |
| Author and coauthor | TAKAGI Ryo†, TANUMA-TAKAHASHI Akiko, AKIYAMA Saeko, KANEKO Wakana, MIURA Chika, YAMATO Masayuki, SHIMIZU Tatsuya, UMEZAWA Akihiko* |
| Authorship | Lead author |
| Publication date | 2021/12 |
| Summary | Introduction:Keratinocytes derived from pluripotent stem cells have a short proliferative lifespan under conventional culture conditions that are optimized for keratinocytes. Recently, a Rho kinase inhibitor, Y-27632, had been used as a standard supplement for culture medium in which the proliferative lifespan of postnatal keratinocytes was markedly expanded. In addition, recombinant human laminin-511 was demonstrated to be an adhesive ligand for promoting proliferation of cultured epidermal keratinocytes. Based on this knowledge, efficacies of Y-27632 and a laminin511-derived recombinant fragment, known as laminin-511 E8 fragment (LN-511-E8), were evaluated for establishing cultivation methods of keratinocyte differentiated from human embryonic stem cells (hESC).Methods:Differentiated cells from hESCs, which were established with clinical grade in previous study, were seeded onto culture dishes coated with LN-511-E8 and co-cultured with a mouse feeder layer in serum-free medium supplemented with Y-27632. Before serial cultivation, hESC-derived keratinocytes were separated from other differentiated cells by trypsinization. The isolated hESC-derived keratinocytes were used for evaluating clonogenicity, gene expression analysis for keratinocyte markers, potency of terminal differentiation by air-lifting culture, and long-term proliferation activity by serial cultivation. Moreover, efficacies of Y-27632, LN-511-E8, and mouse feeder layer were evaluated on proliferation of hESC-derived keratinocytes.Results:hESC-derived keratinocytes with activity of clonal growth were successfully isolated by trypsinization and exhibited potency of differentiation to form stratified epidermal equivalents with expressions of progenitor and differentiation markers of epidermal keratinocyte. Y-27632 and LN-511-E8 were required for maintaining the proliferative activity of the hESC-derived keratinocytes in serially cultivation using mouse feeder layer with stable doubling time during logar |
| DOI | 10.1016/j.reth.2021.07.004 |
| PMID | 34409136 |