Laminin-511-derived recombinant fragment and Rho kinase inhibitor Y-27632 facilitate serial cultivation of keratinocytes differentiated from human embryonic stem cells

Abstract

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 logarithmic growth phase. Conclusions These results indicate the utility of Y-27632 and LN-511-E8 for serial cultivation of hESC-derived keratinocytes, which have a potential for fabricating allogeneic cellular products in clinical situations for regeneration of stratified epithelial tissues.

Human intestinal organoid-derived PDGFRα+ mesenchymal stroma empowers LGR4+ epithelial stem cells

“…We employed a medium containing TGF-β inhibitor, Cholera toxin, Nicotinamide, EGF, Noggin, Wnt3a and R-spondin1 that support the proliferative expansion of a diverse array of human epithelial cells. 19,[21][22][23][24] This study demonstrates that it is also efficacious for intestinal epithelial cells.…”

Laminin-221-derived recombinant fragment facilitates isolation of cultured skeletal myoblasts

“…Laminin-derived recombinant fragments have been utilized as culture substrates for various cell types including human induced pluripotent stem cells and embryonic stem cells [ 7 ]. Besides, they have been utilized for differentiation of various cell types such as keratinocytes [ 21 ], neuronal cells [ 22 ], cardiac [ 7 ] and skeletal myocytes [ 23 ], and ocular cells [ 24 ]. However, this is the first report to utilize these recombinant fragments for cell isolation.…”

Functional and long-lived melanocytes from human pluripotent stem cells with transient ectopic expression of JMJD3

“…We have prepared bio-artificial organs with functional cells in humans (10, 11). Pigmented cells appeared with a certain probability during hepatic differentiation of human pluripotent stem cells.…”