Keratinocyte Differentiation in iPS cells cultured without feeder layer

Objective: Induced pluripotent stem (iPS) cells behaved like pluripotent embryonic stem (ES) cells, which can be derived from a wide range of somatic cells via overexpression of a set of specific genes. Comparison of all expression patterns identified a subtle difference between iPS and ES cells. The iPS cells have excellent prospects for use in substitutive cell therapy; however, the methods of iPS cell directed differentiation have not yet been sufficiently elaborated. Retionic acid (RA) and BMP-4 induces keratinocyte differentiation of ES cells with a feeder layer. We examined whether RA and BMP-4 induced keratinocyte differentiation of iPS cells without a feeder layer. Methods: Mouse iPS cells (Nakagawa et al.; Nat Biotech, 2008.), iPS-MEF-Ng-178B-5 (RIKEN BioResource Center) were feeder-free-cultured in ESF-C MEDIUM (NIPRO), which contained 1000 Units/ml LIF (Wako) on gelatin-coated dish. After the second passages, Embryoid body (EB) at the cell concentration of 4000 cells/well was formed in 96 well plate U-bottom (Lipidure®-Coat Plate NOF) in ESF-C MEDIUM without LIF for three days.In order to induce the keratinocyte differentiation in EBs, the cells were transferred to a new dish and treated with differentiation medium, KBM-Gold [Keratinocyte Cell Basal Medium (LONZA) contained a combination of DMSO, 1 μM all-trans RA (Sigma) and 25ng/ml BMP-4 (R&D System Inc.)] on type Ⅳ collagen-coated dish, and were cultured for 5 days. The expression levels of Nanog, Oct3/4, klf-4, Sox2, β-TubllinⅢ, ΔNp63 and K14 mRNAs in the cells were observed by RT-PCR and quantitative RT-PCR using TaqMan probes. Results: Expression level of ΔNp63 and K14 in the cells incubated with both RA and BMP-4 was significantly higher than iPS cells . Conclusion: The results indicate that RA and BMP-4 may induce keratinocyte differentiation in iPS cells.