Keratinocyte Differentiation of iPS cells by Retinoic acid and BMP-4

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. The present study examined whether retionic acid and BMP-4 induced keratinocyte differentiation of iPS cells. Methods:Human iPS cells (Nakagawa et al. Nat Biotech 26:101-106, 2008; 253G1, RIKEN BioResource Center) were cultured on a feeder layer of mouse embryonic fibroblasts (MEF; ReproCell, Japan) in Primate ES cell culture medium (ReproCell). After the second passages, expression of Oct3/4 and Nanog mRNAs were observed by RT-PCR to confirm their pluripotency. A combination of DMSO, 1mM all-trans RA (Sigma) and/or BMP-4 (R&D System Inc.) was added to the culture systems for 0,3,7 and 14 days. Immunocytochemical staining was performed using anti-p63 and anti-K14 antibodies (Lab Vision, CA) as primary antibodies. Expression levels of p63, K14 and K18 mRNAs were evaluated by RT-PCR and quantitative RT-PCR using TaqMan probes. The significance of the differences was analyzed using by Student's t-test (n=5). Results:Expression level of p63, K14 and K18 in the cells incubated with RA for 7days was significantly higher than in those with DMSO alone (control)(p<0.05). Expression level of K14 and K18 in the cells incubated with both RA and BMP-4 was significantly higher than in those with RA (p<0.05). Positive staining for p63 and K14 was observed in the cells incubated with both RA alone and RA plus BMP-4 for 7days. Conclusion:The results indicate that retionic acid and BMP-4 may induce keratinocyte differentiation in iPS cells.