The Rho-associated kinase inhibitor fasudil can replace Y-27632 for use in human pluripotent stem cell research
Fig 4
Characterization of hPSCs following fasudil treatment for 2 to 3 months.
A: Quantitative RT-PCR analysis of OCT4, SOX2, and NANOG expression relative to GAPDH. ROCK inhibitors were present only on the first day of passage. The difference between the two treatments was not significant (3 biological replicates, 3 technical replicates). B: Quantitative RT-PCR analysis of expression of markers of the 3-germ layer, AFP, TBXT, and PAX6, relative to GAPDH (*: p < 0.05; versus EBs, 2 biological replicates, 2 technical replicates). EBs were derived from hiPS1 and cultured for 7 days. C: Immunofluorescence analysis examining OCT4, SSEA-4, and TRA-1-60 expression, following fasudil treatment for 3 months. All 5 lines of PSC expressed the pluripotency markers. Nuclei were stained with DAPI (blue). Scale bars = 100 μm. D: G-banding karyotypes of hPSCs cultured with fasudil for 3 months. None of the five lines of PSC had any abnormality of karyotype. E: Ectodermal, mesodermal, and endodermal tissue associated with teratoma formation by five lines of hPSCs after 3 months in fasudil-treated culture. All three germ layers were formed. Scale bars = 200 μm. hPSC: human pluripotent stem cell, EBs: embryoid bodies, hiPS: human induced pluripotent stem cell.