Table 1.
List of hPSC lines used.
Fig 1.
Molecular characteristics of fasudil and Y-27632.
A: Characteristics of Y-27632 and fasudil. Y-27632 is more difficult to synthesize than fasudil. B: Price comparison of Y-27632 and fasudil. Y-27632 is 10 to 30 times more expensive than fasudil, depending on the providing company. C: Products ion mass spectrum (top) and stability comparisons (bottom) of fasudil and Y-27632 in media in the refrigerator (2–8°C) or in PSC culture condition (5% CO2, 37°C) using LC-ESI/MS/MS. The stability of fasudil was similar to that of Y-27632 (3 biological replicates). D: Typical colony morphologies of hPSCs (hES2 and hiPS1) cultured using Y-27632 or fasudil for 7 days after thawing. When treated with fasudil, colonies were larger than the control group. Scale bars = 200 μm. E: Fold increases in Y-27632- or fasudil-supplemented cultures for 7 days after thawing. Both fasudil- and Y-27632-treatment stimulated growth (*: p < 0.05, 3 biological replicates, 2 technical replicates). The fasudil-treated hES2 group had significantly higher growth than the Y-27632 treated group (#: p < 0.05, 3 biological replicates, 2 technical replicates). LC-ESI/MS/MS: liquid chromatography-electrospray ionization tandem mass spectrometry, hPSC: human pluripotent stem cell, hES: human embryonic stem cell, hiPS: human induced pluripotent stem cell.
Fig 2.
Fasudil enhances the cell growth of hPSCs.
A: Growth of hES3 cells for 7 days according to fasudil concentration. The growth of hES3 cells at fasudil concentrations of 1, 5, and 10 μM was concentration-independent, and significantly faster than the untreated control (*: p < 0.05, 3 biological replicates). B: ALP staining of hES3 and hiPS2 cells cultured with Y-27632 or fasudil. Treatment with fasudil or Y-27632 increased the size of PSC colonies. Scale bar = 10 mm. C, D: Fold increase of initially seeded hESCs (hES1, 2, and 3) and hiPSCs (hiPS1 and 2) using commercial reagent C or lab-made reagent D. Growth of all hPSC lines except hES1 increased in response to fasudil or Y-27632 (*: p < 0.05, 3 biological replicates). E: Increase of hES3 cell numbers with time. After three passages, treatment with fasudil and Y-27632 significantly increased cell numbers compared with the untreated group (*: p < 0.05, 3 technical replicates). hPSC: human pluripotent stem cell, hES: human embryonic stem cell, ALP: Alkaline Phosphatase, hiPS: human induced pluripotent stem cell.
Fig 3.
A: Analysis of live and apoptotic hES3 and hiPS2 cells (%) 24 hours after seeding at 1 × 104 cells/cm2. Compared with the control group, apoptotic cells decreased when treated with fasudil (*: p < 0.05; 3 biological replicates). B: Cell proliferation measured using immunofluorescence (left) and flow cytometry (right) to examine Ki-67 expression in hES3 and hiPS2 cells 2 days after seeding. There were no significant differences between the groups (3 biological replicates). Scale bar = 100 μm. hPSC: human pluripotent stem cell, hES: human embryonic stem cell, hiPS: human induced pluripotent stem cell.
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.
Fig 5.
Fasudil promotes differentiation of PSCs.
A: Expression of RPE-specific markers; PAX6 and MITF examined by immunocytochemistry (ICC) at 13 days. B: PAX6 and MITF positive cell populations analyzed by flow cytometry at 13 days. The percentage of PAX6 was similar in the groups, while MITF-positive cells were more frequent in the treated groups than in the untreated control (*: p < 0.05, 3 replicates). Fasudil did not affect cell number in RPE differentiation. C: The expression of NCC markers; PAX3 and SOX10 by ICC. D: PAX3 and SOX10 positive cell population analysis using flow cytometry at 6 days. Fasudil promoted cell survival during NCC differentiation of hiPSCs (*: p < 0.05, 3 replicates). RPE: retinal pigment epithelium, NCC: neural crest cell.
Fig 6.
Other applications of fasudil.
A: Supportive effect of fasudil on hES3 survival and growth in suspension culture. The fasudil-treated groups and Y-27632-treated groups were similar in the rate of formation of cell aggregates and their size; the control cells did not form aggregates. Scale bar = 200 μm (*: p < 0.05, 2 biological replicates, 7 technical replicates). B: Seven-day cultures of single hES3 cells on vitronectin-coated culture plates in the presence of 10 μM Y-27632 or fasudil. Scale Bar = 200 μm. The growth of hPSCs was faster with Y-27632 or fasudil than in the untreated control (*: p < 0.05, 3 technical replicates). hES: human embryonic stem cell, hPSC: human pluripotent stem cell.