Rho kinase inhibitor Y27632 promotes survival of human induced pluripotent stem cells during differentiation into functional midbrain dopaminergic progenitor cells in vitro

Objective

To enhance the efficiency of differentiating primitive neural epithelial cells (NECs), derived from human induced pluripotent stem cells (hiPSCs), into functional midbrain dopaminergic progenitor cells (DAPs).

Methods

HiPSCs were cultured in mTeSR™ medium containing DMH1 (10 μmol/L), SB431542 (10 μmol/L), SHH (200 ng/mL), FGF8 (100 ng/mL), purmorphamine (2 μmol/L), CHIR99021 (3 μmol/L), and 1% N2 supplement for 12 days to induce differentiation into primitive neuroepithelial cells (NECs). The resulting hiPSCs-NECs were digested with collagenase IV and then transferred to neurobasal medium containing 1% N2, 2% B27-A, BDNF (10 ng/mL), GDNF (10 ng/mL), ascorbic acid (AA), TGF-β, cAMP, 1% GlutaMax, and varying concentrations of the Rho kinase inhibitor Y27632. After 24 hours, Y27632 was removed by changing the medium. Continuous induction was maintained until day 28 to generate DAPs.

Results

HiPSCs expressed key pluripotency markers (OCT4, SOX2, Nanog, and SSEA1) and showed positive alkaline phosphatase staining. By day 13, NECs formed characteristic neural rosettes expressing neuroepithelial markers (SOX2, nestin, and PAX6). The addition of 5 μmol/L Y27632 significantly improved the survival of digested NECs, enhanced cell viability, increased the proportion of S-phase cells (P < 0.01), and reduced apoptotic cell rates (P < 0.05). By day 28, the differentiated cells expressed key DAP markers, including TH, FOXA2, NURR1, and Tuj1.

Conclusion

A 24-hour treatment with 5 μmol/L Y27632 significantly enhances the survival of hiPSCs-NECs during their differentiation into DAPs without impairing their developmental potential. This improved survival indirectly boosts the overall efficiency of the differentiation process.