Inhibition of Rho‑associated protein kinase improves the survival of human induced pluripotent stem cell‑derived cardiomyocytes after dissociation

Abstract

Heart disease remains the leading cause of morbidity and mortality worldwide. Induced pluripotent stem cells (iPSCs) have the ability to differentiate into cardiomyocytes (CMs), rendering this cell type to be a promising precursor of cardiomyocytes for cell-based cardiac regeneration. Obtaining CMs with a high yield and purity coupled with improved subsequent survival could prove to be invaluable for the future cell replacement therapeutic strategies. Rho-associated protein kinase (ROCK) is involved in a wide range of fundamental cellular functions and serves significant roles in cardiac physiology. In the present study, human (h)iPSC-CMs were generated from iPSCs by including glycogen synthase kinase 3β and Wnt inhibitors in the basal culture media. The possible effect of Y27632, a ROCK inhibitor, on hiPSC-CMs was then investigated. hiPSC-CMs of high purity were harvested with >96% of cells expressing cardiac troponin T. Additionally, treatment with 10 µM Y27632 significantly improved the viability of dissociated hiPSC-CMs. The effects of ROCK inhibitors Y27632 and fasudil, on the proliferation and apoptosis of hiPSC-CMs were also examined. Treatment with ROCK inhibitors markedly enhanced hiPSC-CM proliferation, by up to 2.5-fold, whilst Y27632 treatment reduced apoptosis in hiPSC-derived CMs under serum starvation and suspension by suppressing the expression of caspase-3. Taken together, data from the present study indicated that ROCK kinase inhibitors effectively improved the cultural system of hiPSC-derived CMs.

ROCK ‘n TOR: An Outlook on Keratinocyte Stem Cell Expansion in Regenerative Medicine via Protein Kinase Inhibition

“…Thereafter, ROCK pharmacological inhibition with Y-27632 has been used extensively for enhancing the survival ex vivo of several stem cell types, including neuronal precursor [ 118 ], and for establishing salivary organoid cultures [ 119 ]. Additionally, ROCK inhibition improves the survival and cultivation of human iPS cell-derived cardiomyocytes after dissociation [ 120 ], suggesting that the protective effect of ROCK inhibitors is not restricted to stem/progenitor cells. From an applicative standpoint, ROCK inhibition is currently used for protecting different stem cell types from suspension-induced death during their manipulations.…”

Cardiac Cell Therapy with Pluripotent Stem Cell-Derived Cardiomyocytes: What Has Been Done and What Remains to Do?

“…Furthermore, the addition of pro-survival factors (anti-apoptotic Bcl-XL, mitochondrial membrane blocker cyclosporine A, IGF-1 and caspase inhibitor, ZVAD-fmk) as well as using insulin-free medium or ROCK inhibitor (Y27632) has been shown to generate a higher proportion and improve survival of cardiomyocytes [69,84]. Other studies have explored the effect of cellular media, cell-cell contact and physical separation to improve cardiomyocyte yield and purity [72,[89][90][91].…”