The novel combination of small-molecule inhibitors increases the survival and colony formation capacity of human induced pluripotent stem cells after single-cell dissociation
Objectives
Human induced pluripotent stem cells (hiPSCs) offer immense potential for regenerative medicine and drug discovery. However, procedures such as single-cell dissociation—critical for genetic modification and clonal selection—often compromise hiPSC viability and colony-forming efficiency. Although strategies involving small molecules and feeder cells have been explored, their effectiveness remains inconsistent. This study aims to enhance post-dissociation survival of hiPSCs using a novel combination of well-characterized small-molecule inhibitors.
Methods
hiPSCs were pretreated for 1 hour with either the ROCK inhibitor Y27632, the SMC4 cocktail (PD0325901, CHIR99021, thiazovivin, SB431542), or a newly formulated SiM5 cocktail (SMC4 + Pifithrin-α). Cells were then dissociated into single cells using accutase and cultured in NutriStem or StemFlex medium supplemented with the corresponding inhibitors. Cell viability, expression of pluripotency markers, colony-forming ability, and karyotypic stability were assessed. The efficacy of SiM5 was further evaluated under hypoxic conditions and following fluorescence-activated cell sorting (FACS).
Results
SiM5 treatment markedly enhanced hiPSC survival—by ~2.5-fold compared to SMC4 and ~25-fold compared to Y27632—across multiple cell lines and media. SiM5 also promoted survival and proliferation under hypoxia and post-FACS. Temporary cell cycle arrest following SiM5 withdrawal did not impede long-term expansion. Importantly, SiM5 preserved pluripotency, colony-forming capacity, and chromosomal stability.
Conclusion
SiM5 significantly improves hiPSC viability and colony formation following single-cell dissociation under various conditions. This method holds promise for optimizing genetic engineering and clonal expansion workflows, with broad applications in stem cell research and regenerative medicine.