The novel combination of small-molecule inhibitors increases the survival and colony formation capacity of human induced pluripotent stem cells after single-cell dissociation.

ObjectivesHuman induced pluripotent stem cells (hiPSCs) hold significant promise in regenerative medicine and drug discovery. However, single-cell dissociation, essential for genetic modification and clonal selection, often reduces hiPSC viability and colony formation. While various methods, including small molecules and feeder cells, have been developed to address this, their outcomes remain inconsistent. This study aims to develop more efficient methods to enhance hiPSC survival post-dissociation using a novel combination of well-characterized small-molecule inhibitors.MethodsHuman induced pluripotent stem cells were pretreated with Rho-associated protein kinase inhibitor (Y27632), SMC4 (PD0325901 + CHIR99021 + thiazovivin + SB431542), or SiM5 (PD0325901 + CHIR99021 + Thiazovivin + SB431542 + Pifithrin-α) for 1 h before subjected to single-cell dissociation by accutase. The dissociated single hiPSCs were then cultured in NutriStem or StemFlex medium supplemented with Y27632, SMC4, or SiM5. Cell viability, pluripotency marker expression, colony formation capacity, and karyotype were then compared between various treatments. The effect of SiM5 treatment on hiPSCs survival and colony formation capacity was also tested under hypoxic conditions and after fluorescence-activated cell sorting.ResultsThe results show that SiM5 treatment significantly increases hiPSCs survival by approximately 2.5 and 25 times compared to those treated with SMC4 and Y27632, respectively. These results were consistently observed across different cell lines and culture media. Furthermore, SiM5 treatment also increased hiPSCs survival and proliferation after single-cell dissociation under hypoxic conditions. The withdrawal of SiM5 after treatment only temporarily hinders hiPSCs cell cycle progression, without impairing their subsequent expansion. Fluorescence-activated cell sorting analysis revealed that SiM5 does not affect the pluripotency of hiPSCs following treatment. Additionally, it was found that SiM5 has no effect on the colony-forming ability or chromosomal stability of hiPSCs.ConclusionSiM5 treatment significantly improves hiPSCs survival and colony formation after single-cell dissociation across various conditions. This approach could enhance the efficiency of genetic manipulation and single-cell cloning, advancing hiPSCs applications in research and clinical settings.