Laowtammathron Chuti, Chingsuwanrote Pimjai, Srisook Pimonwan, Klaihmon Phatchanat, Meaunpim Patcharee, Lorthongpanich Chanchao, Kheolamai Pakpoom, Issaragrisil Surapol
Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Flow Cytometry Laboratory, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
Sci Prog. 2025 Apr-Jun;108(2):368504251330956. doi: 10.1177/00368504251330956. Epub 2025 May 13.
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.
目标
人类诱导多能干细胞（hiPSC）在再生医学和药物研发领域具有巨大潜力。然而，对于基因编辑和克隆筛选至关重要的单细胞解离过程，常常会降低hiPSC的活力和集落形成能力。尽管已经开发了包括小分子和饲养层细胞在内的多种方法来解决这一问题，但其结果仍不一致。本研究旨在通过使用一组经过充分表征的新型小分子抑制剂组合，开发更有效的方法来提高hiPSC解离后的存活率。
方法
在通过胰蛋白酶进行单细胞解离前1小时，用Rho相关蛋白激酶抑制剂（Y27632）、SMC4（PD0325901 + CHIR99021 +噻唑维文+ SB431542）或SiM5（PD0325901 + CHIR99021 +噻唑维文+ SB431542 +匹非尼酮-α）预处理人类诱导多能干细胞。然后将解离后的单个hiPSC接种于添加了Y27632、SMC4或SiM5的NutriStem或StemFlex培养基中培养。随后比较不同处理组之间的细胞活力、多能性标志物表达、集落形成能力和核型。还在低氧条件下以及荧光激活细胞分选后测试了SiM5处理对hiPSC存活和集落形成能力的影响。
结果
结果表明，与SMC4和Y27632处理组相比，SiM5处理分别使hiPSC的存活率显著提高了约2.5倍和25倍。在不同细胞系和培养基中均一致观察到这些结果。此外，SiM5处理还提高了低氧条件下单细胞解离后hiPSC的存活和增殖能力。处理后撤除SiM5仅会暂时阻碍hiPSC细胞周期进程，而不会损害其随后的扩增。荧光激活细胞分选分析显示，SiM5处理后不会影响hiPSC的多能性。此外，发现SiM5对hiPSC的集落形成能力或染色体稳定性没有影响。
结论
SiM5处理显著提高了各种条件下单细胞解离后hiPSC的存活和集落形成能力。这种方法可以提高基因操作和单细胞克隆的效率，推动hiPSC在研究和临床领域的应用。
