Matsumoto Takaki, Kim Mee-Hae, Kino-Oka Masahiro
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Osaka, Japan.
Research Base for Cell Manufacturability, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Osaka, Japan.
Bioengineering (Basel). 2022 Oct 25;9(11):613. doi: 10.3390/bioengineering9110613.
Rho-associated protein kinase (ROCK) inhibitors are used for the survival of single-dissociated human induced pluripotent stem cells (hiPSCs); however, their effects on the growth behaviors of hiPSCs in suspension culture are unexplored. Therefore, we investigated the effect of ROCK inhibitor on growth behaviors of two hiPSC lines (Tic and 1383D2) with different formation of aggregate that attached between single cells in suspension culture. The apparent specific growth rate by long-term exposure to Y-27632, a ROCK inhibitor, was maintained throughout the culture. Long-term exposure to ROCK inhibitor led to an increase in cell division throughout the culture in both lines. Immunofluorescence staining confirmed that hiPSCs forming spherical aggregates showed localization of collagen type I on its periphery. In addition, phosphorylated myosin (pMLC) was localized at the periphery in culture under short-term exposure to ROCK inhibitor, whereas pMLC was not detected at whole the aggregate in culture under long-term exposure. Scanning electron microscopy indicated that long-term exposure to ROCK inhibitor blocked the structural alteration on the surface of cell aggregates. These results indicate that pMLC inhibition by long-term ROCK inhibition leads to enhanced growth abilities of hiPSCs in suspension culture by maintaining the structures of extracellular matrices.
Rho相关蛋白激酶（ROCK）抑制剂用于单离的人诱导多能干细胞（hiPSC）的存活；然而，它们对悬浮培养中hiPSC生长行为的影响尚未得到研究。因此，我们研究了ROCK抑制剂对两种hiPSC系（Tic和1383D2）生长行为的影响，这两种细胞系在悬浮培养中单个细胞之间形成的聚集体不同。在整个培养过程中，长期暴露于ROCK抑制剂Y-27632后的表观比生长速率得以维持。长期暴露于ROCK抑制剂导致两个细胞系在整个培养过程中的细胞分裂增加。免疫荧光染色证实，形成球形聚集体的hiPSC在其周边显示I型胶原的定位。此外，短期暴露于ROCK抑制剂的培养中，磷酸化肌球蛋白（pMLC）定位于周边，而长期暴露的培养中在整个聚集体中未检测到pMLC。扫描电子显微镜表明，长期暴露于ROCK抑制剂会阻止细胞聚集体表面的结构改变。这些结果表明，长期ROCK抑制对pMLC的抑制通过维持细胞外基质的结构，导致悬浮培养中hiPSC的生长能力增强。
