Chang Jessica, Kim Mee-Hae, Agung Eviryanti, Senda Sho, Kino-Oka Masahiro
Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, 210-8681 Japan.
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Regen Ther. 2018 Nov 20;10:27-35. doi: 10.1016/j.reth.2018.10.004. eCollection 2019 Jun.
Understanding how extracellular matrix (ECM) protein composition regulates the process of human induced pluripotent stem cell (hiPSC) colony formation may facilitate the design of optimal cell culture environments. In this study, we investigated the effect of migratory behaviors on hiPSC colony formation on various ECM-coated surfaces.
To quantify how different ECM proteins affect migratory behavior during the colony formation process, single cells were seeded onto surfaces coated with varying concentrations of different ECM proteins. Cell behavior was monitored by time-lapse observation, and quantitative analysis of migration rates in relation to colony formation patterns was performed. Actin cytoskeleton, focal adhesions, and cell-cell interactions were detected by fluorescence microscopy.
Time-lapse observations revealed that different mechanisms of colony formation were dependent upon the migratory behavior of cells on different ECM surfaces. HiPSCs formed tight colonies on concentrated ECM substrates, while coating with dilute concentrations of ECM yielded more motile cells and colonies capable of splitting into single cells or small clusters. Enhanced migration caused a reduction of cell-cell contacts that enabled splitting or merging between cells and cell clusters, consequently reducing the efficiency of clonal colony formation. High cell-to-cell variability in migration responses to ECM surfaces elicited differential focal adhesion formation and E-cadherin expression within cells and colonies. This resulted in variability within focal adhesions and further loss of E-cadherin expression by hiPSCs.
Migration is an important factor affecting hiPSC colony-forming patterns. Regulation of migratory behavior can be an effective way to improve the expansion of hiPSCs while improving the process of clonal colony formation. We believe that this investigation provides a valuable method for understanding cell phenotypes and heterogeneity during colony formation in culture.
了解细胞外基质（ECM）蛋白组成如何调节人诱导多能干细胞（hiPSC）集落形成过程，可能有助于设计最佳的细胞培养环境。在本研究中，我们调查了迁移行为对不同ECM包被表面上hiPSC集落形成的影响。
为了量化不同ECM蛋白在集落形成过程中如何影响迁移行为，将单细胞接种到涂有不同浓度不同ECM蛋白的表面上。通过延时观察监测细胞行为，并对与集落形成模式相关的迁移率进行定量分析。通过荧光显微镜检测肌动蛋白细胞骨架、粘着斑和细胞间相互作用。
延时观察表明，不同的集落形成机制取决于细胞在不同ECM表面上的迁移行为。hiPSC在浓缩的ECM底物上形成紧密的集落，而用稀释浓度的ECM包被则产生更多可移动的细胞和能够分裂成单细胞或小簇的集落。增强的迁移导致细胞间接触减少，从而使细胞和细胞簇之间能够分裂或合并，进而降低了克隆集落形成的效率。细胞对ECM表面迁移反应的高变异性导致细胞和集落内粘着斑形成和E-钙粘蛋白表达的差异。这导致粘着斑内的变异性以及hiPSC进一步丧失E-钙粘蛋白表达。
迁移是影响hiPSC集落形成模式的重要因素。调节迁移行为可能是改善hiPSC扩增同时改善克隆集落形成过程的有效方法。我们认为，这项研究为理解培养过程中集落形成期间的细胞表型和异质性提供了一种有价值的方法。
