Metabolic patterns predispose human pluripotent stem cells to spatial organization of cell fate

Chunhao Deng^1,2Zhaoying Zhang¹Xia Xiao¹Carlos Godoy-Parejo¹Faxiang Xu¹Chengcheng Song¹Huanyi Lin²Qinru Li²Shicai Fang¹Weiwei Liu¹Guokai Chen^1,2*

• ¹University of Macau, Taipa, China
• ²University of Macau Zhuhai UM Science and Technology Research Institute, Zhuhai, China

During embryogenesis, tissue pattern formation is induced by specific morphogen gradients. In two-dimensional (2D) human pluripotent stem cell (hPSC) culture, distinct patterns can emerge within hPSC colonies in the absence of morphogen gradients, implying that critical intrinsic factors may induce the spatial organization. However, it is difficult to study the mechanism of pattern formation due to the lack of efficient labels to spatially track cells in a colony under various cell culture manipulations. In this report, we reveal that mitochondrial membrane potential probe JC-1 can stain and track cells in hPSC colonies, and is a valuable tool for studying pattern formation. Using JC-1 staining, we discovered clear metabolic patterning in hPSC colonies, which is significantly affected by cell culture coating materials. Distinct metabolic patterns emerge in hPSC colonies on integrin-stimulating matrices versus E-cadherin coated surface, and this difference correlates with spatial patterns of mesodermal cell fate under BMP4 induction. mTOR pathway modulators can alter the metabolic pattern, which is followed by changes in mesoderm induction. This study reveals novel mechanisms regulating pattern formation, and highlights JC-1 as a potent spatial marker to study fundamental mechanisms of tissue patterning.