Location dependent differential gene expression within human embryonic stem cell (H9) colony.

We and others found that human pluripotent stem cells (hPSCs) grown as colonies exhibits different cell polarity, cytoskeletal structures and mechanical dynamics depending on their location within a colony. With our novel fluorescent dye labeling method, we could isolate high-labeled edge cells and low-labeled center cells in hPSC colonies cultured in micropattern by FACS based on dye intensity. This approach enabled us to compare transcriptome profile between edge and center cells by mRNA sequencing. Five center samples and 4 edge samples obtained from 5 independent experiments were sequenced and 16,742 genes were included in the analysis by edgeR. Unpaired comparison between edge and center samples revealed that 272 genes were significantly different in these two cell populations. And KEGG and GO analyses demonstrated these differentially expressed genes are mostly related with cell polarization, migration, actin dynamics and endocytosis. These RNA sequencing data were confirmed by several assays including qPCR, immunostaining and in situ hybridization. This study suggests that position-dependent differences in cellular structure and function in hPSC colonies might induce differential gene expression profiles, leading to position-dependent differential response of hPSCs to extracellular stimuli.