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The potential applications of human pluripotent stem cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells in cell therapy and regenerative medicine have been widely studied. The precise definition of pluripotent stem cell status during culture using biomarkers is essential for basic research and regenerative medicine. Culture conditions, including extracellular matrices, influence the balance between self-renewal and differentiation. Accordingly, to explore biomarkers for defining and monitoring the pluripotent substates during culture, we established different substates in H9 human ES cells by changing the extracellular matrix from vitronectin to Matrigel. The substate was characterised by low and high expression of the pluripotency marker R-10G epitope and the mesenchymal marker vimentin, respectively. Immunohistochemistry, induction of the three germ layers, and exhaustive expression analysis showed that the substate was ectoderm-biased, tended to differentiate into nerves, but retained the potential to differentiate into the three germ layers. Further integrated analyses of mRNA and miRNA microarrays and qPCR analysis showed that nine genes ( COL9A2 ,  DGKI ,  GBX2 ,  KIF26B ,  MARCH1 ,  PLXNA4 ,  SLC24A4 ,  TLR4 , and  ZHX3 ) were upregulated in the ectoderm-biased cells as ectoderm-biased biomarker candidates in pluripotent stem cells. Our findings provide important insights into ectoderm-biased substates of human pluripotent stem cells in the fields of basic research and regenerative medicine.

mRNA and miRNA expression profiles in an ectoderm-biased substate of human pluripotent stem cells