Expression profiling of cell growth on different layers

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) hold great promise for applications in drug discovery and regenerative medicine. While tremendous ongoing efforts are focused on establishing more optimal cellular microenvironments for hESCs/hiPSCs to facilitate their differentiation into the specific phenotypical functions of interests, it is still challenging to systematically determine the optimal environment and the circumstances supporting each environment, due to a lack of control over the microenvironmental factors in a three-dimensional (3D) space. In this study, we developed a 3D cellular microenvironment plate (3D-CEP), which consisted of a polydimethylsiloxane (PDMS) microfluidic device combined with a thermo-responsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) (PNIPAAm-β-PEG) hydrogel (HG), as a platform for hESC/hiPSC culture and analysis. The HG/3D-CEP system enables tuning of both chemical and physical environmental cues as well as in situ monitoring of cells. H9-derived hESCs cultured in the HG/3D-CEP system maintained pluripotent marker expression for octamer-binding transcription factor 4 (OCT4), (sex determining region Y)-box 2 (SOX2), NANOG, stage-specific embryonic antigen 4 (SSEA4), and TRA-1-60, but did not express the differentiation marker SSEA1 under self-renewing conditions. Small variations between different test environments and their influence on the development of biological networks could be further examined using global gene expression techniques. We envision that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines derived from hESCs/hiPSCs and facilitate advances in more precise drug screening and regenerative medicine H9-derived hESCs cultured in the HG/3D-CEP system – high mebil gel, low mebil gel, matrigel, sphere culture, controls, 3 replicates per condition.