The chemical and mechanical microenvironments are crucial in determining cellular behavior. The chemical microenvironment, which is composed mainly of extracellular matrix proteins (ECMPs) and growth factors (GFs), can affect cell fate in a complex manner. There is recent evidence that mechanical factors such as shear stress, stretch, and substrate rigidity can also play significant roles in modulate cell fate. Experimental manipulation of the microenvironmental factors to modulate proliferation and differentiation of embryonic stem cells (ESCs) is central to developing strategies for the production of defined cell types to treat a variety of disorders in regenerative medicine. In order to effectively screen the vast combinations of factors that may influence hESC fate, we have developed an array platform for the real-time, simultaneous screening of hundreds of physiochemical parameters on hESC attachment, proliferation, and gene and protein expressions. We have used this novel approach to systematically assess and probe the complex relationships between hESCs and their microenvironment. The application of such combinatorial techniques has shown that the effects of combinations of ECMPs cannot be predicted from their individual actions, demonstrating the importance of their interactions.

Combinations of ECMPs, GFs, and glycans were monitored for their effects on (Hues 1 and Hues 9) growth and survival of human ESCs (hESCs). Using real-time microscopy and hESC lines designed to express fluorescent proteins, we are able to monitor growth and survival over a period of 5 days. Statistical analysis showed that specific, defined microenvironments were able to support hESC proliferation at levels exceeding that obtained under current undefined culture conditions. A subset of these microenvironments was assayed for their ability to support the long-term maintenance (> 10 passages) of these hESC lines for defining the efficacy of the fully defined and optimized conditions to maintain hESC pluripotency and differentiation.

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Shear stresses and mechanical stretches have been shown to modulate ESC growth and differentiation. Mechanical rigidity of the ECM substrate has been shown to direct the differentiation of multipotent ESCs to cell types with rigidities matching those of the substrate.

The elucidation of the chemical and mechanical factors in modulating ESC fate will facilitate the identification of the optimum conditions for the growth and controlled differentiation in regenerative medicine.