Year 2

To establish the stem cell-based regenerative therapy, it is necessary to generate sufficient number of cells with specific functions. The maintenance of stem cells with pluripotency as well as the control of differentiation under well defined condition without the support of the mouse feeder cells is essential for the safe use of stem cells in therapies. The aim of our study is to develop a new platform for the comprehensive screening of the well-defined parameters and protocols for determining the optimum conditions to expand cultured stem cells to large cell numbers and to differentiate them into specific cell types to generate desired tissues. Experimental manipulation of the environmental cues, such as chemical signals and physical forces, to which stem cells are exposed, will lead to the discovery of conditions that specifically direct hESC growth and differentiation.

Based on our extracellular matrix protein (ECMPs) microarray platform, we have screened the combinations of 5 different proteins, and the data suggest that the combination of collagen 1, collagen 4 [Isn’t laminin also in here?] and fibronectin provides excellent support for the long-term growth of the two hESC lines (HUES1 and HUES9) tested with prolonged pluripotency. In addition to ECMPs, testing of growth factors indicates that three factors (WNT5A, WNT3A, and FGF) have positive effects on cell pluripotency maintenance, and two factors (BMP4 and RA) affect hESC differentiation. Furthermore, our tests also indicate that mechanical loading can promote cell differentiation. Our systematic study of the roles of microenvironmental factors by using a combinatorial approach can replace the undefined cell culture conditions with the use of mouse feeder cells.

In summary, our results provide new insight on defining the optimum stem cell culture condition, which can be used to customize the parameters for expansion of different types of stem cell culture. Hence, this project has fundamental importance and broad applications. This is a most cost-effective way to pursue hESC research for the improvement of human health and quality of life, and the resulting technology advances would provide financial gain for the people in California.