Human embryonic stem (hES) cells are being studied as potential source of cells for the treatment of many diseases (e.g. diabetes, spinal cord injury, Parkinson's, leukemia, congestive heart failure, etc.). These same cells are also being touted an ideal cell source for ex vivo tissue engineering or in situ regenerative medicine. The successful integration of hES cell into such therapies will hinge upon three critical steps: 1) stem cell expansion in number without differentiation (i.e., self-renewal);2) directed differentiation into a specific cell type or collection of cell types;and, 3) cell survival and promotion of their functional integration into existing tissue. Precisely controlling each of these steps will be essential to maximize the hES cell's therapeutic efficacy. However, it is difficult to precisely control the behavior of hES cells, since environmental conditions for self-renewal and differentiation are poorly understood. We propose to develop a tunable completely synthetic surface and chemically defined media to control the self-renewal/expansion of hES cells. If hES cells can be derived and maintained within a completely synthetic environment, then it will be possible to eliminate pathogen transmission associated with animal-derived materials, provide a scalable basis for large-scale production of hES cells, and provide a precise base for further development to control hES cell differentiation. This application will develop materials to address the hypothesis that the contractile state of a hES cell, manifested by nuclear shape morphology via integrin engagement, regulates hES cell self-renewal. Our hypothesis is centered on a common mechanism by which cells respond differentially to either materials with variable moduli or materials that spatially confine a cell's shape via adhesion site distribution. We propose that a common mechanism that controls hES cell self-renewal and cell fate determination is the contractile state of the cell manifested by nuclear morphology, and integrin engagement and clustering. Thus, we wish to explore the spatial arrangement of cell adhesion domains (i.e., their size, number/cell body, and spatial arrangement) and assess their effect on the self-renewal of hES cells. We propose that altering the physical state of a pluripotent hES cell, via spatial clustering of its adhesions with a surface, will influence self-renewal and differentiation to a specific phenotype. The following specific aims are proposed.
Specific Aim 1 : To develop and characterize nanopatterned cell culture substrata where the size, peptide ligand density, number/cell body, and spatial arrangement of integrin-engaging domains will be varied to control cell and colony morphology.
Specific Aim 2 : To evaluate the nanopatterned substrata to support the long-term growth (5-10 passages) of human ES cells in chemically-defined media.
This application will focus specifically on engineering a tunable and well-defined environment presenting hES cells with a completely synthetic cell culture surface and chemically-defined media to promote self-renewal. The result will be a synthetic microenvironment that can both serve as a regenerative medicine technology platform for large scale hES cell expansion, as well as provide a novel and highly modular system for dissecting basic signaling mechanisms underlying hES cell self-renewal.
