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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085754-03
Application #
8058790
Study Section
Special Emphasis Panel (ZRG1-BST-G (02))
Program Officer
Deatherage, James F
Project Start
2009-04-01
Project End
2013-03-31
Budget Start
2011-04-01
Budget End
2012-03-31
Support Year
3
Fiscal Year
2011
Total Cost
$325,671
Indirect Cost
Name
University of California Berkeley
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Jeon, Hojeong; Koo, Sangmo; Reese, Willie Mae et al. (2015) Directing cell migration and organization via nanocrater-patterned cell-repellent interfaces. Nat Mater 14:918-23
Schmidt, Ray C; Healy, Kevin E (2013) Effect of avidin-like proteins and biotin modification on mesenchymal stem cell adhesion. Biomaterials 34:3758-62
Jeon, Hojeong; Schmidt, Ray; Barton, Jeremy E et al. (2011) Chemical patterning of ultrathin polymer films by direct-write multiphoton lithography. J Am Chem Soc 133:6138-41
Irwin, Elizabeth F; Gupta, Rohini; Dashti, Derek C et al. (2011) Engineered polymer-media interfaces for the long-term self-renewal of human embryonic stem cells. Biomaterials 32:6912-9
Keung, Albert J; Healy, Kevin E; Kumar, Sanjay et al. (2010) Biophysics and dynamics of natural and engineered stem cell microenvironments. Wiley Interdiscip Rev Syst Biol Med 2:49-64
Meng, Ying; Eshghi, Shawdee; Li, Ying J et al. (2010) Characterization of integrin engagement during defined human embryonic stem cell culture. FASEB J 24:1056-65
Kohen, Naomi T; Little, Lauren E; Healy, Kevin E (2009) Characterization of Matrigel interfaces during defined human embryonic stem cell culture. Biointerphases 4:69-79