Dissecting Self-renewal Mechanisms of iPS Cells on Defined Synthetic Substrates Abstract ) We developed a synthetic polymer matrix substrate (PMEDSAH) that supports induced pluripotent stem (iPS) cell and human embryonic stem (hES) cell expansion in an undifferentiated state (self-renewal) in defined culture conditions that are free from xenogeneic contamination. We have thus overcome the undefined growth conditions that typically depend on the support of mouse embryonic feeder cells (MEFs) or an undefined matrix such as MatrigelTM and that until now have severely limited our ability to perform unimpeded mechanistic studies and hindered our ability to use these stem cells to treat debilitating human diseases. Our goals for this proposal are to define the molecular mechanisms that maintain iPS cells in an undifferentiated state, the derivation of iPS cell lines in our xeno-free and fully defined culture system, and the controlled differentiation of these cells towards a mesenchymal stem cell (MSC) phenotype. We will take full advantage of our unique, xeno-free and fully defined culture system, which consists of PMEDSAH as the substrate and serum-free, defined culture medium to elucidate the mechanisms responsible for self-renewal on this substrate. Accomplishing these goals is an important prerequisite to the development of therapeutic protocols using pluripotent stem cells to regenerate human tissues. The projects outlined in this competing renewal proposal are designed to directly address these goals and our success should have a significant impact in methods to regenerate human tissues using pluripotent stem cells. It is well recognize that the microenvironment influences the fate of stem cells, thus in Specific Aim 1 we will define the structural and/or physico-chemical properties of PMEDSAH that lead to iPS cell self-renewal and maintenance of the undifferentiated state.
In Specific Aim 2 we will determine the cell receptor mechanisms that direct adhesion and maintain iPS cells in an undifferentiated state on synthetic polymer substrates, testing the hypothesis that pluripotent iPS cells use more than one cell adhesion system to adhere to and support self-renewal on a defined polymer substrate.
In Specific Aim 3 we will demonstrate that patient-specific iPS cells can be derived on defined substrates free of xenogeneic contamination and are able to differentiate into mesenchymal stem cells capable of regenerating craniofacial skeletal defects, as a proof of concept that our system has the potential of getting the medical and scientific community closer to "clinical-grade" pluripotent stem cells. Our approach is unique and fundamentally different than the state of the art because it uses synthetic components as the structural motifs in cell-substrate interactions. By accomplishing our goal, we will make significant contributions to the understanding one of the major unresolved issues in pluripotent stem cell biology;that is, learning how pluripotent stem cells interact with their extracellular environment to: 1) remain in a unique undifferentiated state and 2) make fate changing lineage decisions. This knowledge is important for both understanding basic stem cell biology and developing consistent and safe regenerative therapies.

Public Health Relevance

The proposed research is relevant to public health because we will determine how to generate of patient- specific puripotent stem cells (iPS) in fully defined and xenogeneic-free conditions that can be differentiated to osteoblasts capable of regenerating clinically relevant skeletal defects. Our innovation is derived from our so- called synthetic biology approach where synthetic PMEDSAH takes on biological functions;for example, supporting cell adhesion, enabling self-renewal and heparin-like binding of growth factors. Because we will derive patient-matched stem cells in completely defined and xengeneic free conditions we will positively impact both the understanding of basic stem cell biology and contribute to the development of consistent and safe regenerative therapies. As such, successful completion of the projects should enable new horizons important to the mission of the NIH.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
2R01DE016530-06
Application #
8240773
Study Section
Skeletal Biology Structure and Regeneration Study Section (SBSR)
Program Officer
Lumelsky, Nadya L
Project Start
2005-04-01
Project End
2017-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
6
Fiscal Year
2012
Total Cost
$383,454
Indirect Cost
$133,959
Name
University of Michigan Ann Arbor
Department
Biology
Type
Schools of Dentistry
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Qian, Xu; Villa-Diaz, Luis G; Kumar, Ramya et al. (2014) Enhancement of the propagation of human embryonic stem cells by modifications in the gel architecture of PMEDSAH polymer coatings. Biomaterials 35:9581-90
Sun, Yubing; Yong, Koh Meng Aw; Villa-Diaz, Luis G et al. (2014) Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells. Nat Mater 13:599-604
Villa-Diaz, L G; Ross, A M; Lahann, J et al. (2013) Concise review: The evolution of human pluripotent stem cell culture: from feeder cells to synthetic coatings. Stem Cells 31:1-7
Qian, X; Villa-Diaz, L G; Krebsbach, P H (2013) Advances in culture and manipulation of human pluripotent stem cells. J Dent Res 92:956-62
Chen, Weiqiang; Villa-Diaz, Luis G; Sun, Yubing et al. (2012) Nanotopography influences adhesion, spreading, and self-renewal of human embryonic stem cells. ACS Nano 6:4094-103
Villa-Diaz, L G; Brown, S E; Liu, Y et al. (2012) Derivation of mesenchymal stem cells from human induced pluripotent stem cells cultured on synthetic substrates. Stem Cells 30:1174-81
Nandivada, Himabindu; Villa-Diaz, Luis G; O'Shea, K Sue et al. (2011) Fabrication of synthetic polymer coatings and their use in feeder-free culture of human embryonic stem cells. Nat Protoc 6:1037-43
Arpornmaeklong, Premjit; Pressler, Michael J; Krebsbach, Paul H (2011) Phenotypic and differentiation stability of human embryonic stem cell-derived osteoblasts. Cells Tissues Organs 194:326-30
Arpornmaeklong, Premjit; Wang, Zhuo; Pressler, Michael J et al. (2010) Expansion and characterization of human embryonic stem cell-derived osteoblast-like cells. Cell Reprogram 12:377-89
Villa-Diaz, Luis G; Nandivada, Himabindu; Ding, Jun et al. (2010) Synthetic polymer coatings for long-term growth of human embryonic stem cells. Nat Biotechnol 28:581-3

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