(616g) Substrate Elasticity Regulates the Biophysical Properties and Lineage Commitment of Hematopoietic Stem and Progenitor Cells

Hematopoietic stem cells (HSCs) are adult stem cells that give rise to the blood and immune cells in the body. These cells reside in a local microenvironment known as the stem cell niche, which consists of stromal cells, extracellular matrix (ECM), and other soluble and growth factors. Evidence suggests that the HSC niche provides critical spatial and temporal extrinsic signals to modulate HSC biology (quiescence, self-renewal, differentiation, and migration (1, 2)) although the underlying mechanisms are still largely unknown. In order to decouple the extrinsic variables (i.e. substrate elasticity, ligand presentation, ligand density, dimensionality) to assess the cell-matrix interactions on HSC biophysical properties and early fate decisions (proliferation, motility, differentiation) in a controlled manner, we fabricated hydrogels with varying mechanical properties that could mimic the naturally occurring range of stiffness in the HSC niche (marrow: <1 kPa; adipose tissue: ~2-3 kPa; bone: >30 kPa) (3). Primary HSCs isolated from 4-8-week-old C57Bl\6 mouse femurs and tibias in absence of any other niche cell types were then cultured on top of the fabricated hydrogels for 24 hours before their biophysical properties and lineage commitment potentials were analyzed. With HSCs cultured on substrates coated with type I collagen, we observed a significant increase in cell spread area with increasing substrate stiffness or ligand coating density (4). To further assess the influence of matrix components on HSC behaviors, we have coated polyacrylamide substrates with other ECM proteins (fibronectin, laminin, vitronectin) and cultured HSCs on top of them. Similar to type I collagen, fibronectin and laminin induced increased spreading and irregular morphology on stiffer substrates. To analyze the lineage commitment of the cultured HSCs, they were re-plated in methylcellulose-based medium and the number of colony-forming units (CFU) arising after 11-14 days were counted. Preliminary data suggests increased macrophage potential in HSCs cultured on collagen and fibronectin-coated substrates with increasing substrate elasticity. Laminin-coated substrates supported erythroid commitment better and to a greater degree on stiffer substrates. Overall, fibronectin-coated substrates led to the greatest total number of colonies as well as maintaining enhanced HSC expansion. Current ongoing work is investigating the lineage commitment further by monitoring changes in HSC surface antigen expression and gene expression levels. Future experiments will incorporate competitive repopulation assays and myosin inhibitors to determine the frequency of HSC differentiation, self-renewal, and quiescence in these defined matrix environments. In order to supplement our findings, we are also designing substrates with a stiffness gradient to monitor HSC mobilization and migration with or without the presence of a chemotactic agent.

References: 
1.         A. Ehninger, A. Trumpp, The bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move in. The Journal of Experimental Medicine 208, 421 (March 14, 2011, 2011).

2.         J. Holst et al., Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells. Nat Biotech 28, 1123 (2010).

3.         D. E. Discher, D. J. Mooney, P. W. Zandstra, Growth factors, matrices, and forces combine and control stem cells. Science 324, 1673 (Jun 26, 2009).

4.         J. S. Choi, B. A. C. Harley, Substrate elasticity and ligand density influence the viability and biophysical properties of hematopoietic stem and progenitor cells. Biomaterials In Press,  (2012).