Substrate Elasticity Regulates the Biophysical Properties of Hematopoietic Stem and Progenitor Cells

Substrate elasticity regulates the biophysical properties of hematopoietic stem and progenitor cells

Ji Sun (Sunny) Choi^*, Tyler Glenn Leonard^*, Brendan Harley^*,#

^* Dept. of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801

^# Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801

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). To elucidate the underlying mechanisms, which still remain largely unknown, it is important to decouple the extrinsic variables in a controlled manner. In this project, we aim to assess the cell-matrix interactions on HSC biophysical properties and early fate decisions (proliferation, motility, differentiation) by using flexible collagen hydrogel systems with varying mechanical properties. As a first step, we fabricated four different 2D collagen-coated polyacrylamide (PA) gels (moduli: 0.71 ± 0.24 kPa, 3.48 ± 0.59 kPa, 8.95 ± 1.40 kPa, 196 ± 54.1 kPa) and two different 3D collagen hydrogels (1.45, 2.9 mg/ml; moduli: 14.8 ± 6.1, 44.2 ± 10.7 Pa) according to published protocols (2, 3). HSCs were then harvested from 4-10-week-old C57Bl\6 mouse femurs and tibias and cultured on top of 2D PA substrates or on top of 3D collagen hydrogels for 24 hours. Alternatively, they were encapsulated in collagen hydrogels to create 3D collagen constructs, and cultured for 24 hours. At the end of culture, cells were assayed for their viability or fixed and stained to visualize F-actin structure and nuclei with a multiphoton laser scanning microscope to create projected 2D images of the cells. Cell spread area and cell shape index (CSI), which is a measure of the circularity of a cell, were then quantified and plotted (Figure 1a). PA gel collagen coating densities were varied and its effect on HSC behavior was also observed (Figure 1b, c). In addition, fluorescence intensity across the cytoplasm revealed differences in F actin organization in cells based on the substrate stiffness. This data was compared to 32D cells (IL-3 dependent murine myeloid progenitor cell line) grown in identical culture conditions, where 32D cells behaved in a different manner due to decreased differentiation potential. In conclusion, differences in cell morphology, spread area, and viability were observed in HSCs cultured on top of or within collagen hydrogels. Ongoing work is investigating the influence of matrix chemistry, integrin blocking, and myosin inhibitors on HSC behavior. Further experiments will incorporate cell division tracing assays, colony forming and competitive repopulation assays to assess the frequency of HSC differentiation, self-renewal, quiescence in these defined matrix environments.

 

Figure 1. Morphological differences in cells cultured on 2D PA substrates with varying stiffness

 

References: 
1. A. Ehninger, A. Trumpp, The Journal of Experimental Medicine 208, 421 (March 14, 2011).
2. J. R. Tse, A. J. Engler, Curr Protoc Cell Biol Chapter 10, Unit 10 16 (Jun, 2010).
3. E. L. Baker, R. T. Bonnecaze, M. H. Zaman, Biophysical Journal 97, 1013 (2009).