(14a) Substrate Elasticity Regulates the Biophysical Properties of Hematopoietic Stem and Progenitor Cells | AIChE

(14a) Substrate Elasticity Regulates the Biophysical Properties of Hematopoietic Stem and Progenitor Cells

Authors 

Choi, J. S. - Presenter, University of Illinois at Urbana-Champaign
Leonard, T. - Presenter, University of Illinois at Urbana-Champaign
Harley, B. A. - Presenter, University of Illinois at Urbana-Champaign


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.



 

UIC Symposium.pngFigure 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).