(136i) The Role of ECM Biomechanics in Liver Progenitor Differentiation

Combinatorial ECM arrays reveal the role of biomechanics in liver
progenitor differentiation

Andreas P. Kourouklis, Kerim B. Kaylan, Gregory H. Underhill

Department
of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL

Recent findings suggest that
biomechanical signals within the liver microenvironment can regulate the differentiation
of mature hepatocytes [1]. However, the role of ECM biomechanics in liver
progenitor differentiation remains primarily unexplored, despites its potential
importance in the processes of liver morphogenesis and regeneration. To examine
these mechanisms, we created high-throughput cellular arrays with the capacity to reiterate combinatorial ECM cues
and characterize the corresponding phenotypic expression. Moreover, we combined
these arrays with substrates of modular stiffness and integrated them with
traction force microscopy (TFM) to assess the associated traction stress. This strategy
provides a novel avenue to examine cell differentiation and elucidate the role
of combinatorial ECM cues in cellular fate.

With this objective, we fabricated high throughput BMEL cell arrays on soft
(4kPa) and stiff (30kPa) substrates presenting all single and pairwise
combinations of 5 ECM molecules (Fig. 1A). We found that the ECM composition along
with the substrate stiffness coordinately control cholangiocytic
differentiation (e.g. OPN+ cell fraction). Most notably, collagen IV (C4) and
fibronectin (FN) exhibited distinct effects (Fig.1B). BMEL cells on FN expressed
stiffness-mediated cholangiocytic differentiation,
indicated by the higher OPN+ fraction on stiff substrates (Fig.1B), while the
cells on C4 exhibited cholangiocytic differentiation
indifferent of substrate stiffness. Moreover, BMEL cells on FN developed higher
traction stress on stiff substrates (Fig.1B), while cells on C4 exhibited
significant traction on both substrates. These data are suggestive of a mechanism
by which liver progenitor differentiation is regulated by traction stress at
the cell-ECM interface.

Figure 1: (A) Successful fabrication of cell arrays with BMEL
domains of a diameter of ~150um. The BMEL arrays are immunostained
for hepatocytic (albumin: ALB) and cholangiocytic (osteopontin: OPN)
markers for high-throughput analysis. Lower raw demonstrates bead embedded gels
for TFM with the corresponding phase contrast and heat maps. OPN expression (B)
and traction stress (C) in response to stiffness and ECM composition.

In conclusion, we integrated
microarray fabrication with TFM in order to reveal the role of cell biomechanics
in the differentiation of liver progenitor cells. We showed that cells
integrate combinatorial ECM cues using traction stress before they commit to
their differentiated phenotype. Future experiments will assess the
intracellular pathways that direct this differentiation response. Overall, this
strategy provides new insight into liver progenitor fate decisions that could
aid the investigation of liver disease mechanisms and the development of
cell-based therapies.

References:

[1] Wells, R.G., Hepatology,
2008. 47(4): p. 1394-400.