Microenvironment Design for Stem Cell Fate Determination - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 249

et al. [ 184 ] cultured hMSC sheets on micro-patterns of controlled shape and
exposed them to a mixture of pro-osteogenic and pro-adipogenic morphogens.
They showed that gradients of mechanical forces can drive a pattern of differen-
tiation dictated by cell spatial arrangement and corresponding cytoskeletal stress.
HMSCs at the edge of multicellular islands differentiated into the osteogenic
lineage, whereas those in the center became adipocytes. Interestingly, changing the
shape of the multicellular sheet modulated the locations of osteogenic versus
adipogenic differentiation. Measuring traction forces revealed gradients of stress
that preceded and mirrored the patterns of differentiation, where regions of high
stress resulted in osteogenesis, whereas stem cells in regions of low stress dif-
ferentiated to adipocytes. These findings demonstrate a role for mechanical forces
created within multicellular organization in spatial cell differentiation. Such geo-
metric control is also useful for controlling cell-cell interactions, as in the
aggregation of ESCs in vitro into embryoid bodies (EBs), a preliminary step
toward their differentiation. The conventional methods of culturing EBs are poorly
controlled and result in the formation of heterogeneous structures with a wide
range of sizes and shapes. Spatial control over the EB formation can lead to a more
homogeneous, and thereby more efficiently controlled differentiation. For instance,
Karp et al. [ 185 ] developed micro-fabricated cell-repellent PEG hydrogel in
micro-wells as templates to initiate the controlled formation of homogeneous EBs.
Their approach resulted in synthetic microenvironments that enhanced the dif-
ferentiation of ESCs and significantly reduced variability in the expression of
differentiation markers. They were also able to pattern EBs into shapes that do not
naturally occur, such a triangles and curves; nevertheless the biological implica-
tions for stem cell fate are not clear [ 185 ].
The effects of matrix physical attributes such as matrix stiffness on stem cell
fate were first examined by Engelr et al. [ 186 ]. In their study, the elasticity of the
matrix was identified as a key factor of the stem cell micro-environment, speci-
fying stem cell commitment. MSCs were cultivated on 2D polyacrylamide (PA)
gels with varying matrix elasticity, set by degree of cross-linking, and adhesion
was provided by coating the gels with collagen I. The researchers showed that
MSCs differentiate into tissues that most closely match the mechanical properties
of the PA substrate upon which they were cultured. MSCs that were cultured on
stiff (bone-like) gels differentiated into osteoblasts, those that were cultured on
medium stiffness (muscle-like) gels differentiated into muscle cells, and those that
were cultured on compliant gels (neural-like) differentiated into neural cells—all
in identical serum conditions. These findings were attributed to the cytoskeleton
tension forces that activated molecular pathways of cell differentiation in a manner
dependent on substrate stiffness [ 186 ].
In another study, the commitment of MSC populations changed in response to
the scaffold rigidity; however, cell fate was not correlated with cell morphology.
Instead, matrix stiffness regulated integrin binding as well as reorganization of
adhesion ligands on the nanoscale, both of which were traction-dependent and
correlated with osteogenic commitment of MSC populations. These findings