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. 2014 Jun 5;4:5188.
doi: 10.1038/srep05188.

Rewiring Mesenchymal Stem Cell Lineage Specification by Switching the Biophysical Microenvironment

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Free PMC article

Rewiring Mesenchymal Stem Cell Lineage Specification by Switching the Biophysical Microenvironment

Junmin Lee et al. Sci Rep. .
Free PMC article

Abstract

The propensity of stem cells to specify and commit to a particular lineage program is guided by dynamic biophysical and biochemical signals that are temporally regulated. However, most in vitro studies rely on "snapshots" of cell state under static conditions. Here we asked whether changing the biophysical aspects of the substrate could modulate the degree of mesenchymal stem cell (MSC) lineage specification. We chose to explore two diverse differentiation outcomes: MSC osteogenesis and trans-differentiation to neuron-like cells. MSCs were cultured on soft (~0.5 kPa) or stiff (~40 kPa) hydrogels followed by transfer to gels of the opposite stiffness. MSCs on soft gels express elevated neurogenesis markers while MSCs on stiff substrates express elevated osteogenesis markers. Transfer of MSCs from soft to stiff or stiff to soft substrates led to a switch in lineage specification. However, MSCs transferred from stiff to soft substrates maintained elevated osteogenesis markers, suggesting a degree of irreversible activation. Transferring MSCs to micropatterned substrates reveal geometric cues that further modulate lineage reversal. Taken together, this study demonstrates that MSCs remain susceptible to the biophysical properties of the extracellular matrix--even after several weeks of culture--and can redirect lineage specification in response to changes in the microenvironment.

Figures

Figure 1
Figure 1. Hydrogel fabrication scheme and experimental strategy.
(a) Protocol for fabricating matrix protein conjugated polyacrylamide hydrogels. (b) Schematic illustration of microenvironment switch between soft (0.5 kPa) and stiff (40 kPa) substrates to monitor the dynamic changes of MSC lineage specification.
Figure 2
Figure 2. Projected cell area and viability are influenced by changing the mechanical properties of the substrate.
(a) – (b) Representative immunofluorescence microscopy images and quantitation of average cell area of MSCs cultured for 10 days and after microenvironment switch (0.5 formula image 40 kPa); scale bar: 120 μm (**P<0.005, ***P<0.0005, Fisher's exact test). (c) – (d) Cell viability of MSCs before and after substrate switch.
Figure 3
Figure 3. Matrix stiffness modulates the degree of MSC lineage specification.
(a) Expression of osteogenic (runx2 and osteopontin) and neurogenic (β-tubulin and MAP2) markers before and after switching the substrate (0.5 formula image 40 kPa) (*P<0.05, **P<0.005, ***P<0.0005, Fisher's exact test). (b) Representative immunofluorescence microscope image of MSCs cultured on the unpatterned fibronectin coated substrates after immunostaining for nuclei, osteopontin, MAP2 and filamentous actin; staining for MSC nuclei (blue), actin (cyan-green), osteopontin (orange), MAP2 (red). Scale bar: 35 μm.
Figure 4
Figure 4. Cell shape directs lineage specification of MSCs after microenvironment change.
(a) Quantitation of osteogenic (runx2 and osteopontin) and neurogenic (β-tubulin and MAP2) markers for a population of cells cultured with and without a microenvironment change from soft to stiff. (b) Representative immunofluorescence images. (c) Expression of osteogenic (runx2 and osteopontin) and neurogenic (β-tubulin and MAP2) markers for a population of cells cultured with and without a microenvironment change from stiff to soft. (d) Representative immunofluorescence images; staining for MSC nuclei (blue), actin (cyan-green), runx2 and osteopontin (orange), β-tubulin and MAP2 (red). Scale bar: 35 μm. (*P<0.05, ***P<0.005, Fisher's exact test).
Figure 5
Figure 5. Geometric cues differentially reprogram early and late markers of neurogenesis and osteogenesis for (a) soft to stiff and (b) stiff to soft.

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