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. 2008 Oct;7(10):816-23.
doi: 10.1038/nmat2269. Epub 2008 Aug 24.

Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells

Danielle S W Benoit  1 , Michael P Schwartz, Andrew R Durney, Kristi S Anseth
Affiliations

Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells

Danielle S W Benoit et al. Nat Mater. 2008 Oct.

Abstract

Cell-matrix interactions have critical roles in regeneration, development and disease. The work presented here demonstrates that encapsulated human mesenchymal stem cells (hMSCs) can be induced to differentiate down osteogenic and adipogenic pathways by controlling their three-dimensional environment using tethered small-molecule chemical functional groups. Hydrogels were formed using sufficiently low concentrations of tether molecules to maintain constant physical characteristics, encapsulation of hMSCs in three dimensions prevented changes in cell morphology, and hMSCs were shown to differentiate in normal growth media, indicating that the small-molecule functional groups induced differentiation. To our knowledge, this is the first example where synthetic matrices are shown to control induction of multiple hMSC lineages purely through interactions with small-molecule chemical functional groups tethered to the hydrogel material. Strategies using simple chemistry to control complex biological processes would be particularly powerful as they could make production of therapeutic materials simpler, cheaper and more easily controlled.

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Figures

Figure 1
Figure 1
Small molecule incorporation alters hMSC protein expression on PEG hydrogels. Chemical structures of functional moieties incorporated (left column) and protein expression of hMSCs (right column, as measured by immunostaining) quantitatively analyzed for collagen II (A), OPN (B), and PPARG (C) after 10 days of culture in control media on unmodified PEG and 0.5, 5, and 50 mM of amino (black bars), t-Butyl (black (top) to white (bottom) gradient bars), phosphate (grey bars), fluoro (white (top) to black (bottom) gradient bars), and acid (white bars). Values are reported as the fluorescent intensity average of 6 samples per composition, relative to the number of cells, as analyzed by propidium iodide counterstaining, and normalized to expression by cells cultured on PEGDM surfaces. Error bars represent one standard deviation. An asterisk (*) denotes statistical significance compared with PEGDM (p<0.05).
Figure 2
Figure 2
Small molecule incorporation alters hMSC gene expression on PEG hydrogels. Gene expression of hMSCs (as measured by in situ hybridization) quantitatively analyzed for Aggrecan (A), CBFA1 (B), and PPARG (C) at days 0 (black bars) , 4 (white bars), and 10 days (grey) of culture on (a) unmodified PEG and 50 mM of (b) amino, (c) t-Butyl, (d) phosphate, (e) fluoro, and (f) acid. Values are reported as the fluorescent intensity average of 6 samples per composition per timepoint, relative to β-actin expression, and normalized to expression by cells cultured on PEGDM surfaces. Error bars represent one standard deviation. An asterisk (*) denotes statistical significance compared with PEGDM (p<0.05).
Figure 3
Figure 3
hMSC morphology is altered in response to small molecule incorporation into PEG hydrogels. Representative light and fluorescent micrographs of TRITC-phalloidin (red) and DAPI (blue)-stained of hMSCs depicting morphology cultured on PEGDM (a and e), acid (b and f), phosphate (c and g), and t-butyl (d and h)-functionalized surfaces (bar = 200 µm for light micrographs, bar = 50 µm for fluorescent micrographs).
Figure 4
Figure 4
Encapsulation of hMSCs in phosphate and t-Butyl functionalized PEG hydrogels alters CBFA1 and PPARG expression. CBFA1, PPARG, and β-actin expression of hMSCs encapsulated in control, t-butyl, and phosphate-functionalized PEG hydrogels and cultured for 0, 4, 10, and 21 days in control media. Immunoblots (A) were quantified with ImageJ software and CBFA1 (B) and PPARG (C) expression levels over the 21-day culture period were normalized to β-actin expression (PEGDM: X; t-butyl functionalized: closed diamonds; phosphate-functionalized: open circles), error bars represent one standard deviation. Differentiation was further verified using histological and immunohistochemistry staining (D) of matrix evolution by encapsulated hMSCs. Masson’s trichrome stains collagen blue (left column) and Oil Red stains intracellular lipid deposits red (third column). OPN and PPARG staining was performed using protein-specific primary antibodies and HRP-labeled secondaries, and visualized with Vector NovaRed Substrate Kit (Vector Labs) (bar = 100 µm).
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