255404 Collagen-Mimetic Hydrogels for Elucidating Mesenchymal Stem Cell Fate Decisions
Introduction: A number of obstacles remain before engineered tissues based on mesenchymal stem cells (MSCs) can be considered viable clinical alternatives. In particular, rational design of scaffolds to elicit desired MSC lineage progression is problematic due, in part, to our incomplete understanding of MSC responses to extracellular matrix (ECM)-mediated stimuli. In the present work, we begin to address this challenge by probing MSC responses to collagen-based biochemical motifs using novel hybrid hydrogels. These hydrogels were generated by covalently crosslinking diacrylate-derivatized poly(ethylene glycol) (PEGDA) and a collagen-mimetic protein, termed Scl2-1. Scl2-1 is unique in that it contains the GXY repeats and stable triple helical structure of native collagen but lacks collagen's array of cell adhesion, cytokine binding, and enzymatic degradation sites. Thus, Scl2-1 provides a “blank-slate” into which desired collagen adhesion sequences can be programmed by site-directed mutagenesis while maintaining the triple helical context natively associated with these motifs. The current work employs modified Scl2-1 proteins to investigate the impact of α1β1 and α2β1 integrin signaling on human bone-marrow derived MSC (hMSC) fate decisions. Specifically, the influence of Scl2-2 (Scl2-1 containing an α1β1/α2β1 binding motif) on hMSC lineage progression was compared to that of Scl2-3 (Scl2-1 containing an α1β1 binding motif).
Materials and Methods: Expression and Mutagenesis of Scl2. Scl2-2 and Scl2-3 were generated by encoding for sequences GFPGER and GFPGEN, respectively, using site-directed mutagenesis of the plasmid encoding for Scl2-1. Associated proteins were recombinantly expressed in E.coli JM101 and were purified by affinity chromatography. Scl2 Conjugation to a PEG Linker. Scl2 proteins were functionalized with photoreactive crosslink sites by reaction with acrylate-PEG-hydroxysuccinimide (Ac-PEG-NHS, MW 3400) per standard protocols. Fabrication of PEGDA-Scl2 Hydrogels. PEGDA-Scl2 gels were fabricated by combining 10 wt% PEGDA (3.4 kDa) with photoinitiator (Irgacure 2959), 1 mg/mL of Ac-PEG-Scl2, Ac-PEG-Scl2-2, or Ac-PEG-Scl2-3, and 1x106 hMSCs (Lonza) per mL. The solutions were then crosslinked via 90 s exposure to 365 nm UV light. In fabricating these gels, a weight ratio of PEGDA to Scl2 of 100:1 was used to ensure that the elastic modulus, mesh size, and degradation rate of each hydrogel network would be dominated by PEGDA. Constructs were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) for 7 days.
Results and Discussion: Following confirmation of the ability of Scl2-2 and Scl2-3 to stimulate distinct focal adhesion kinase (FAK) signaling (Fig. 1A), hMSC-laden PEGDA-Scl2 hydrogels were prepared and cultured for 7 days. Media without differentiation supplements was utilized in order to focus on the influence of integrin-mediated signaling on hMSC fate decisions. Endpoint samples were harvested and transcription factors indicative of early lineage progression were analyzed by competitive ELISA. No differences in myoD or Runx2 levels were noted among hydrogel formulations, suggesting that the selected integrin adhesion motifs did not significantly stimulate myogenic or osteogenic differentiation, at least for the gel formulations probed. However, significant differences in PPARg (adipogenic) and sox9 (chondrogenic) levels were observed (Fig. 1B). Specifically, PPARg expression appeared to be elevated by α2β1 signaling but not by α1β1 adhesion (Scl2-2 vs Scl2-3). In contrast, α1β1 signaling (Scl2-3) was associated with increased sox9 expression, but not when in the presence of simultaneous α2β1 adhesion (Scl2-2). Given that α2β1 binding activates p38 whereas α1β1 does not (Fig. 1A), the p38 MAPK signaling cascade may play an important role in hMSC adipogenic differentiation.
Conclusions: The present results demonstrate our ability to achieve a controlled 3D environment that can be used to probe MSC responses to highly defined collagen-based adhesion signals while maintaining collagen's triple helical context. This controlled hydrogel platform enables more precise examination of the signaling underlying observed cell responses and should significantly advance our understanding of the processes associated with MSC lineage progression. Future studies will incorporate gene silencing to elucidate causative cell signaling.