Abstract
Extracellular matrix (ECM) proteins are structural elements of tissue and also potent signaling molecules. Previously, our laboratory showed that ECM of 2D coatings can trigger differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into mesodermal lineages in an ECM-specific manner over 14 days, in some cases comparable to chemical induction. To test whether a similar effect was possible in a 3D, tissue-like environment, we designed a synthetic-natural biomaterial composite. The composite can present whole-molecule ECM proteins to cells, even those that do not spontaneously form hydrogels ex vivo, in 3D. To this end, we entrapped collagen type I, laminin-111, or fibronectin in ECM composites with MSCs and directly compared markers of mesodermal differentiation including cardiomyogenic (ACTC1), osteogenic (SPP1), adipogenic (PPARG), and chondrogenic (SOX9) in 2D versus 3D. We found the 3D condition largely mimicked the 2D condition such that the addition of type I collagen was the most potent inducer of differentiation to all lineages tested. One notable difference between 2D and 3D was pronounced adipogenic differentiation in 3D especially in the presence of exogenous collagen type I. In particular, PPARG gene expression was significantly increased ∼16-fold relative to chemical induction, in 3D and not in 2D. Unexpectedly, 3D engagement of ECM proteins also altered immunomodulatory function of MSCs in that expression of IL-6 gene was elevated relative to basal levels in 2D. In fact, levels of IL-6 gene expression in 3D composites containing exogenously supplied collagen type I or fibronectin were statistically similar to levels attained in 2D with tumor necrosis factor-α (TNF-α) stimulation and these levels were sustained over a 2-week period. Thus, this novel biomaterial platform allowed us to compare the biochemical impact of whole-molecule ECM proteins in 2D versus 3D indicating enhanced adipogenic differentiation and IL-6 expression of MSC in the 3D context. Exploiting the biochemical impact of ECM proteins on MSC differentiation and immunomodulation could augment the therapeutic utility of MSCs.
Highlights
Utilization of mesenchymal stem cells (MSCs) in the clinic and attempts to drive mesodermal differentiation of MSCs in vitro would benefit from enhanced understanding of the impact of 3D, exogenous extracellular matrix (ECM) on MSC state
Results Human MSC (hMSC) in ECM composites exhibit morphologies influenced by ECM engagement in 3D ECM proteins, provided exogenously at the time of composite formation, distinctively impacted the distribution and morphology of hMSCs in 3D
ECM proteins exogenously supplied in 3D composites were able to modulate hMSC-ECM engagement
Summary
Utilization of mesenchymal stem cells (MSCs) in the clinic and attempts to drive mesodermal differentiation of MSCs in vitro would benefit from enhanced understanding of the impact of 3D, exogenous extracellular matrix (ECM) on MSC state. MSCs are a heterogenous lot, primed to mature to several distinct mesodermal states or to remain multipotent depending on the local environment (e.g., native tissue, recipient tissue or tissue culture scenario). The local environment contains soluble signaling factors, other cells or cell types, and ECM, and the bulk of past literature reports pertain to soluble factor stimulation with respect to MSC state. An appreciation of the role of ECM in MSC specification has emerged including work of our laboratory in 2009 showing the presentation of ECM a Jangwook P.
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