Heterogeneous Differentiation of Human Mesenchymal Stem Cells in 3D Extracellular Matrix Composites

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.

Keywords: biomaterials; extracellular matrix; stem cells.

FIG. 1.

Schematic for production of extracellular matrix (ECM) composites with either collagen type I, laminin-111, or fibronectin and mesecnhymal stem cell (MSC) in four-armed poly(ethylene glycol) (PEG) hydrogels. ECM protein, MSC, and PEG-precursor are the core components of a 3D ECM composite in the article. PEG-precursors are functionalized with either cysteine (Cys) or thioester, forming native amide bond (CONH) (i.e., native chemical ligation). A representative z stack obtained from a collagen type I composite after 21 days of human MSC culture (right).

FIG. 2.

Morphology and engagement of human MSCs (hMSCs) with ECM of 3D composites over time. Multi-photon laser scanning microscopy (MPLSM) was used to optically section (at 1 μm interval) microenvironments of LN composites containing hMSCs to convey the three-dimensionality of the microenvironments within the composite (a–c, 161 planes). Z stacks of images were reconstructed from raw images (a, b) to solid objects using Imaris software to best view cell–ECM interactions (c); scale bar 80 μm. Single optical sections of ECM composites at days 1, 14, and 28 after composite formation. ColI (collagen type I) composites (d–f), LN (laminin-111) composites (g–i), and FN (fibronectin) composites (j–l). ECM proteins (green), nuclei (blue), and actin filaments (red); Scale bar 50 μm.

FIG. 3.

Multi-lineage differentiation of hMSCs on 2D ECM films and in 3D ECM composites. The expression of ACTC1 (cardiomyogenic), SPP1 (osteogenic), PPARG (adipogenic), and SOX9 (chondrogenic) on 2D ECM films was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Chemical induction (CI) without 2D ECM films, performed in parallel with cultures on 2D ECM films (a–d). The expression of ACTC1, SPP1, PPARG, and SOX9 in 3D composites was probed by qRT-PCR (e–h). The relative expression of each lineage marker was further normalized to the relative expression of composites containing hMSCs and PEG only at each corresponding time point (i–l). CI in 3D composite was performed in tissue culture flasks (e–h). mean ± SD, n = 3. ANOVA Tukey's HSD post hoc test, ##p < 0.01 and #p < 0.05. ANOVA Fisher's LSD post hoc or Student's t-test, **p < 0.01 and *p < 0.05. * or ** without brackets between the time points in the group.

FIG. 4.

Expression of FABP4 and PPAR-γ proteins in 3D ColI composites after 28 days of culture. Immunofluorescence was visualized by MPLSM. FABP4 (a–c) and PPAR-γ (d–f). Adipogenic protein markers (green) and nuclei (blue); scale bar 50 μm.

FIG. 5.

The expression of IL-6 and IDO genes by hMSCs was significantly upregulated both in 2D cytokine stimulation and in 3D ECM composites. Both indoleamine 2,3-dioxygenase (IDO) and interleukin 6 (IL-6) were significantly upregulated 24 h after IFN-γ (a) and TNF-α (b) stimulation in 2D cultures, respectively. IL-6 genes were distinctively upregulated in ECM-specific manner over 14 and 28 days in 2D and 3D culture, respectively (c, d), while IDO genes were undetectable. Mean ± SD, n = 3. Student's t-test or ANOVA Fisher's LSD post hoc test, **p < 0.01 and *p < 0.05. ANOVA Tukey's HSD post hoc test, ##p < 0.01. * or ** without brackets between the time points in the group.