PMC

Macromolecular crowding (MMC) increases the accumulation of lipid droplets of mesenchymal stem cells (MSCs) during adipogenic differentiation for 21 days. (A) Quantitative analysis of the fluorescence area normalized to cell number showed significantly more lipid storage when MSCs are adipogenically induced +MMC. Scale bar: 500 μm. (B) Flow cytometry gating shows no significant difference between percentage of lipid droplet–laden cells in conventionally induced (69.7%±4.4%) and induced +MMC (58.9%±0.2%) MSCs. This run was performed in duplicate. Student's t-test *p<0.05, **p<0.01 and ***p<0.001. Color images available online at www.liebertpub.com/tea

MMC up-regulates the expression of early to late adipogenic genes of MSCs undergoing adipogenesis for 21 days. mRNA analyses demonstrated commitment of MSCs to adipogenic lineage, with induced +MMC expressing differentially up-regulated gene response throughout the entire differentiation period. In particular, PPARγ, the master regulator of adipogenic differentiation, shows an increased expression under induced +MMC condition as early as day 4. Late adipogenic genes GLUT4, FABP4, and LEP followed with significant up-regulation under induced +MMC conditions at day 21. PPARγ, peroxisome proliferator-activated receptor gamma; GLUT4, glucose transporter type 4; FABP4, fatty acid binding protein 4; LEP, leptin. ^‡Borderline significance at p=0.05. *p<0.05, **p<0.01 and ***p<0.001.

Enhanced presence of MMP-2 in cell layer of adipogenically induced MSCs and enhanced collagenase activity in the presence of MMC. (A) Gelatin zymography of MSCs undergoing adipogenic induction at various time points to assess amount of MMP evident as white bands of size ∼60–65 kDa. At day 7, MMC-induced samples had a significant 29% increase in the amount of MMP in comparison to conventionally induced cells. (B) Amount of MMP as a percentage of day 0 quantified by densitometric quantitation of zymographic bands adjusted for cell number. (C) Enzyme activity assay of bacterial collagenase type IV. In the presence of MMC, digestion velocity is increased by 22%. *p<0.05.

MMC induces pronounced changes of various extracellular matrix (ECM) components en route to an adipogenic matrix during 21 days of adipogenic differentiation of MSCs. Representative immunocytochemistry images throughout the 21-day period showed an increase in deposition of (A) Col IV and (B) heparin sulfate proteoglycan (HSPG), with this increase visibly more enhanced in induced +MMC conditions. On the other hand, (C) under induced +MMC condition, reduction in fibronectin (FN) deposition was observed on d21, which correlated with the literature demonstrating FN reduction during adipogenesis. Corresponding quantitative analyses show fluorescence area normalized to cell number. Scale bar: 200 μm. *p<0.05, **p<0.01 and ***p<0.001. Color images available online at www.liebertpub.com/tea

Enhanced state of deposition and remodeling of adipogenic ECM under MMC after 21 days of adipogenic differentiation. Representative immunocytochemistry images of (A) Col IV, (B) HSPG, (C) Col I, and (D) FN of MSCs that underwent adipogenic differentiation±MMC compared to nondifferentiated (-induction) controls. Induced +MMC conditions showed the stoichiometric increase in Col IV and HSPG with the concomitant decrease of Col I and FN compared with its noninduced controls [gray bars vs. white bars of (+MMC group)]. The dynamic remodeling of the various matrix proteins from a fibrillar to a recticular conformation as a result of adipogenic differentiation (left images to right images) was more evident under +MMC conditions (bottom panel). Corresponding quantitative analyses show fluorescence area normalized to cell number. Scale bar: 200 μm. *p<0.05, **p<0.01 and ***p<0.001. Color images available online at www.liebertpub.com/tea

Schematic representation of adipogenic cell-matrix reciprocity in MSCs as driven by a microenvironment enhanced by MMC. MMC increases ECM deposition by spindle-shaped undifferentiated MSCs, including collagen I, IV, fibronectin, and heparan sulfate proteoglycans (HSPG), here perlecan. Collagen IV, a major component of the basement membrane, is predominant in the matrix of adipose tissue, providing structural support to lipid-laden adipocytes. As differentiation ensues, the geometry and composition of these matrices are remodeled, which is mirrored by a rounded cell morphology. Increased presence of HSPG in the adipocyte matrix suggests increased sequestration of adipogenically relevant growth factors such as fibroblast growth factor-2 (FGF-2), which are secreted by MSC in an autocrine fashion. This results in a positive feedback loop (as shown by the arrows) guiding differentiating MSCs deeper into adipogenic maturation. Color images available online at www.liebertpub.com/tea

Adipocyte-derived ECM generated under MMC substantially augments chemically induced adipogenesis. Undifferentiated MSCs were seeded on decellularized ECM generated by undifferentiated MSCs (MSC-ECM) and adipogenically differentiated MSCs (Adipo-ECM)±MMC and were chemically induced into adipogenesis for 21 days. Lipid droplets were then stained with Nile Red. (A) Fluorescent images show most lipid droplet accumulation in cells differentiated on Adipo-ECM (+MMC). Scale bar: 200 μm. (B) Quantitation of fluorescent lipid area normalized to cell number demonstrated a 60% increase in lipid droplet accumulation for MSCs differentiated on Adipo-ECM (+MMC) compared with 10% on Adipo-ECM matrices relative to TCPS controls. In contrast, decellularized naïve-state MSC-ECM (±MMC) led to a substantial reduction of lipid droplet accumulation representing 50% of that seen on TCPS controls. (C) This was also reflected by relative mRNA expression levels for PPARγ showing a similar increase for MSCs seeded on Adipo-ECM (+MMC). (D) Without chemical induction, re-seeded naïve-state MSCs did not exhibit significant lipid droplet accumulation after 21 days except for those seeded on Adipo-ECM (+MMC), which exhibited a five-fold increase in lipid droplet accumulation suggesting spontaneous adipogenic differentiation. *p<0.05, **p<0.01 and ***p<0.001. Color images available online at www.liebertpub.com/tea

Collagen IV is remodeled extensively from a fibrillar to a reticular honeycomb structure in adipogenically induced MSCs under MMC. (A) Rose plots of Col IV directional alignment after 21 days of adipogenic differentiation. Inserts show corresponding Col IV images. (i) & (ii) For non-induced conditions, rose plots exhibited a smaller range of angles with smaller amplitudes, corresponding to a more fibrillar network. When adipogenically induced (iii), a shift toward a fan-shaped pattern indicated a transition to a reticular network, (iv) but was more pronounced under +MMC with a larger amplitude. (B) Similarly, rose plots during the course of adipogenic differentiation reveal that (i) both range of angles and amplitude increased over time, and (ii) the onset of remodeling occurred earlier (day 7) for the induced +MMC condition. (C) (i) Space analysis of Col IV at day 21 reveals an increased number of spaces under induced conditions, and enhanced with MMC (gray bars), indicative of the development of a reticular ECM meshwork. (ii) In addition, the average number of spaces during adipogenesis exhibited parallel dynamics seen in ; with a faster and steeper increase under +MMC conditions. *p<0.05, **p<0.01 and ***p<0.001. Color images available online at www.liebertpub.com/tea