Geometric cues for directing the differentiation of mesenchymal stem cells

Abstract

Significant efforts have been directed to understanding the factors that influence the lineage commitment of stem cells. This paper demonstrates that cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature-and with each occupying the same area-display different adipogenesis and osteogenesis profiles. The results reveal that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. Taken together, this work points to the role that geometric shape cues can play in orchestrating the mechanochemical signals and paracrine/autocrine factors that can direct MSCs to appropriate fates.

Fig. 1.

(A) Percentage of cells captured on rectangles of varying aspect ratio differentiating to adipocyte or osteoblast lineage. (B) Percentage of cells differentiating to either lineage when captured on fivefold symmetric shapes. (C) Comparison of color intensity histograms generated by measuring the red and purple channels of cells in flower and star shapes (n = 393). The dotted line represents thresholds used to define lineage specification. The bar graph summarizes lineage assignment from these cells using this method (R = red channel, P = purple channel). Error bars are standard deviations from four separate experiments with over 200 cells per shape, p-value < 0.005. (Scale bar, 50 μm).

Fig. 2.

(A)-(C) Immunofluorescent images of cells in flower and star shapes stained for F-actin (green), vinculin (red) and nuclei (blue). (D) Immunoflourescent images of cells in flower and star shapes stained for myosin IIa. (E) Fluorescent heatmaps of > 80 cells stained for myosin IIa as a quantitative measure of contractility. (Scale bar, 20 μm).

Fig. 3.

(A) Percentage of cells differentiating to adipocytes or osteoblasts when cultured on patterns of the same shape, p-value < 0.01. (B)-(E) Effect on shape-promoted differentiation in the presence of cytoskeleton disruptors and (F) integrin blocking antibodies.

Fig. 4.

Hierarchical clustering of (A) osteogenic and adipogenic transcripts expressed in cells captured to both shapes and (B) MAP kinase and Wnt signaling transcripts: Left—cells in flower and star shapes cultured in standard growth media for 1 week. Right (blue outline)—after 1 week exposure to mixed adipogenic and osteogenic media. (C) Change in differentiation from control when exposed to MAPK inhibitors. (D) Change in differentiation of cells in the presence of Wnt antagonists. (E) Speculative pathway for shape directed differentiation of adherent MSCs.

Fig. 5.

(A) Myosin IIa immunofluorescent heat maps of > 80 cells cultured in circles or holly shapes. (B) Corresponding differentiation of cells in both shapes after exposure to mixed media, p-value < 0.005.