Control of stem cell fate and function by engineering physical microenvironments

Abstract

The phenotypic expression and function of stem cells are regulated by their integrated response to variable microenvironmental cues, including growth factors and cytokines, matrix-mediated signals, and cell–cell interactions. Recently, growing evidence suggests that matrix-mediated signals include mechanical stimuli such as strain, shear stress, substrate rigidity and topography, and these stimuli have a more profound impact on stem cell phenotypes than had previously been recognized, e.g. self-renewal and differentiation through the control of gene transcription and signaling pathways. Using a variety of cell culture models enabled by micro and nanoscale technologies, we are beginning to systematically and quantitatively investigate the integrated response of cells to combinations of relevant mechanobiological stimuli. This paper reviews recent advances in engineering physical stimuli for stem cell mechanobiology and discusses how micro- and nanoscale engineered platforms can be used to control stem cell niche environments and regulate stem cell fate and function.

Figure 1

Schematic diagram of biomechanical regulation of stem cell behaviors. Mechanical stimuli such as mechanical strain, substrate stiffness, shear stress and topography affect on stem cell phenotypes in a combinatorial fashion.

Figure 2

Representative examples of biomechanical regulation of stem cell behaviors.(A) Actin filaments (green) and vWF (red) in the absence and presence of shear, respectively. Arrows indicate direction of flow; Scale bars = 50 μm. Reprinted with permission from John Wiley and Sons. (B) Immunofluorescent staining of MAP2 and nestin of hMSCs cultured on nano-patterned, and unpatterned PDMS. Nestin, the neuronal differentiation marker is shown in red, MAP2 in green. The direction of the gratings on the nano-patterned PDMS is indicated with a white arrow. Reprinted with permission from Elsevier Publishing. (C) Immunohistochemical assessment of gels either seeded with bovine bone marrow-derived progenitor cells. Mechanically stimulated bovine gels (Left) compared with static controls (Right) cultured for 21 days showed stimulated cells containedordered collagen type I fiber bundles (arrowheads) in the direction of load, i.e., along the longitudinal axis of the ligaments (double-arrow); Static controls exhibited absence of fuber bundle organization. Reprinted with permission from FASEB. (D) The response of human fibroblasts to rigid (Left; Young’s modulus (E)=100 kPa) or soft (Right; E=10 kPa) fibronectin-coated PDMS substrates. Organization of paxillin-GFP-labelled focal adhesions (green) and phalloidin-labelled filamentous actin (red), as well as overall cell shape, strongly differ in cells that are plated onto the two substrates. Reprinted with permission from Nature Publishing Company.

Figure 3

Effects of substrate stiffness on stem cell phenotypes. (A) Solid tissues exhibit a range of stiffness, as measured by the elastic modulus, E. (B) The in vitro gel system allows for control of E through crosslinking, control of cell adhesion by covalent attachment of collagen-I, and control of thickness, h. Naive MSCs of a standard expression phenotype are initially small and round but develop increasingly branched, spindle, or polygonal shapes when grown on matrices respectively in the range typical of brain (0.1–1 kPa), muscle (8–17 kPa), or stiff crosslinked-collagen matrices (25–40 kPa). Scale bar =20 μm. A, B reprinted with permission from Elsevier. (C) Substrate stiffness influences adhesion structures and dynamics, cytoskeleton assembly and cell spreading, and differentiation processes such as striation of myotubes. A cell-on-cell layering demonstration; Lower layer is attached first to glass followed by the upper layer, derived from myoblasts that are added later allowing myoblasts to perceive a softer, cellular surface.

Figure 4

Influences of substrate topographical cues on stem cell phenotypes. (A) Top row shows images of nanotopographical geometries fabricated by electron beam lithography. All have 120-nm-diameter pits (100 nm deep, absolute or average 300 nm centre–centre spacing) with hexagonal (2), square (3), displaced square (4)(50 nm from true centre) and random placements (5). Osteoprogenitors cultured on the control (1) exhibit lack of positive OPN and OCN stain; on hexagonal arrangment (2) show loss of cell adhesion; and on square (3) show decreased cell adhesion but increase in OPN and OCN stain; on displaced square geometry (4) show bone nodule formation; and on random arrangement (5) an OCN and OPN positive cell subpopulations. Actin=red, OPN/OCN=green. Reprinted with permission from Nature Publishing Group. (B) Immunofluorescent images of OPN (Right), as well as actin (Left) on 100-nm diameter TiO2 nanotubes after 3 weeks of culture. (C) Schematic illustration of the overall trends of nano cue effects on hMSC fate and morphology after a 24-h culture. The change in hMSC cell adhesion and growth without differentiation (solid red line) has the same trend as protein particle density (broken red line), whereas that of differentiation (solid blue line) has the same trend as hMSC elongation (broken blue line). Reprinted with permission from National Academy of Sciences.