Biomechanical, biophysical and biochemical modulators of cytoskeletal remodelling and emergent stem cell lineage commitment

Abstract

Across complex, multi-time and -length scale biological systems, redundancy confers robustness and resilience, enabling adaptation and increasing survival under dynamic environmental conditions; this review addresses ubiquitous effects of cytoskeletal remodelling, triggered by biomechanical, biophysical and biochemical cues, on stem cell mechanoadaptation and emergent lineage commitment. The cytoskeleton provides an adaptive structural scaffold to the cell, regulating the emergence of stem cell structure-function relationships during tissue neogenesis, both in prenatal development as well as postnatal healing. Identification and mapping of the mechanical cues conducive to cytoskeletal remodelling and cell adaptation may help to establish environmental contexts that can be used prospectively as translational design specifications to target tissue neogenesis for regenerative medicine. In this review, we summarize findings on cytoskeletal remodelling in the context of tissue neogenesis during early development and postnatal healing, and its relevance in guiding lineage commitment for targeted tissue regeneration. We highlight how cytoskeleton-targeting chemical agents modulate stem cell differentiation and govern responses to mechanical cues in stem cells' emerging form and function. We further review methods for spatiotemporal visualization and measurement of cytoskeletal remodelling, as well as its effects on the mechanical properties of cells, as a function of adaptation. Research in these areas may facilitate translation of stem cells' own healing potential and improve the design of materials, therapies, and devices for regenerative medicine.

Fig. 1. The mechanome, a map of volume (dilational) and shape (deviatoric) changing stresses, relates to exposure to biochemical induction factors, and exposure time, and to lineage commitment (as depicted by data point shape) of stem cells.

During prenatal development and postnatal healing, stem cell differentiation and tissue neogenesis are inextricably tied to the mechanadaptation of stem cells, which can be observed in the remodeling of the actin and microtubule cytoskeleton. a The actin and microtubule cytoskeleton remodel in response to the spatiotemporal presentation of biophysical and biochemical cues within the tissue. The transition between epithelial (sheet-like) and mesenchymal (globular) tissue templates is itself mechanically modulated and enables the growth and specialization of tissue structure and function (adapted with permission from ref. ). b Cytoskeletal remodeling underpins the epithelial-to-mesenchymal transition (EMT), including the formation of lamellipodia and the rapid polymerization of parallel stress fibers, resulting in front–back polarity of the mesenchymal cell, itself a mechanoadaption. The remodeling is necessary to drive EMT from tensional balance between epithelial cells where adhesion force equals contractile force, to higher contractility at the interface of ingressing and non-ingressing cells. c The mechanome map of the cells’ actual stress–strain data upon introduction of mechanical and biochemical cues and the corresponding fates. Cells experience intrinsic mechanical cues during development such as volume-changing (dilatational) stress (red plane, x axis) (i.e., hydrostatic compression and tension [log₁₀ Pa]) and shape-changing shear stress (blue plane, y axis) (shear stress magnitudes [dyn/cm² or 0.1 Pa], that dictate their lineage commitment over time (z axis) (adapted with permission from ref. , ref. ). Stem cells’ emergent mechano- and differentiation responses, presented as different shape data points, demonstrate the spatiotemporal adaptation to the magnitude of mechanical cues and the interplay with biochemical cues (i.e., induction medium, indicated by orange filled data point shapes). The yellow plane with opaque to transparent gradient represent the ranges of stress predicted during early development in utero^,,,,,,,–.

Fig. 2. The remodeling of actin and microtubules due to tensional cues delivered as stretch or barriers to stretch via substrate rigidity, and their multiscale translation to the structural and mechanical properties of cells and tissues.

Culture of tissue on soft substrates (a) reduces the intrinsic tension of stress fibers (SF) and thus increases cell relaxation (b); this in turn permits cofilin binding to actin and promotes actin disassembly or severing of actin (c). Cofilin binding increases the rate of actin subunit loss from the pointed end of actin filament, releases profilin from the filament and increases the number of free barbed ends where subunits can be added. The growing ends of microtubules sense the periphery of cell and thus maintain filament growth. Microtubules do not necessarily target focal adhesion, but their end-binding protein (EB1) forms complexes with microtubule actin cross-linking factor 1, MACF1, that is enriched at focal adhesion complex. Increased tension due to culture on stiff substrates (d) increases intrinsic tensile stress of the cell, and thus actin reinforcement (e). Tension enhances actin polymerization as it recruits profilin and α-actinin actin polymerization factors (f). The formin FH2 domain-bound barbed end of actin enhances the rapid addition of actin subunit-bound profilin via interaction with the formin FH1 domain. SF localization provides templates for focal adhesion maturation, reduces the pool of free G-actin monomers, and inhibits binding of cofilin. ROCK and Rho signaling facilitates GTP to GDP exchange for actin polymerization. Actin forms thicker SF bundles via complexes with myosin and α-actinin to adapt to increasing tension. Tension stabilizes microtubule structure and promotes alignment with actin in the direction of tension. Microtubule growth facilitates SF assembly and elongation via transport of actin polymerase proteins.

Fig. 3. The unique remodeling of the cytoskeleton represents the adaptiveness of stem cells to their environment, shaping their differentiation responses.

Similar to tracking of genetic markers that provide a fingerprint in time for processes of stem cell differentiation^,, remodeling of actin (green) (a) and tubulin (red) cytoskeleton (b) correlates to incipient differentiation of mesenchymal stem cells (hMSCs). Regulation of actin and tubulin de-/polymerization results in specific spatiotemporal patterns of their fibers (shape, size, distribution, organization), reflecting the cells’ adaptation of structure and function to their prevailing mechanical and biochemical milieux (refs. ^{; ; ; ; ;} ; and ). In turn, changes in gene regulation and cytoskeletal remodeling scale up to create spatiotemporal patterns of tissues with specific architectures and functions.

Fig. 4. Multiscale mechanoadaptation as a rationale for design of engineering tissue templates, materials or devices for regenerative medicine and for physical therapy protocols in the context of cytoskeletal remodeling.

Transfer of forces  experienced by an organism to its constituent organs, tissues, and cells (left, top to bottom) drives the multiscale structure–function adaptation of the cytoskeleton, cells, tissues and organs, to the dynamic mechanical environment of the organism, which is essential to healing (right, bottom to top). At the molecular level, focal adhesion complexes mediate outside-in signaling and mechanically link tissue rigidity to cytoskeleton dynamics, regulating cytoskeletal interactions with adapter proteins, and force transduction to the nucleus. The mechanical signals, transduced as actin and microtubule dynamics, determine cell shape and force generation, resulting in tension equilibrium at tissue and other length scales (adapted with permission from ref. ).

Fig. 5. Quantitative imaging protocols for monitoring spatiotemporal changes in cytoskeletal structure during stem cell adaptation to cues driving proliferation and differentiation.

Live cell, fixed cell, and combined imaging methods have proven to be useful in monitoring cytoskeletal changes indicative of lineage commitment as a function of time, with time resolution from within minutes to hours (live cell imaging), and or days, e.g. 24 h (fixed cell imaging). a Dynamic changes in actin and tubulin cytoskeleton (quantified as filament length (b) and volume (c)) are observed as early as 15 min during neural differentiation of induced pluripotent stem cells (iPSCs)-derived neurons via time-lapse imaging. Simultaneous imaging and introduction of volume-changing stress (seeding density) and shape-changing stress (flow)^, (d) enable elucidation of spatiotemporal cytoskeletal adaptation to controlled mechanical cues. Spatial distribution of actin (e) and tubulin (f) are measured at 30 min intervals as fluorescence intensity within the thickness of the cell and the total cell volume. g Combining live and fixed cell labeling and imaging of actin using fluorescent-conjugated SiR-actin (SA) and phalloidin, respectively, SiR-actin-based measurement of actin turnover (SMAT) analysis distinguishes MSC differentiation (h) toward adipogenic (red line), chondrogenic (green line), and osteogenic (blue line) lineages, at timepoints as early as 1 h. Reduction in probe intensity (i) is observable within a few hours of switching from adipogenic induction medium (AD) to basal medium (BA), as shown qualitatively (j). Actin cytoskeleton morphometric descriptors, including shapes, intensities, and spatial distribution, represent the apparent changes that are readily detected within 24 h of hMSC differentiation. k The lineage commitment propensity of hMSCs cultured in respective differentiation induction media on various substrates could be parsed using descriptor-based computational modeling. The resulting confocal images are processed using Gaussian filter, enhanced and segmented for each single cell (l) to generate 43 descriptors (m). n Multidimensional scaling reduces the combination of descriptors into 3D space in a nonlinear fashion. Scatter plots in this 3D space show clear, time-dependent segmentation of adipogenic and osteogenic differentiation on non-treated glass (o–r), which can be observed after 72 h (q). Microtubules mediate nuclear deformations, invagination, volume increase (s, t) through imposing constraints on the swelling nucleus during hPSC early differentiation into myeloid progenitors. This can be monitored within 24–72 h and quantified via 3D reconstruction of confocal images.