The plausible interplay between cell behavioral dynamics and intracellular mechanical regulation in cell fate modulation. In the culture microenvironments, cells sense and respond to extrinsic mechanical stimuli from the culture substrate, surrounding cells, and fluid dynamics exerted on cellular membranes and different biomechanosensors, such as integrins and E-cadherins. The alteration of the integrin-mediated cell–substrate and E-cadherin-mediated cell–cell adhesion behaviors triggered by the extrinsic forces can stimulate a wide range of intracellular biochemical signaling cascades and induce intrinsic force generation by regulating the actomyosin cytoskeletal contractility. The distribution of the intrinsic cytoskeletal tension mediates the nuclear structural remodeling and cytoplasmic-nuclear shuttling of several mechanosensitive transcription regulators, influencing the intranuclear events, such as epigenetic modifications and gene transcriptional activity, and consequently altering cell fates and functions.
The binding of cell adhesion molecules promotes the recruitment of multiple structural and signaling proteins at the cytoplasmic domains. The association of adhesion protein complexes primarily facilitates a direct force transmission from adhesion contacts towards internal cell compartments and nuclei via physical cytoskeletal connections
[36,37][36][37]. Moreover, the exerted forces on cell adhesion can be converted into biochemical signals by altering the compositions and activities of regulatory proteins at the adhesion complexes, which generates a sequence of signaling pathways and changes in the cytoskeletal alignment and tensional dynamics
[38,39][38][39]. The adhesion-mediated activation of Rho A, a member of the Rho GTPase family, and ROCK signaling promotes the phosphorylation of motor protein myosin II and inhibits the activity of myosin II phosphatase
[40,41][40][41]. The kinetics of the assembly between the phosphorylated myosin II and filamentous actin termed actomyosin complex produces alternating cycles of actin cyto-skeletal contraction and relaxation
[42]. Active Rho protein at cell–cell adherens junctions produces signals through its effectors to establish apical actomyosin networks
[17,38][17][38]. Whereas the localization of p120-catenin elicits the recruitment and activation of other Rho GTPases, Rac and Cdc42, inducing actin polymerization and suppressing the Rho/ROCK-dependent actomyosin contractility
[43]. The antagonism of the Rho GTPase members in controlling contraction or elongation of actin bundles serves as a molecular switch to manipulate the balance between cell adhesive and migratory behaviors and direct the intra- and intercellular tensional homeostasis.
3. Emerging Methods for Enhancing PSC Expansion through the Regulation of Cell Behaviors
Spatiotemporal differences in cellular microenvironments and structural self-organization along the culture of PSCs potentially contribute to cell-to-cell phenotypic variability
[14,16][14][16]. Fine-tuning three key components of cell mechanical transducers at the cell-microenvironment interface (cell–substrate interaction, cell–cell interaction, and cell migration) by applying alternative culture substrates and biochemical molecules has been recently introduced by several groups
[35,67,79,80,81][35][49][50][51][52]. The interaction of PSCs with their surrounding ECM plays a role in coordinating the balances of force generation at the cell-ECM contacts and the overall strength of the intracellular and intercellular contraction
[27]. Recent advancements in the field of biomaterials provide a wide range of surface coating agents, including recombinant ECM proteins and synthetic biomimetic matrices
[35,82][35][53]. Differences in the biochemical composition, molecular structure, and mechanical characteristics of culture matrices strongly influence cell behaviors and pluripotent capacity
[53][54]. The distinct structural isoforms of the ECM adhesion molecules, such as laminin-511, -521, -332, -211, and -111, have been shown to affect the efficiency of proliferation and differentiation of PSCs
[53,83,84][54][55][56]. Previous research demonstrated that E8 fragments of laminin-511 and -332, which are the minimal forms retaining the integrin-binding specificity, successfully maintained iPSCs in an undifferentiated state with a normal karyotype and pluripotency for more than 30 passages
[83][55]. However, differences in the binding affinity to E8 fragments of laminin-511, -332, and -211 determine the degree of cell colony compaction and actomyosin contractility, consequently switching the differentiation propensity of cultured iPSCs towards distinct ocular lineages involving Wnt and YAP signaling modulation
[53][54].
The availability of synthetic polymer- and peptide-based matrices with tunable mechanical properties allows users to regulate an optimal strength of cell–substrate adhesion in a target cell-specific manner, thereby promoting an efficient generation of desired cells
[35,79,85,86,87,88][35][50][57][58][59][60]. Stiffness represents a key mechanical property of coating material, critically dictating the subcellular allocation and activity of the integrin-mediated adhesion molecules, and further modifying the cell interactions with neighboring cells
[89][61]. Cultivating ESCs on tunable decellularized fibroblast-derived matrices indicated that the extent of substrate stiffness modulates their cell–substrate adhesive potential and cell motility, mediating either induction or inhibition of the epithelial to mesenchymal transition program and controlling the activity of pluripotent gene expression
[28]. The ranges of optimal stiffness should be considered when developing culture matrices to facilitate pluripotency maintenance and long-term cell expansion
[90][62]. Recent research on synthetic hydrogel systems has introduced a concept of cell behavioral control through the in situ modifying of structural and adhesive microenvironments
[91][63]. Combined hydrogel matrices have been optimized to switch between pluripotency-permissive and differentiation-permissive states via ionic de-cross-linking
[91][63]. Interestingly, controlling the timing of matrix switching can regulate the ESCs to differentiate into ectoderm or mesendoderm lineages
[91][63]. These culture matrices have been used successfully to generate an integrated platform for growing undifferentiated ESCs and subsequently differentiating them into terminally specialized cells
[91][63]. Additionally, hydrogel-based matrices have been applied to fabricate labile substrates with patterned islands, which restrict the cell–substrate adhesion to designated areas and induce the self-assembly cell aggregation for producing size- and shape-controlled 3D cell aggregates
[78][64]. Regulating the aggregation kinetics by adjusting the labile substrate ligand density allows for the controlling of the porous structure of cell aggregates and indirectly determining stem cell fate
[78][64].
The exogenous regulation of integrin- or E-cadherin-mediated adhesion can attune the properties and functions of cultured PSCs
[27,31,92][27][31][65]. Applying a uniform mode of integrin- or E-cadherin-based adhesion regulation attenuates the spatial cell heterogeneity in culture
[31]. A non-colony culture system based on recombinant E-cadherin-immobilized surfaces has been proposed to grow undifferentiated PSCs in a more homogeneous microenvironments with moderation of cell–cell contacts
[93][66]. The cultures of ESCs and iPSCs on an E-cadherin-coated substrate, which retain their E-cadherin-based interaction, have been found to increase cell proliferation and maintain cell viability and pluripotency during subculture
[93,94][66][67]. Furthermore, to modulate the cell–cell interaction, the E-cadherin function-blocking agent botulinum hemagglutinin (HA) has been used as a culture tool to selectively remove cells that deviate from an undifferentiated state during the expansion of iPSCs
[67][49]. Due to the weakened E-cadherin-mediated cell–cell adhesion in deviated cells, HA-induced E-cadherin disruption causes the detachment of deviated cells from cell colonies; however, the undifferentiated cells can restore the E-cadherin-mediated cell–cell interaction and retain their pluripotency following HA removal
[67][49]. Moreover, routine HA treatment in serial passages has been shown to facilitate the long-term maintenance of the iPSC population in an undifferentiated state
[81][52]. It has been suggested that the temporal relaxation of cell–cell junctions by HA can stimulate cell migratory behaviors and cytoskeletal rearrangement, resulting in a relatively uniform dispersion of cells in colonies
[81][52]. In addition, the HA-mediated temporal cell–cell adhesion disruption has been adopted to establish an in situ cell aggregate break-up method for high-density suspension expansion
[95][68]. In cell aggregate growth in conventional culture, large-size aggregates enhance collagen type I accumulation on the aggregate periphery, restricting the homogeneous microenvironments and consequently resulting in undesirable cell proliferation and cell necrosis within the aggregate. The HA-mediated dissociation of cell–cell adhesion facilitates the break-up of aggregates into small sizes, allowing a significant increase in the expansion fold of cells with no adverse effect on maintaining pluripotency
[95][68]. These studies represent current progress in tailoring cell behaviors in PSC cultures. The use of emerging culture strategies that integrate the precise control of culture microenvironments and cell behavioral dynamics may ultimately contribute to regulating the maintenance of undifferentiated state and pluripotent ability of cultured PSCs along the expansion process.