Cell Behavioral Dynamics in Pluripotent Stem Cells Bioprocessing

Cell Behavioral Dynamics in Pluripotent Stem Cells Bioprocessing: Comparison

Please note this is a comparison between Version 1 by Naruchit Thanuthanakhun and Version 2 by Vivi Li.

Pluripotent stem cells (PSCs) are important for future regenerative medicine therapies. However, in the production of PSCs and derivatives, the control of culture-induced fluctuations in the outcome of cell quality remains challenging. A detailed mechanistic understanding of how PSC behaviors are altered in response to biomechanical microenvironments within a culture is necessary for rational bioprocessing optimization.

pluripotent stem cells
cell behaviors
culture microenvironments
stem cell bioprocessing
Waddington’s epigenetic landscape

1. Introduction

Pluripotent stem cells (PSCs), which can be isolated from embryos as embryonic stem cells (ESCs) or reprogrammed from somatic cells as induced pluripotent stem cells (iPSCs), are remarkable for their unlimited self-renewal in vitro and ability to differentiate into all cell types of the three embryonic germ layers, including the ectoderm, mesoderm, and endoderm [1]. These cellular capacities make them highly attractive candidates for regenerative medicine. They have been investigated for various clinical complications associated with globally high morbidity rates, including age-related macular degeneration, neurological disorders, and type 1 diabetes ^[2][3][4][2,3,4]. Despite an extensive exploration of the therapeutic potential of PSCs, there remain challenges toward real-world implementation of these cells in terms of cell bioprocessing and manufacturability.

Cell-based therapies require a high cell density for downstream differentiation and transplantation. Therefore, the development and optimization of robust, scalable, and cost-effective bioprocessing systems are needed to provide an adequate quantity of cells with stable quality ^[5][6][5,6]. Different cell culture systems have emerged for propagating and differentiating PSCs to generate various target cell types. However, the cell variability and suboptimal quality observed within and between batches reflect the existence of fluctuation in the bioprocessing of PSCs (Figure 1) ^{[7][8][9][10]}[7,8,9,10]. Typically, the variation in cell quality has been identified to be dependent on the cell origin (origin-induced) and the culture environment (culture-induced) [11]. During in vitro culture adaptation, PSCs accumulate epigenetic changes and potentially undergo phenotypic transition, influencing their clonal self-renewal and lineage differentiation propensity ^[9][12][13][9,12,13]. It has been shown that variable input factors applied to the culture process, including raw culture materials, culture conditions, and operational parameters, determine the actual microenvironments of the growing cells and how they behave in culture [14]. The dynamic alteration of the cell–microenvironment interactions can profoundly impact many intracellular biological events and cell pluripotency features ^[10][15][16][10,15,16]. Previously, cellular characteristics and functional variation were investigated among culture platforms. The growth-dependent self-organization within cell colonies under two-dimensional (2D) culture conditions is associated with inter- and intra-colony heterogeneity and spatial differences in the transcription of genes involved in cell cycle regulation, pluripotency, and epithelial function ^[17][18][19][17,18,19]. The distinct patterns of cell behaviors and growth phase transition during expansion have been further linked with the subsequent preferential differentiation ^[18][20][18,20]. Moreover, in a three-dimensional (3D) aggregate culture system, the growth and survival of PSCs, as well as the cell aggregate structural integrity, are variable, depending on their intercellular interactions and endogenous extracellular matrix (ECM) secretion capability ^[21][22][23][21,22,23]. After prolonged expansion, the increasing local heterogeneity in ECM accumulation and cytoskeletal alignment, in concert with the uneven distribution of nutrients and essential biochemicals, likely exacerbates growth limitation and spontaneous cell differentiation within 3D cell aggregates ^[22][24][25][22,24,25]. These prior studies demonstrated that multifactorial variables over courses of culture processes play a role in modulating cell behaviors and regulating the PSC properties and potential. From the perspective of bioprocess engineering, how to efficiently stabilize this fluctuation during PSC culture has become a key challenge in boosting process performance and final cell product quality.

Figure 1. Culture-induced fluctuation in the cell preparation bioprocess. The expansion process contributes to producing an adequate quantity of undifferentiated cells with stable quality for further differentiation and clinical translation. The existence of cell quality variability has been recognized to be influenced by intrinsic cellular properties and extrinsic variables in the cell culture, including raw material adaptability, culture conditions, and the operator’s skills and sense. Differences in the culture inputs and spatiotemporal changes in the cell structural organization during culture determine the biomechanical properties of the cellular microenvironments. The interactions between cells and their microenvironments may result in fluctuations in the phenotypic features of the PSC products.

2. Mechanistic Roles of Cell Behavioral Dynamics in Modulating Cell Fate Decision

Recent evidence has indicated that complex reciprocity between cells and culture microenvironments produces dynamic changes in cell behaviors and intracellular mechanics, contributing to the spatiotemporal variations in cell state and functional regulation within culture ^{[26][27][28][29]}[26,27,28,29]. In addition to biochemical cues, cultured cells continually perceive extrinsic stimuli through direct interactions with their surroundings and relay them intracellularly to regulate the multitudes of molecular pathways and gene transcriptional networks. This process is called mechanotransduction (Figure 2) ^[28][30][28,30]. At the cell surface, the anchorage of cells to the local ECM components and the neighboring cells, mediated by specialized adhesion molecules, such as integrin and cadherin, conjointly exerts a mechanosensation role and coordinates cell migration ^[31][32][33][31,32,33]. The strength of the cell adhesion interactions depends on the magnitude of mechanical constraints sensed by the cells, eliciting a graded mechanosignaling output from the adhesion sites ^[34][35][34,35].

Figure 2. 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.

The actomyosin-induced contraction propagates mechanical cues to the nucleus, which are directly transmitted to the chromatin domains via a linker of nucleoskeleton and cytoskeleton (LINC) complexes and further modulates the cytoplasmic-to-nuclear translocation of epigenetic modifiers and transcription regulators ^[27][44][45][27,44,45]. The direct tethering of actomyosin to the nucleoskeleton and associated chromatin influences the dynamics of nuclear deformation and nuclear lamina–genome interactions based on increased or decreased actomyosin activities [46]. Studies demonstrated that actomyosin-induced nuclear flattening and chromatin condensation in the mechanical stress-applied cells involve the alteration of histone methylation and the transcriptional upregulation of mechanoresponsive and quiescent genes ^[27][47][48][27,47,48].

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][49][50][51][52]}[35,67,79,80,81]. 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][53][35,82]. Differences in the biochemical composition, molecular structure, and mechanical characteristics of culture matrices strongly influence cell behaviors and pluripotent capacity ^[54][53]. 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 ^[54][55][56][53,83,84]. 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 ^[55][83]. 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 ^[54][53]. 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][50][57][58][59][60]}[35,79,85,86,87,88]. 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 ^[61][89]. 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 ^[62][90]. Recent research on synthetic hydrogel systems has introduced a concept of cell behavioral control through the in situ modifying of structural and adhesive microenvironments ^[63][91]. Combined hydrogel matrices have been optimized to switch between pluripotency-permissive and differentiation-permissive states via ionic de-cross-linking ^[63][91]. Interestingly, controlling the timing of matrix switching can regulate the ESCs to differentiate into ectoderm or mesendoderm lineages ^[63][91]. These culture matrices have been used successfully to generate an integrated platform for growing undifferentiated ESCs and subsequently differentiating them into terminally specialized cells ^[63][91]. 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 ^[64][78]. 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 ^[64][78]. The exogenous regulation of integrin- or E-cadherin-mediated adhesion can attune the properties and functions of cultured PSCs ^[27][31][65][27,31,92]. 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 ^[66][93]. 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 ^[66][67][93,94]. 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 ^[49][67]. 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 ^[49][67]. Moreover, routine HA treatment in serial passages has been shown to facilitate the long-term maintenance of the iPSC population in an undifferentiated state ^[52][81]. 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 ^[52][81]. 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 ^[68][95]. 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 ^[68][95]. 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.