Novel strategy to improve hepatocyte differentiation stability through synchronized behavior-driven mechanical memory of iPSCs.

Cellular homeostasis is assumed to be regulated by the coordination of dynamic behaviors. Lack of efficient methods for synchronizing large quantities of cells makes studying cell culture strategies for bioprocess development challenging. Here, we demonstrate a novel application of botulinum hemagglutinin (HA), an E-cadherin function-blocking agent, to synchronize behavior-driven mechanical memory in human induced pluripotent stem cell (hiPSC) cultures. Application of HA to hiPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration-and time-dependent manner. Interestingly, cytoskeleton rearrangement in cells with prolonged exposure to HA resulted in mechanical memory synchronization with Yes-associated protein, which increased pluripotent cell homogeneity. Synchronized hiPSCs have higher capability to differentiate into functional hepatocytes than unsynchronized hiPSCs, resulting in improved efficiency and robustness of hepatocyte differentiation. Thus, our strategy for cell behavior synchronization before differentiation induction provides an approach against the instability of differentiation of pluripotent cells.

The impact of culture dimensionality on behavioral epigenetic memory contributing to pluripotent state of iPS cells.

Three-dimensional (3D) culture platforms have been explored to establish physiologically relevant cell culture environment and permit expansion scalability; however, little is known about the mechanisms underlying the regulation of pluripotency of human induced pluripotent stem cells (hiPSCs). This study elucidated epigenetic modifications contributing to pluripotency of hiPSCs in response to 3D culture. Unlike two-dimensional (2D) monolayer cultures, 3D cultured cells aggregated with each other to form ball-like aggregates. 2D cultured cells expressed elevated levels of Rac1 and RhoA; however, Rac1 level was significantly lower while RhoA level was persisted in 3D aggregates. Compared with 2D monolayers, the 3D aggregates also exhibited significantly lower myosin phosphorylation. Histone methylation analysis revealed remarkable H3K4me3 upregulation and H3K27me3 maintenance throughout the duration of 3D culture; in addition, we observed the existence of naïve pluripotency signatures in cells grown in 3D culture. These results demonstrated that hiPSCs adapted to 3D culture through alteration of the Rho-Rho kinase-phospho-myosin pathway, influencing the epigenetic modifications and transcriptional expression of pluripotency-associated factors. These results may help design culture environments for stable and high-quality hiPSCs.

Mechanobiological conceptual framework for assessing stem cell bioprocess effectiveness.

Fully realizing the enormous potential of stem cells requires developing efficient bioprocesses and optimizations founded in mechanobiological considerations. Here, we emphasize the importance of mechanotransduction as one of the governing principles of stem cell bioprocesses, underscoring the need to further explore the behavioral mechanisms involved in sensing mechanical cues and coordinating transcriptional responses. We identify the sources of intrinsic, extrinsic, and external noise in bioprocesses requiring further study, and discuss the criteria and indicators that may be used to assess and predict cell-to-cell variability resulting from environmental fluctuations. Specifically, we propose a conceptual framework to explain the impact of mechanical forces within the cellular environment, identify key cell state determinants in bioprocesses, and discuss downstream implementation challenges.

Designing a blueprint for next-generation stem cell bioprocessing development.

The creation of a blueprint for stem cell bioprocess development that it is easily readable and shareable among those involved in the construction of the bioprocess is a necessary step toward full-fledged bioprocess integration. The blueprint provides the culturing tools and methodologies, designed to highlight knowledge gaps within biological sciences and bioengineering. This review highlights a blueprint for stem cell bioprocessing development using a landscape architecture approach that can aid the development of culture technologies and tools that satisfy the demands for stem cell-derived products for use in clinical and industrial applications. This work is intended to provide insights to cell biologists, geneticists, bioengineers, and clinicians seeking knowledge outside of their field of expertise and fosters a leap from a reductionist approach to one, that is, globally integrated in stem cell bioprocessing.

Maintenance of human chondrogenic phenotype on a dendrimer-immobilized surface for an application of cell sheet engineering.

BACKGROUND: Dedifferentiation of chondrocytes during cell expansion is one of the barriers in tissue construction for cartilage repair. To understand chondrocyte behavior and improve cell expansion in monolayer culture, this study investigated the effects of morphological changes and cellular aggregation on the maintenance of chondrogenic capacity by observing the expression patterns of chondrogenic (collagen type II and aggrecan) and dedifferentiation (collagen type I) markers. Primary human chondrocytes were cultured on either a polystyrene surface (PS) or a polyamidoamine dendrimer surface with a fifth-generation (G5) dendron structure to create a one-step process of cell expansion and the maintenance of chondrogenic activities prior to the construction of cell sheets. RESULTS: During the first two passages (P0 - P2), the relative mRNA level of collagen type II decreased in all cultures, while that of collagen type I increased. Remarkably, the level of collagen type II was higher and aggrecan was retained in the chondrocytes, forming cell aggregates and showing some round-shaped cells with less production of stress fibers on the G5 surface compared to fibroblast-like chondrocytes with abundant stress fibers on the PS surface. The numbers of P2 chondrocytes on the G5 and PS surfaces were nearly the same and sufficient for construction of chondrocyte sheets using a temperature-responsive plate. Without a supporting material during cell sheet manipulation, chondrocyte sheets spontaneously detached and exhibited a honeycomb-like structure of stress fibers. Unlike the chondrocyte sheets constructed from cells on the PS surface, the chondrocyte sheets from cells on the G5 surface had higher chondrogenic activities, as evidenced by the high expression of chondrogenic markers and the low expression of dedifferentiation markers. CONCLUSIONS: The one-step process of cell expansion and maintenance of chondrogenic activity could be obtained using the G5 surface. Human chondrocyte sheets were successfully constructed with high chondrogenic activity. These findings may lead to an alternative cultivation technique for human chondrocytes that offers high clinical potential in autologous chondrocyte implantation.

Botulinum hemagglutinin-mediated in situ break-up of human induced pluripotent stem cell aggregates for high-density suspension culture.

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high-density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break-up using botulinum hemagglutinin (HA), which specifically bound with E-cadherin and disrupted cell-cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E-cadherin-mediated cell-cell connections to facilitate the break-up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 106 cells ml-1 was obtained by aggregate break-up into small ones, which was three times higher than that with the conventional culture without aggregate break-up. Therefore, the temporary activity of HA for disrupting E-cadherin-mediated cell-cell connection was key to establishing a simple in situ method for hiPSC aggregate break-up in bioreactors, leading to high cell density in suspension culture.

A Novel Strategy for Simple and Robust Expansion of Human Pluripotent Stem Cells Using Botulinum Hemagglutinin.

Clinical and industrial application of human pluripotent stem cells (hPSCs) has been hindered by the lack of robust strategies to sustain cultures in an undifferentiated state. Here, we describe a simple and robust method to culture and propagate hPSCs, which we anticipate will remove major roadblocks in investigating the basic properties of undifferentiated hPSCs and accelerate cell-based manufacturing. We also provide an overview of the use of botulinum hemagglutinin, an inhibitor of E-cadherin, to maintain and expand various hPSC lines in an undifferentiated state in different culture conditions. Hemagglutinin selectively removes cells that have lost the undifferentiated state, dissociates aggregates in situ, and is easy to use, scalable, and reproducible.

Chondrogenesis and hypertrophy in response to aggregate behaviors of human mesenchymal stem cells on a dendrimer-immobilized surface.

OBJECTIVES: To investigate the behaviors of aggregates of human mesenchymal stem cells (hMSCs) on chondrogenesis and chondrocyte hypertrophy using spatiotemporal expression patterns of chondrogenic (type II collagen) and hypertrophic (type X collagen) markers during chondrogenesis. RESULTS: hMSCs were cultured on either a polystyrene surface or polyamidoamine dendrimer surface with a fifth generation (G5) dendron structure in chondrogenic medium and growth medium. At day 7, cell aggregates without stress fibers formed on the G5 surface and triggered differentiation of hMSCs toward the chondrogenic fate, as indicated by type II collagen being observed while type X collagen was undetectable. In contrast, immunostaining of hMSCs cultured on polystyrene, which exhibited abundant stress fibers and did not form aggregates, revealed no evidence of either type II and or type X collagen. At day 21, the morphological changes of the cell aggregates formed on the G5 surface were suppressed as a result of stress fiber formation. Type II collagen was observed throughout the aggregates whereas type X collagen was detected only at the basal side of the aggregates. Change of cell aggregate behaviors derived from G5 surface alone regulated chondrogenesis and hypotrophy, and this was enhanced by chondrogenic medium. CONCLUSIONS: Incubation of hMSCs affects the expression of type II and X collagens via effects on cell aggregate behavior and stress fiber formation.

Kinetic analysis of deviation from the undifferentiated state in colonies of human induced pluripotent stem cells on feeder layers.

Understanding of the fundamental mechanisms that govern unintentional differentiation of human induced pluripotent stem cells (hiPSCs) provides key strategies to maintain their undifferentiated state during cell expansion. This study focused on deviation from the undifferentiated state in hiPSC colonies during culture with feeder cells. Deviated cells from the undifferentiated state of hiPSCs in cultures with SNL and MEF feeder cells were observed at the center and periphery of the colonies, respectively, accompanied by dramatic changes in the cell morphology from small to large flattened shapes. It was found that the deviation of undifferentiated hiPSCs in culture with SNL feeder cells caused deviated cells in the center of the colony through spontaneous occurrence in a colony size-dependent manner, whereas the deviation of undifferentiated hiPSCs in culture with MEF feeder cells caused deviated cells in the periphery of the colonies through accidental events during migration in a colony size-independent manner. Based on a kinetic analysis of time-lapse images of single hiPSC colonies, the specific growth rate for replication of deviated cells from the undifferentiated state in culture with SNL feeder cells was 1.83 and 3.57 times higher than those of undifferentiated cells and transformation, respectively, meaning that the deviation of undifferentiated hiPSCs dramatically expanded through replication of deviated cells from the undifferentiated state and transformation once deviation from the undifferentiated state had occurred. In the case of MEF feeder cells, the specific growth rates for replication of deviated cells from the undifferentiated state was 3.12 times higher than that of undifferentiated cells, whereas the rate by transformation exhibited a negligible level compared with the rates of replication for undifferentiated cells and deviated cells from undifferentiated state, meaning that deviation of undifferentiated hiPSCs dramatically expanded only through replication of deviated cells from the undifferentiated state. These results suggest that once deviation has occurred in a colony, the deviated cells from undifferentiated state undertake dramatic invasion to occupy the colony. Maintenance of the undifferentiated state in subcultures inevitably requires vigilant care to remove any colonies that include deviated cells from the undifferentiated state.

A simple tool for the synchronous differentiation of human induced pluripotent stem cells into pancreatic progenitors.

Efficient differentiation of human induced pluripotent stem cells (hiPSCs) into functional pancreatic cells holds great promise for diabetes research and treatment. However, a robust culture strategy for producing pancreatic progenitors with high homogeneity is lacking. Here, we established a simple differentiation strategy for generating synchronous iPSC-derived pancreatic progenitors via a two-step method of sequential cell synchronization using botulinum hemagglutinin (HA), an E-cadherin function-blocking agent. Of the various methods tested, the first-step synchronization method with HA exposure induces a synchronous switch from E- to N-cadherin and N- to E-cadherin expression by spatially controlling heterogeneous cell distribution, subsequently improving their competency for directed differentiation into definitive endodermal cells from iPSCs. The iPSC-derived definitive endodermal cells can efficiently generate PDX1+ and NKX6.1+ pancreatic progenitor cells in high yields. The PDX1+ and PDX1+ /NKX6.1+ cell densities showed 1.6- and 2.2-fold increases, respectively, compared with those from unsynchronized cultures. The intra-run and inter-run coefficient of variation were below 10%, indicating stable and robust differentiation across different cultures and runs. Our approach is a simple and efficient strategy to produce large quantities of differentiated cells with the highest homogeneity during multistage pancreatic progenitor differentiation, providing a potential tool for guided differentiation of iPSCs to functional insulin-producing cells.

Growth prolongation of human induced pluripotent stem cell aggregate in three-dimensional suspension culture system by addition of botulinum hemagglutinin.

Human induced pluripotent stem cells (hiPSCs) can be used in regenerative therapy as an irresistible cell source, and so the development of scalable production of hiPSCs for three-dimensional (3D) suspension culture is required. In this study, we established a simple culture strategy for improving hiPSC aggregate growth using botulinum hemagglutinin (HA), which disrupts cell-cell adhesion mediated by E-cadherin. When HA was added to the suspension culture of hiPSC aggregates, E-cadherin-mediated cell-cell adhesion was temporarily disrupted within 24 h, but then recovered. Phosphorylated myosin light chain, a contractile force marker, was also recovered at the periphery of hiPSC aggregates. The cell aggregates were suppressed the formation of collagen type I shell-like structures at the periphery by HA and collagen type I was homogenously distributed within the cell aggregates. In addition, these cell aggregates retained the proliferation marker Ki-67 throughout the cell aggregates. The apparent specific growth rate with HA addition was maintained continuously throughout the culture, and the final cell density was 1.7-fold higher than that in the control culture. These cells retained high expression levels of pluripotency markers. These observations indicated that relaxation of cell-cell adhesions by HA addition induced rearrangement of the mechanical tensions generated by actomyosin in hiPSC aggregates and suppression of collagen type I shell-like structure formation. These results suggest that this simple and readily culture strategy is a potentially useful tool for improving the scalable production of hiPSCs for 3D suspension cultures.

Stable and efficient generation of functional iPSC-derived neural progenitor cell rosettes through regulation of collective cell-cell behavior.

Although the potential of stem cells to differentiate into several cell types has shown promise in regenerative medicine, low differentiation efficiency and poor reproducibility significantly limit their practical application. We developed an effective and robust differentiation strategy for the efficient and robust generation of neural progenitor cell rosettes from induced pluripotent stem cells (iPSCs) incorporating botulinum hemagglutinin (HA). Treatment with HA suppressed the spontaneous differentiation of iPSCs cultured under undirected differentiation conditions, resulting in the preservation of their pluripotency. Moreover, treatment with HA during neural progenitor differentiation combined with dual SMAD inhibition generated a highly homogeneous population of PAX6-and SOX1-expressing neural progenitor cells with 8.4-fold higher yields of neural progenitor cells than untreated control cultures. These neural progenitor cells formed radially organized rosettes surrounding the central lumen. This differentiation method enhanced the generation of functional iPSC-derived neural progenitor cell rosettes throughout the culture vessel, suggesting that the regulation of collective cell-cell behavior using HA plays a morphogenetically important role in rosette formation and maturation. These findings show the significance of HA in the suppression of spontaneous differentiation through spatial homogeneity. The study proposes a novel methodology for the efficient derivation of functional iPSC-derived neural progenitor cell rosettes.

An in vitro culture platform to study the extracellular matrix remodeling potential of human mesenchymal stem cells.

The ability of mesenchymal stem cells (MSCs) to synthesize and degrade extracellular matrix (ECM) is important for MSC-based therapies. However, the therapeutic effects associated with ECM remodeling in cultured MSCs have been limited by the lack of a method to assess the ability of cultured cells to degrade ECM in vitro. Here, we describe a simple in vitro culture platform for studying the ECM remodeling potential of cultured MSCs using a high-density collagen (CL) surface. Cells on the CL surface have remarkable ability to degrade collagen fibrils by secreting matrix metalloproteinase (MMP); to study this, the marker collagen hybridizing peptide (CHP) was used. Confirming the ECM remodeling potential of MSCs with different population doublings (PDs), young and healthy γ-H2AX-negative cells, a marker of DNA damage and senescence, showed more extensive collagen degradation on the CL surface, whereas damaged cells of γ-H2AX-positive cells showed no collagen degradation. The frequency of γ-H2AX-/CHP + cells at PD = 0 was 49%, which was 4.9-fold higher than that at PD=13.07, whereas the frequency of γ-H2AX+/CHP- at PD=13.07 was 50%, which was 6.4-folds higher than that at PD=0. Further experimentation examining the in vitro priming effect of MSCs with the pro-inflammatory cytokine interferon-γ treatment showed increased frequency of cells with ECM remodeling potential with higher MMP secretion. Thus, this culture surface can be used for studying the ECM remodeling capacity of ex vivo-expanded MSCs in vitro and may serve as a platform for prediction in vivo ECM remodeling effect. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) remodeling potential of cultured mesenchymal stem cells (MSCs) is important for assessing the effectiveness of MSC-based therapy. However, methods to assess the ability of cultured cells to degrade ECM in vitro are still lacking. Here, we developed a simple in vitro culture platform to study the ECM remodeling potential of cultured MSCs using high-density collagen surfaces. This platform was used to evaluate the ECM remodeling potential of long-term ex vivo-expanded MSCs in vitro.

An in vitro culture platform for studying the effect of collective cell migration on spatial self-organization within induced pluripotent stem cell colonies.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) provide an in vitro system to identify the impact of cell behavior on the earliest stages of cell fate specification during human development. Here, we developed an hiPSC-based model to study the effect of collective cell migration in meso-endodermal lineage segregation and cell fate decisions through the control of space confinement using a detachable ring culture system. RESULTS: The actomyosin organization of cells at the edge of undifferentiated colonies formed in a ring barrier differed from that of the cells in the center of the colony. In addition, even in the absence of exogenous supplements, ectoderm, mesoderm, endoderm, and extraembryonic cells differentiated following the induction of collective cell migration at the colony edge by removing the ring-barrier. However, when collective cell migration was inhibited by blocking E-cadherin function, this fate decision within an hiPSC colony was altered to an ectodermal fate. Furthermore, the induction of collective cell migration at the colony edge using an endodermal induction media enhanced endodermal differentiation efficiency in association with cadherin switching, which is involved in the epithelial-mesenchymal transition. CONCLUSIONS: Our findings suggest that collective cell migration can be an effective way to drive the segregation of mesoderm and endoderm lineages, and cell fate decisions of hiPSCs.

Effect of Rho-Associated Kinase Inhibitor on Growth Behaviors of Human Induced Pluripotent Stem Cells in Suspension Culture.

Rho-associated protein kinase (ROCK) inhibitors are used for the survival of single-dissociated human induced pluripotent stem cells (hiPSCs); however, their effects on the growth behaviors of hiPSCs in suspension culture are unexplored. Therefore, we investigated the effect of ROCK inhibitor on growth behaviors of two hiPSC lines (Tic and 1383D2) with different formation of aggregate that attached between single cells in suspension culture. The apparent specific growth rate by long-term exposure to Y-27632, a ROCK inhibitor, was maintained throughout the culture. Long-term exposure to ROCK inhibitor led to an increase in cell division throughout the culture in both lines. Immunofluorescence staining confirmed that hiPSCs forming spherical aggregates showed localization of collagen type I on its periphery. In addition, phosphorylated myosin (pMLC) was localized at the periphery in culture under short-term exposure to ROCK inhibitor, whereas pMLC was not detected at whole the aggregate in culture under long-term exposure. Scanning electron microscopy indicated that long-term exposure to ROCK inhibitor blocked the structural alteration on the surface of cell aggregates. These results indicate that pMLC inhibition by long-term ROCK inhibition leads to enhanced growth abilities of hiPSCs in suspension culture by maintaining the structures of extracellular matrices.

Cell Behavioral Dynamics as a Cue in Optimizing Culture Stabilization in the Bioprocessing of Pluripotent Stem Cells.

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. In this review, we discuss recent insights into the role of cell behavioral and mechanical homeostasis in modulating the states and functions of PSCs during culture processes. We delineate promising ways to manipulate the culture variability through regulating cell behaviors using currently developed tools. Furthermore, we anticipate their potential implementation for designing a culture strategy based on the concept of Waddington's epigenetic landscape that may provide a feasible solution for tuning the culture quality and stability in the bioprocessing space.

Effect of initial seeding density on cell behavior-driven epigenetic memory and preferential lineage differentiation of human iPSCs.

Understanding the cellular behavioral mechanisms underlying memory formation and maintenance in human induced pluripotent stem cell (hiPSC) culture provides key strategies for achieving stability and robustness of cell differentiation. Here, we show that changes in cell behavior-driven epigenetic memory of hiPSC cultures alter their pluripotent state and subsequent differentiation. Interestingly, pluripotency-associated genes were activated during the entire cell growth phases along with increased active modifications and decreased repressive modifications. This memory effect can last several days in the long-term stationary phase and was sustained in the aspect of cell behavioral changes after subculture. Further, changes in growth-related cell behavior were found to induce nucleoskeletal reorganization and active versus repressive modifications, thereby enabling hiPSCs to change their differentiation potential. Overall, we discuss the cell behavior-driven epigenetic memory induced by the culture environment, and the effect of previous memory on cell lineage specification in the process of hiPSC differentiation.

Novel approach to enhance aggregate migration-driven epigenetic memory which induces cardiomyogenic differentiation on a dendrimer-immobilized surface.

The dynamic migratory behavior of human mesenchymal stem cells (hMSCs) has a significant impact on the epigenetic profiles that determine fate choice and lineage commitment during differentiation. Here we report a novel approach to enhance repeated migration-driven epigenetic memory which induces cardiomyogenic differentiation on a dendrimer surface with fifth generation (G5). Cells exhibited the formation of cell aggregates on the G5 surface through active migration with morphological changes, and these aggregates showed strong expression of the cardiac-specific marker cardiac troponin T (cTnT) at 10 days. When cell aggregates were passaged onto a fresh G5 surface over three passages of 40 days, the expression levels of the multiple cardiac-specific markers including GATA4, NKX2.5, MYH7, and TNNT2 were higher compared to those passaged as single cells. To investigate whether cardiomyogenic differentiation of hMSCs was enhanced by repeated aggregate migration-driven epigenetic memory, cells on the G5 surface were reseeded onto a fresh G5 surface during three passages using aggregate-based and single cell-based passage methods. Analyses of global changes in H3 histone modifications exhibited pattern of increased H3K9ac and H3K27me3, and decreased H3K9me3 in aggregate-based passage cultures during three passages. However, the pattern of their histone modification on the PS surface was repeated after the initialization and reformation during three passages in single cell-based passage cultures. Thus, repetitive aggregate migratory behavior during aggregate-based passage led to a greater degree of histone modification, as well as gene expression changes suggestive of cardiomyogenic differentiation.

Bioengineering Considerations for a Nurturing Way to Enhance Scalable Expansion of Human Pluripotent Stem Cells.

Understanding how defects in mechanotransduction affect cell-to-cell variability will add to the fundamental knowledge of human pluripotent stem cell (hPSC) culture, and may suggest new approaches for achieving a robust, reproducible, and scalable process that result in consistent product quality and yields. Here, the current state of the understanding of the fundamental mechanisms that govern the growth kinetics of hPSCs between static and dynamic cultures is reviewed, the factors causing fluctuations are identified, and culture strategies that might eliminate or minimize the occurrence of cell-to-cell variability arising from these fluctuations are discussed. The existing challenges in the development of hPSC expansion methods for enabling the transition from process development to large-scale production are addressed, a mandatory step for industrial and clinical applications of hPSCs.

Muscle lineage switching by migratory behaviour-driven epigenetic modifications of human mesenchymal stem cells on a dendrimer-immobilized surface.

Understanding of the fundamental mechanisms of epigenetic modification in the migration of human mesenchymal stem cells (hMSCs) provides surface design strategies for controlling self-renewal and lineage commitment. We investigated the mechanism underlying muscle lineage switching of hMSCs by cellular and nuclear deformation during cell migration on polyamidoamine dendrimer surfaces. With an increase in the dendrimer generation number, cells exhibited increased nuclear deformation and decreased lamin A/C and lamin B1 expression. Analysis of two repressive modifications (H3K9me3 and H3K27me3) and one activating modification (H3K9ac) revealed that H3K9me3 was suppressed, and H3K9ac and H3K27me3 were upregulated in the cultures on a higher-generation dendrimer surface. This induced significant hMSC lineage switching to smooth, skeletal, and cardiac muscle lineages. Thus, reorganizations of the nuclear lamina and cytoskeleton related to migration changes on dendrimer surfaces are responsible for the integrated regulation of histone modifications in hMSCs, thereby shifting the cells from the multipotent state to muscle lineages. These findings improve our understanding of the role of epigenetic modification in cell migration and provide new insights into how designed surfaces can be applied as cell-instructive materials in the field of biomaterial-guided differentiation of hMSCs to different cell types. STATEMENT OF SIGNIFICANCE: Stem cell engineering strategies currently applied the mechanical cues that emerge from cellular microenvironment to regulate stem cell behaviour. This study significantly improved our understanding of the mechanotransduction mechanism involving cell-ECM and cytoskeleton-nucleoskeleton interactions, and of nuclear genome regulation based on cellular responses to biomaterial modifications. The new insights into how the physical environment on a culture surface influences cell behaviour improve our understanding of mechanical control mechanisms of the interactions of cells with the extracellular environment. Our findings are also expected to contribute to and play an essential role in the development of future material strategies for creating artificial cell-instructive niches.