Biophysical perspectives to understanding cancer-associated fibroblasts.

The understanding of cancer has evolved significantly, with the tumor microenvironment (TME) now recognized as a critical factor influencing the onset and progression of the disease. This broader perspective challenges the traditional view that cancer is primarily caused by mutations, instead emphasizing the dynamic interaction between different cell types and physicochemical factors within the TME. Among these factors, cancer-associated fibroblasts (CAFs) command attention for their profound influence on tumor behavior and patient prognoses. Despite their recognized importance, the biophysical and mechanical interactions of CAFs within the TME remain elusive. This review examines the distinctive physical characteristics of CAFs, their morphological attributes, and mechanical interactions within the TME. We discuss the impact of mechanotransduction on CAF function and highlight how these cells communicate mechanically with neighboring cancer cells, thereby shaping the path of tumor development and progression. By concentrating on the biomechanical regulation of CAFs, this review aims to deepen our understanding of their role in the TME and to illuminate new biomechanical-based therapeutic strategies.

High-resolution assessment of multidimensional cellular mechanics using label-free refractive-index traction force microscopy.

A critical requirement for studying cell mechanics is three-dimensional assessment of cellular shapes and forces with high spatiotemporal resolution. Traction force microscopy with fluorescence imaging enables the measurement of cellular forces, but it is limited by photobleaching and a slow acquisition speed. Here, we present refractive-index traction force microscopy (RI-TFM), which simultaneously quantifies the volumetric morphology and traction force of cells using a high-speed illumination scheme with 0.5-Hz temporal resolution. Without labelling, our method enables quantitative analyses of dry-mass distributions and shear (in-plane) and normal (out-of-plane) tractions of single cells on the extracellular matrix. When combined with a constrained total variation-based deconvolution algorithm, it provides 0.55-Pa shear and 1.59-Pa normal traction sensitivity for a 1-kPa hydrogel substrate. We demonstrate its utility by assessing the effects of compromised intracellular stress and capturing the rapid dynamics of cellular junction formation in the spatiotemporal changes in non-planar traction components.

Surface tension-induced biomimetic assembly of cell-laden fibrous bundle construct for muscle tissue engineering.

The field of tissue engineering has been long seeking to develop functional muscle tissue that closely resembles natural muscle. This study used a bio-inspired assembly based on the surface tension mechanism to develop a novel method for engineering muscle tissue. This approach enabled uniaxially ordered electrospun fibers to naturally collide into an aligned bundle without the need for manual handling, thereby reducing cell damage during the cell culture procedure. During the assembly procedure, C2C12 myoblasts were cultured in a viscous collagen hydrogel that caused wetting while providing adequate structural stability for the cell-fiber construct. In addition, gene expression analysis of the resulting muscle-like fibril bundle revealed improved myogenic differentiation. These findings highlight the potential of using a collagen hydrogel and the surface tension mechanism to construct biologically relevant muscle tissue, offering a promising strategy that may outperform existing approaches. Overall, this study contributes to the development of advanced tissue engineering methods and brings us a step closer to creating functional muscle tissue for therapeutic and regenerative medicine applications.

Shear-induced phenotypic transformation of microglia in vitro.

The brain cells are affected by continuous fluid shear stress that is driven by varying hydrostatic and osmotic pressure conditions, depending on the brain's pathophysiological conditions. Although all brain cells are sensitive to the subtle changes in various physicochemical factors in the microenvironment, microglia, the resident brain immune cells, exhibit the most significant morphodynamic transformation. However, little is known about the phenotypic alterations in microglia in response to changes in fluid shear stress. In this study, we established a flow-controlled microenvironment to investigate the effects of shear flow on microglial phenotypes, including morphology, motility, and activation states. We observed two distinct morphologies of microglia in a static condition: bipolar cells that oscillate along their long axis and unipolar cells that migrate persistently. When exposed to flow, a significant fraction of bipolar cells showed unstable oscillation with an increased amplitude of oscillation and a decreased frequency, which consequently led to the phenotypic transformation of oscillating cells into migrating cells. Furthermore, we observed that the level of proinflammatory genes increased in response to shear stress, although there were no significant changes in the level of antiinflammatory genes. Our findings suggest that an interstitial fluid-level stimulus can cause a dramatic phenotypic shift in microglia toward proinflammatory states, shedding light on the pathological outbreaks of severe brain diseases. Given that the fluidic environment in the brain can be locally disrupted in pathological circumstances, the mechanical stimulus by fluid flow should also be considered a crucial element in regulating the immune activities of the microglia in brain diseases.

Light-stimulated insulin secretion from pancreatic islet-like organoids derived from human pluripotent stem cells.

Optogenetic techniques permit non-invasive, spatiotemporal, and reversible modulation of cellular activities. Here, we report a novel optogenetic regulatory system for insulin secretion in human pluripotent stem cell (hPSC)-derived pancreatic islet-like organoids using monSTIM1 (monster-opto-Stromal interaction molecule 1), an ultra-light-sensitive OptoSTIM1 variant. The monSTIM1 transgene was incorporated at the AAVS1 locus in human embryonic stem cells (hESCs) by CRISPR-Cas9-mediated genome editing. Not only were we able to elicit light-induced intracellular Ca2+ concentration ([Ca2+]i) transients from the resulting homozygous monSTIM1+/+-hESCs, but we also successfully differentiated them into pancreatic islet-like organoids (PIOs). Upon light stimulation, the ß-cells in these monSTIM1+/+-PIOs displayed reversible and reproducible [Ca2+]i transient dynamics. Furthermore, in response to photoexcitation, they secreted human insulin. Light-responsive insulin secretion was similarly observed in monSTIM1+/+-PIOs produced from neonatal diabetes (ND) patient-derived induced pluripotent stem cells (iPSCs). Under LED illumination, monSTIM1+/+-PIO-transplanted diabetic mice produced human c-peptide. Collectively, we developed a cellular model for the optogenetic control of insulin secretion using hPSCs, with the potential to be applied to the amelioration of hyperglycemic disorders.

Influence of Transforming Growth Factors beta 1 and beta 3 in the Scar Formation Process.

BACKGROUND: Transforming growth factor-beta (TGF-ß) plays an instrumental role in forming scars and keloids. TGF-ß isoforms exhibit differential expression, indicating distinct wound healing and scar formation functions. However, the role of TGF-ß1 and TGF-ß3 in wound healing and scar formation remains unclear. This study aimed to compare the specific roles of TGF-ß1 and TGF-ß3 in wound healing and scar formation by biomolecular analysis. MATERIALS AND METHODS: The study was conducted by cell isolation and culture cells from a total of 20 human samples. Normal human fibroblasts (NHF) were isolated from normal human samples and myofibroblasts from the different scar types, namely hypertrophic (HT) and keloid (K) scars. NHF and cells from the HT, and K scar, each of which were divided into 3 sample groups: the untreated control, TGF-ß1 (10 µg/mL)-treated group, and TGF-ß3 (10 µg/mL)-treated group. The results of confocal microscopy and fluorescence-activated cell sorting experiments were compared. RESULTS: Both the HT and K groups had higher α-smooth muscle actin (α-SMA) expression than the NHF group in the untreated control group. In comparison with the untreated group, NHFs showed a significant increase in α-SMA expression in the TGF-ß1-treated group. HT showed a high α-SMA level, which was statistically significant compared with the normal fibroblasts. In the TGF-ß3-treated group, α-SMA expression was slightly increased in NHF as compared with the untreated group. TGF-ß3 treated HT exhibited a greater reduction in α-SMA expression than in the TGF-ß1 treated HT. K, on the other hand, had only a minimal effect on the treatment of TGF-ß1 and TGF-ß3. CONCLUSIONS: The findings suggest that TGF-ß3 may play a regulatory role in the wound repair process, which could be useful in the development of scar-reducing therapies for patients with scar-related cosmetic concerns.

Fibrous Matrix Architecture-Dependent Activation of Fibroblasts with a Cancer-Associated Fibroblast-like Phenotype.

Cancer-associated fibroblasts (CAFs) are one of the most prevalent cell types within the tumor microenvironment (TME). While several physicochemical cues from the TME, including growth factors, cytokines, and ECM specificity, have been identified as essential factors for CAF activation, the precise mechanism of how the ECM architecture regulates CAF initiation remains elusive. Using a gelatin-based electrospun fiber mesh, we examined the effect of matrix fiber density on CAF activation induced by MCF-7 conditioned media (CM). A less dense (3D) gelatin mesh matrix facilitated better activation of dermal fibroblasts into a CAF-like phenotype in the CM than a highly dense (3D) gelatin mesh matrix. In addition, it was discovered that CAF activation on the less dense (LD) matrix is dependent on the cell size-related AKT/mTOR signaling cascade, accompanied by an increase in intracellular tension within the well-spread fibroblasts.

ECM Architecture-Mediated Regulation of ß-Cell Differentiation from hESCs via Hippo-Independent YAP Activation.

Changes in the extracellular matrix (ECM) influence stem cell fate. When hESCs were differentiated on a thin layer of Matrigel coated onto PDMS (Matrigel_PDMS), they exhibited a substantial increase in focal adhesion and focal adhesion-associated proteins compared with those cultured on Matrigel coated onto TCPS (Matrigel_TCPS), resulting in YAP/TEF1 activation and ultimately promoting the transcriptional activities of pancreatic endoderm (PE)-associated genes. Interestingly, YAP activation in PE cells was mediated through integrin α3-FAK-CDC42-PP1A signaling rather than the typical Hippo signaling pathway. Furthermore, pancreatic islet-like organoids (PIOs) generated on Matrigel_PDMS secreted more insulin than those generated from Matrigel_TCPS. Electron micrographs revealed differential Matrigel architectures depending on the underlying substrate, resulting in varying cell-matrix anchorage resistance levels. Accordingly, the high apparent stiffness of the unique mucus-like network structure of Matrigel_PDMS was the critical factor that directly upregulated focal adhesion, thereby leading to better maturation of the pancreatic development of hESCs in vitro.

Ceiling culture chip reveals dynamic lipid droplet transport during adipocyte dedifferentiation via actin remodeling.

Adipocyte dedifferentiation has recently gained attention as a process underpinning adipocyte plasticity; however, a lack of suitable experimental platforms has hampered studies into the underlying mechanisms. Here, we developed a microscope-mountable ceiling culture chip that provides a stable yet tunable culture environment for long-term live-imaging of dedifferentiating adipocytes. A detailed spatiotemporal analysis of mature adipocyte dedifferentiation utilizing the culture platform and Cre-recombinase tracers revealed the involvement of dynamic actin remodeling for lipid droplet (LD) secretion during adipocyte dedifferentiation. Additionally, Hippo, Hedgehog, and PPARγ signaling pathways were identified as potent regulators of adipocyte dedifferentiation. Contrary to the belief that adult adipocytes are relatively static, we show that adipocytes are very dynamic, relying on actin-driven mechanical forces to execute LD extrusion and intercellular LD transfer processes.

Cell segregation via differential collision modes between heterotypic cell populations.

In tissue development and regeneration, the establishment of sharp boundaries between heterotypic cells is essential for the differentiation of tissue functions. During the dynamic rearrangements of constituent cells that result from cell division and collective migration, the segregation boundary encounters various challenges. Several studies have suggested that cortical actomyosin structures play a crucial role in the maintenance of the boundary interface of segregated cell populations, implicating actin-mediated stresses. Examining physical cellular properties such as motility, traction, and intercellular stress, we investigated the formation and maintenance of the stable segregation between epithelial and mesenchymal cell populations devoid of heterotypic adhesions. At the contact boundary, the homotypic adhesion-mediated epithelial aggregates exerted collision-mediated compression against the surrounding mesenchymal cells. Our results demonstrated that heterotypic cell populations established a robust interfacial boundary by accumulating stress from active collisions and repulsions between two dissimilar cell types. Furthermore, the moment of the heterotypic collisions was identified by the existence of a sharp rise in maximum shear stress within the cell cluster.

Fibroblasts Close a Void in Free Space by a Purse-String Mechanism.

The mechanism by which stromal cells fill voids in injured tissue remains a fundamental question in regenerative medicine. While it is well-established that fibroblasts fill voids by depositing extracellular matrix (ECM) proteins as they migrate toward the wound site, little is known about their ability to adopt an epithelial-like purse-string behavior. To investigate fibroblast behavior during gap closure, we created an artificial wound with a large void space. We discovered that fibroblasts could form a free-standing bridge over deep microvoids, closing the void via purse-string contraction, a mechanism previously thought to be unique to epithelial wound closure. The findings also revealed that myosin II mediated contractility and intercellular adherent junctions were required for the closure of the fibroblast gap in our fabricated three-dimensional artificial wound. To fulfill their repair function under the specific microenvironmental conditions of wounds, fibroblasts appeared to acquire the structural features of epithelial cells, namely, contractile actin bundles that span over multiple cells along the boundary. These findings shed light on a novel mechanism by which stromal cells bridge the 3D gap during physiological processes such as morphogenesis and wound healing.

Vimentin intermediate filaments and filamentous actin form unexpected interpenetrating networks that redefine the cell cortex.

The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryo-electron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin.

Pillar-Based Mechanical Induction of an Aggressive Tumorigenic Lung Cancer Cell Model.

Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 µm diameter and 16 µm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 µm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and in vivo tumor colonization assays. Moreover, the pillar-induced morphological deformation in nuclei was accompanied by cell-cycle arrest in the S phase, leading to suppressed proliferation. While these marked traits of morphology-migration-proliferation support more aggressive characteristics of metastatic cancer cells, typical indices of epithelial-mesenchymal transition were not found, but instead, remarkable traces of amoeboidal transition were confirmed. Our study also emphasizes the importance of mechanical stimuli from the microenvironment during pathogenesis and how gained traits can be passed onto subsequent generations, ultimately affecting their pathophysiological behavior. Furthermore, this study highlights the potential use of pillar-based mechanical stimuli as an in vitro cell culture strategy to induce more aggressive tumorigenic cancer cell models.

Engineering 3D Cortical Spheroids for an In Vitro Ischemic Stroke Model.

Three-dimensional (3D) spheroids composed of brain cells have shown great potential to mimic the pathophysiology of the brain. However, a 3D spheroidal brain-disease model for cerebral ischemia has not been reported. This study investigated an ultralow attachment (ULA) surface-mediated formation of 3D cortical spheroids using primary rat cortical cells to recapitulate the cerebral ischemic responses in stroke by oxygen-glucose deprivation-reoxygenation (OGD-R) treatment. Comparison between two-dimensional (2D) and 3D cell culture models confirmed the better performance of the 3D cortical spheroids as normal brain models. The cortical cells cultured in 3D maintained their healthy physiological morphology of a less activated state and suppressed mRNA expressions of pathological stroke markers, S100B, IL-1ß, and MBP, selected based on in vivo stroke model. Interestingly, the spheroids formed on the ULA surface exhibited striking aggregation dynamics involving active cell-substrate interactions, whereas those formed on the agarose surface aggregated passively by the convective flow of the media. Accordingly, ULA spheroids manifested a layered arrangement of neurons and astrocytes with higher expressions of integrin ß1, integrin α5, N-cadherin, and fibronectin than the agarose spheroids. OGD-R-induced stroke model of the ULA spheroids successfully mimicked the ischemic response as evidenced by the upregulated mRNA expressions of the key markers for stroke, S100B, IL-1ß, and MBP. Our study suggested that structurally and functionally distinct cortical spheroids could be generated by simply tuning the cell-substrate binding activities during dynamic spheroidal formation, which should be an essential factor to consider in establishing a brain-disease model.

Physical analysis reveals distinct responses of human bronchial epithelial cells to guanidine and isothiazolinone biocides.

Changes in the physical state of the cells can serve as important indicators of stress responses because they are closely linked with the changes in the pathophysiological functions of the cells. Physical traits can be conveniently assessed by analyzing the morphological features and the stresses at the cell-matrix and cell-cell adhesions in both single-cell and monolayer model systems in 2D. In this study, we investigated the mechano-stress responses of human bronchial epithelial cells, BEAS-2B, to two functionally distinct groups of biocides identified during the humidifier disinfectant accident, namely, guanidine (PHMG) and isothiazolinone (CMIT/MIT). We analyzed the physical traits, including cell area, nuclear area, and nuclear shape. While the results showed inconsistent average responses to the biocides, the degree of dispersion in the data set, measured by standard deviation, was remarkably higher in CMIT/MIT treated cells for all traits. As mechano-stress endpoints, traction and intercellular stresses were also measured, and the cytoskeletal actin structures were analyzed using immunofluorescence. This study demonstrates the versatility of the real-time imaging-based biomechanical analysis, which will contribute to identifying the temporally sensitive cellular behaviors as well as the emergence of heterogeneity in response to exogenously imposed stress factors. This study will also shed light on a comparative understanding of less studied substance, CMIT/MIT, in relation to a more studied substance, PHMG, which will further contribute to more strategic planning for proper risk management of the ingredients involved in toxicological accidents.

Enriching neural stem cell and anti-inflammatory glial phenotypes with electrical stimulation after traumatic brain injury in male rats.

Traumatic brain injury (TBI) by an external physical impact results in compromised brain function via undesired neuronal death. Following the injury, resident and peripheral immune cells, astrocytes, and neural stem cells (NSCs) cooperatively contribute to the recovery of the neuronal function after TBI. However, excessive pro-inflammatory responses of immune cells, and the disappearance of endogenous NSCs at the injury site during the acute phase of TBI, can exacerbate TBI progression leading to incomplete healing. Therefore, positive outcomes may depend on early interventions to control the injury-associated cellular milieu in the early phase of injury. Here, we explore electrical stimulation (ES) of the injury site in a rodent model (male Sprague-Dawley rats) to investigate its overall effect on the constituent brain cell phenotype and composition during the acute phase of TBI. Our data showed that a brief ES for 1 hr on day 2 of TBI promoted anti-inflammatory phenotypes of microglia as assessed by CD206 expression and increased the population of NSCs and Nestin+ astrocytes at 7 days post-TBI. Also, ES effectively increased the number of viable neurons when compared to the unstimulated control group. Given the salience of microglia and neural stem cells for healing after TBI, our results strongly support the potential benefit of the therapeutic use of ES during the acute phase of TBI to regulate neuroinflammation and to enhance neuroregeneration.

Electrospun Microvasculature for Rapid Vascular Network Restoration.

BACKGROUND: Sufficient blood supply through neo-vasculature is a major challenge in cell therapy and tissue engineering in order to support the growth, function, and viability of implanted cells. However, depending on the implant size and cell types, the natural process of angiogenesis may not provide enough blood supply for long term survival of the implants, requiring supplementary strategy to prevent local ischemia. Many researchers have reported the methodologies to form pre-vasculatures that mimic in vivo microvessels for implantation to promote angiogenesis. These approaches successfully showed significant enhancement in long-term survival and regenerative functions of implanted cells, yet there remains room for improvement. METHODS: This paper suggests a proof-of-concept strategy to utilize novel scaffolds of dimpled/hollow electrospun fibers that enable the formation of highly mature pre-vasculatures with adequate dimensions and fast degrading in the tissue. RESULT: Higher surface roughness improved the maturity of endothelial cells mediated by increased cell-scaffold affinity. The degradation of scaffold material for functional restoration of the neo-vasculatures was also expedited by employing the hollow scaffold design based on co-axial electrospinning techniques. CONCLUSION: This unique scaffold-based pre-vasculature can hold implanted cells and tissue constructs for a prolonged time while minimizing the cellular loss, manifesting as a gold standard design for transplantable scaffolds.

Remodeling of Adhesion Network within Cancer Spheroids via Cell-Polymer Interaction.

3D spheroids are considered as the improved in vitro model to mimic the distinct arrangements of the cells in vivo. To date, low-attachment surfaces have been most widely used to induce the spontaneous aggregation of cells in suspension by simply tuning the relative strength of the cell-cell adhesion over cell-substrate adhesion. However, aggregating cancer cells into 3D clusters should mean more than just adjoining the cells in the physical proximity. The tumor cell functionality is strongly affected by the adhesion networks between cancer cells and extracellular matrix (ECM). Here, we performed an in-depth analysis of how the nonmetastatic breast cancer cells (MCF7) can be transformed to gain invasive phenotypes through compact aggregation into 3D spheroids on a functional polymer film surface, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4). By comparing the adhesion networks and invasion dynamics between 3D spheroids cultured on the pV4D4 surface with those cultured on conventional ultra-low-attachment (ULA) dishes, we report that only spheroids on the pV4D4 display active and sporadic cell-surface binding activities via dynamic protrusions, which correlates strongly with an increase in integrin ß1. Moreover, localized laminin expression at the core of the pV4D4-cultured spheroids confirms the prominence of the intimate integrin-laminin interactions prompted by the exposure to pV4D4. This study suggests that structurally and functionally dissimilar 3D spheroids can be generated from the same type of cells on the surfaces of different physicochemical properties without any chemical treatment or genetic manipulation.

Super-Resolution Three-Dimensional Imaging of Actin Filaments in Cultured Cells and the Brain via Expansion Microscopy.

Actin is an essential protein in almost all life forms. It mediates diverse biological functions, ranging from controlling the shape of cells and cell movements to cargo transport and the formation of synaptic connections. Multiple diseases are closely related to the dysfunction of actin or actin-related proteins. Despite the biological importance of actin, super-resolution imaging of it in tissue is still challenging, as it forms very dense networks in almost all cells inside the tissue. In this work, we demonstrate multiplexed super-resolution volumetric imaging of actin in both cultured cells and mouse brain slices via expansion microscopy (ExM). By introducing a simple labeling process, which enables the anchoring of an actin probe, phalloidin, to a swellable hydrogel, the multiplexed ExM imaging of actin filaments was achieved. We first showed that this technique could visualize the nanoscale details of actin filament organizations in cultured cells. Then, we applied this technique to mouse brain slices and visualized diverse actin organizations, such as the parallel actin filaments along the long axis of dendrites and dense actin structures in postsynaptic spines. We examined the postsynaptic spines in the mouse brain and showed that the organizations of actin filaments are highly diverse. This technique, which enables the high-throughput 60 nm resolution imaging of actin filaments and other proteins in cultured cells and thick tissue slices, would be a useful tool to study the organization of actin filaments in diverse biological circumstances and how they change under pathological conditions.

Three-Dimensional Spheroid Culture on Polymer-Coated Surface Potentiate Stem Cell Functions via Enhanced Cell-Extracellular Matrix Interactions.

The aggregation of mesenchymal stem cells (MSCs) into three-dimensional (3D) spheroids has emerged as a promising therapeutic candidate for the treatment of a variety of diseases. In spite of the numerous 3D culture methods suggested recently for MSC spheroid generation, it is still elusive to fully reflect real stem cell niches; this effort majorly suffers from a lack of cell-extracellular matrix (ECM) interactions within the 3D spheroids. In this study, we develop a simple but versatile method for generating human MSC (hMSC) spheroids by culturing the cells on a functional polymer film surface, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4). Interestingly, the pV4D4-coated surface allows a dynamic cell adhesion to the polymer surface while developing the formation of 3D spheroids. The corresponding mechanotransduction promotes the expression of the endogenous ECM and, in turn, results in a remarkable improvement in self-renewal abilities, pro-angiogenic potency, and multilineage differentiation capabilities. This observation highlights the significance of our method compared to the conventional spheroid-generating methods in terms of recreating the ECM-rich microenvironment. We believe the developed surface can serve as a versatile but reliable method for stem cell-based tissue engineering and regenerative medicine.