Spatial cell fate manipulation of human pluripotent stem cells by controlling the microenvironment using photocurable hydrogel.

Human pluripotent stem cells (hPSCs) dynamically respond to their chemical and physical microenvironment, dictating their behavior. However, conventional in vitro studies predominantly employ plastic culture wares, which offer a simplified representation of the in vivo microenvironment. Emerging evidence underscores the pivotal role of mechanical and topological cues in hPSC differentiation and maintenance. In this study, we cultured hPSCs on hydrogel substrates with spatially controlled stiffness. The use of culture substrates that enable precise manipulation of spatial mechanical properties holds promise for better mimicking in vivo conditions and advancing tissue engineering techniques. We designed a photocurable polyethylene glycol-polyvinyl alcohol (PVA-PEG) hydrogel, allowing the spatial control of surface stiffness and geometry at a micrometer scale. This versatile hydrogel can be functionalized with various extracellular matrix proteins. Laminin 511-functionalized PVA-PEG gel effectively supports the growth and differentiation of hPSCs. Moreover, by spatially modulating the stiffness of the patterned gel, we achieved spatially selective cell differentiation, resulting in the generation of intricate patterned structures.

Super-resolution imaging reveals nucleolar encapsulation by single-stranded DNA.

In eukaryotic cell nuclei, specific sets of proteins gather in nuclear bodies and facilitate distinct genomic processes. The nucleolus, a nuclear body, functions as a factory for ribosome biogenesis by accumulating constitutive proteins, such as RNA polymerase I and nucleophosmin 1 (NPM1). Although in vitro assays have suggested the importance of liquid-liquid phase separation (LLPS) of constitutive proteins in nucleolar formation, how the nucleolus is structurally maintained with the intranuclear architecture remains unknown. This study revealed that the nucleolus is encapsulated by a single-stranded (ss)DNA-based molecular complex inside the cell nucleus. Super-resolution lattice-structured illumination microscopy (lattice-SIM) showed that there was a high abundance of ssDNA beyond the 'outer shell' of the nucleolus. Nucleolar disruption and the release of NPM1 were caused by in situ digestion of ssDNA, suggesting that ssDNA has a structural role in nucleolar encapsulation. Furthermore, we identified that ssDNA forms a molecular complex with histone H1 for nucleolar encapsulation. Thus, this study illustrates how an ssDNA-based molecular complex upholds the structural integrity of nuclear bodies to coordinate genomic processes such as gene transcription and replication.

Spatiotemporal analysis of multi-scale cell structure in spheroid culture reveals hypertrophic chondrocyte differentiation.

3D cell culture has emerged as a promising approach to replicate the complex behaviors of cells within living organisms. This study aims to analyze spatiotemporal behavior of the morphological characteristics of cell structure at multiscale in 3D scaffold-free spheroids using chondrogenic progenitor ATDC5 cells. Over a 14-day culture period, it exhibited cell hypertrophy in the spheroids regarding cellular and nuclear size as well as changes in morphology. Moreover, biological analysis indicated a signification up-regulation of normal chondrocyte as well as hypertrophic chondrocyte markers, suggesting early hypertrophic chondrocyte differentiation. Cell nuclei underwent changes in volume, sphericity, and distribution in spheroid over time, indicating alterations in chromatin organization. The ratio of chromatin condensation volume to cell nuclear volume decreased as the cell nuclei enlarged, potentially signifying changes in chromatin state during hypertrophic chondrocyte differentiation. Our image analysis techniques in this present study enabled detailed morphological measurement of cell structure at multi-scale, which can be applied to various 3D culture models for in-depth investigation.

Pluripotency state of mouse ES cells determines their contribution to self-organized layer formation by mesh closure on microstructured adhesion-limiting substrates.

Assembly of pluripotent stem cells to initiate self-organized tissue formation on engineered scaffolds is an important process in stem cell engineering. Pluripotent stem cells are known to exist in diverse pluripotency states, with heterogeneous subpopulations exhibiting differential gene expression levels, but how such diverse pluripotency states orchestrate tissue formation is still an unrevealed question. In this study, using microstructured adhesion-limiting substrates, we aimed to clarify the contribution to self-organized layer formation by mouse embryonic stem cells in different pluripotency states: ground and naïve state. We found that while ground state cells as well as sorted REX1-high expression cells formed discontinuous cell layers with limited lateral spread, naïve state cells could successfully self-organize to form a continuous layer by progressive mesh closure within 3 days. Using sequential immunofluorescence microscopy to examine the mesh closure process, we found that KRT8+ cells were particularly localized around unfilled holes, occasionally bridging the holes in a manner suggestive of their role in the closure process. These results highlight that compared with ground state cells, naïve state cells possess a higher capability to contribute to self-organized layer formation by mesh closure. Thus, this study provides insights with implications for the application of stem cells in scaffold-based tissue engineering.

Effect of chemically induced osteogenesis supplements on multicellular behavior of osteocytic spheroids.

Understanding in multicellular behaviors in three-dimensional (3D) culture models such as organoids is important to help us better comprehend the mechanisms of the morphogenesis and functions of diverse organs in vivo cellular environment. In this study, we elucidated the multicellular behaviors of the osteocytic spheroids in response to the chemically induced osteogenesis supplements (OS). Particularly, we conducted 1) size change measurement, 2) fusion experiment, and 3) collagen embedding experiment of spheroids, in response to the OS. We found out that the OS alters the multicellular behaviors of the spheroid by greater reduction in the size change measurement and slowing down the speed of fusion experiment and collagen embedding experiment of the spheroids. We also highlighted that the driving force of these changes was the tight actin filaments generated on the surface of the spheroids. Hence, the results altogether indicate that the spheroid model exerted the different multicellular behaviors against the differentiation capability. This study will contribute to understanding the multicellular behaviors of the 3D culture model reconstructed by the cells with greater cell-cell interaction force.

Polarized cellular mechano-response system for maintaining radial size in developing epithelial tubes.

Size control in biological tissues involves multicellular communication via mechanical forces during development. Although fundamental cellular behaviours in response to mechanical stimuli underlie size maintenance during morphogenetic processes, the mechanisms underpinning the cellular mechano-response system that maintains size along an axis of a polarized tissue remain elusive. Here, we show how the diameter of an epithelial tube is maintained during murine epididymal development by combining quantitative imaging, mechanical perturbation and mathematical modelling. We found that epithelial cells counteract compressive forces caused by cell division exclusively along the circumferential axis of the tube to produce polarized contractile forces, eventually leading to an oriented cell rearrangement. Moreover, a mathematical model that includes the polarized mechano-responsive regime explains how the diameter of proliferating tubes is maintained. Our findings pave the way for an improved understanding of the cellular response to mechanical forces that involves collective multicellular behaviours for organizing diverse tissue morphologies.

Spheroid culture for chondrocytes triggers the initial stage of endochondral ossification.

Endochondral ossification is the process of bone formation derived from growing cartilage duringskeletal development. In previous studies, we provoked the osteocyte differentiation of osteoblast precursor cells under a three-dimensional (3D) culture model. To recapitulate the endochondral ossification, the present study utilized the self-organized scaffold-free spheroid model reconstructed by pre-chondrocyte cells. Within 2-day cultivation in the absence of the chemically induced chondrogenesis supplements, the chondrocyte marker was greatly expressed in the inner region of the spheroid, whereas the hypertrophic chondrocyte marker was strongly detected in the surface region of the spheroid. Notably, we found out that the gene expression levels of osteocyte markers were also greatly upregulated compared to the conventional 2D monolayer. Moreover, after long-term cultivation for 28 days, it induced morphological changes in the spheroid, such as cellular hypertrophy and death. In this study, in order to recapitulate the initial stage of the endochondral ossification, we highlighted the potentials of the 3D culture method to drive the hypertrophic chondrocyte differentiation of the pre-chondrocyte cells.

Elasticity-based boosting of neuroepithelial nucleokinesis via indirect energy transfer from mother to daughter.

Neural progenitor cells (NPCs), which are apicobasally elongated and densely packed in the developing brain, systematically move their nuclei/somata in a cell cycle-dependent manner, called interkinetic nuclear migration (IKNM): apical during G2 and basal during G1. Although intracellular molecular mechanisms of individual IKNM have been explored, how heterogeneous IKNMs are collectively coordinated is unknown. Our quantitative cell-biological and in silico analyses revealed that tissue elasticity mechanically assists an initial step of basalward IKNM. When the soma of an M-phase progenitor cell rounds up using actomyosin within the subapical space, a microzone within 10 µm from the surface, which is compressed and elastic because of the apical surface's contractility, laterally pushes the densely neighboring processes of non-M-phase cells. The pressed processes then recoil centripetally and basally to propel the nuclei/somata of the progenitor's daughter cells. Thus, indirect neighbor-assisted transfer of mechanical energy from mother to daughter helps efficient brain development.

Mechanotransduction via the Piezo1-Akt pathway underlies Sost suppression in osteocytes.

Osteocytes function as critical regulators of bone homeostasis by coordinating the functions of osteoblasts and osteoclasts, and are constantly exposed to mechanical force. However, the molecular mechanism underlying the mechanical signal transduction in osteocytes is not well understood. Here, we found that Yoda1, a selective Piezo1 agonist, increased intracellular calcium mobilization and dose-dependently decreased the expression of Sost (encoding Sclerostin) in the osteocytic cell line IDG-SW3. We also demonstrated that mechanical stretch of IDG-SW3 suppressed Sost expression, a result which was abrogated by treatment with the Piezo1 inhibitor GsMTx4, and the deficiency of Piezo1. Furthermore, the suppression of Sost expression was abolished by treatment with an Akt inhibitor. Taken together, these results indicate that the activation of the Piezo1-Akt pathway in osteocytes is required for mechanical stretch-induced downregulation of Sost expression.

Functional Adaptation of the Fibrocartilage and Bony Trabeculae at the Attachment Sites of the Anterior Cruciate Ligament.

The direct insertion of an enthesis is composed of uncalcified fibrocartilage (FC) and calcified FC, believed to function as buffers for multidirectional forces applied to the ligament. This study was performed to investigate the relationship between the FC thickness and bony trabecular orientation of the anterior cruciate ligament (ACL) on both the femoral and tibial sides. Six cadavers were examined (age at death: 73-92 years). Both femoral and tibial insertions of the ACL were harvested and used to make 0.5 mm interval semi-serial sections. Microdigital images were taken and used to measure the maximum thickness of both the uncalcified FC and Calcified FC. Two-dimensional discrete Fourier analysis was also performed to determine the orientation angle and orientation intensity of bony trabeculae directly under the FC. The mean thicknesses of the uncalcified FC at the femoral and tibial insertions were 0.98 mm and 0.49 mm, respectively. The mean thicknesses of the calcified FC were 0.47 mm and 0.38 mm, respectively. There was a significant difference in the uncalcified FC thickness, but not in the calcified FC thickness. The bony trabeculae showed a prominent orientation parallel to the ACL fiber on both sides, but it was more intense on the tibial side than on the femoral side. The trabecular orientation was prominent in the proximal-posterior part of the femoral side and in the anteromedial part of the tibial side, suggesting that mechanical stress is greater in these parts. The dominant bony trabecular angle was 69.0° on the femoral side and 59.8° on the tibial side when the tidemark was set at 0°. These findings suggest that the femoral side receives multidirectional stresses, while relatively unidirectional stress is loaded on the tibial side. Furthermore, stress is considered to be greater in the proximal-posterior part of the femoral insertion and in the anteromedial part of the tibial insertion. At the time of ACL reconstruction, attention should be paid to assign a robust portion of the graft to the posterior part of the femoral insertion and anteromedial part of the tibial insertion. Clin. Anat., 33:988-996, 2020. © 2019 Wiley Periodicals, Inc.

Mobility of Molecular Motors Regulates Contractile Behaviors of Actin Networks.

Cells generate mechanical forces primarily from interactions between F-actin, cross-linking proteins, myosin motors, and other actin-binding proteins in the cytoskeleton. To understand how molecular interactions between the cytoskeletal elements generate forces, a number of in vitro experiments have been performed but are limited in their ability to accurately reproduce the diversity of motor mobility. In myosin motility assays, myosin heads are fixed on a surface and glide F-actin. By contrast, in reconstituted gels, the motion of both myosin and F-actin is unrestricted. Because only these two extreme conditions have been used, the importance of mobility of motors for network behaviors has remained unclear. In this study, to illuminate the impacts of motor mobility on the contractile behaviors of the actin cytoskeleton, we employed an agent-based computational model based on Brownian dynamics. We find that if motors can bind to only one F-actin like myosin I, networks are most contractile at intermediate mobility. In this case, less motor mobility helps motors stably pull F-actins to generate tensile forces, whereas higher motor mobility allows F-actins to aggregate into larger clustering structures. The optimal intermediate motor mobility depends on the stall force and affinity of motors that are regulated by mechanochemical rates. In addition, we find that the role of motor mobility can vary drastically if motors can bind to a pair of F-actins. A network can exhibit large contraction with high motor mobility because motors bound to antiparallel pairs of F-actins can exert similar forces regardless of their mobility. Results from this study imply that the mobility of molecular motors may critically regulate contractile behaviors of actin networks in cells.

Talin is required to increase stiffness of focal molecular complex in its early formation process.

For cellular adaptation in mechanical environments, it is important to consider transmission of forces from the outside to the inside of cells via a focal molecular complex. The focal molecular complex, which consists of integrin, talin, vinculin and actin, is known to form in response to a force applied via the extra-cellular matrix (ECM). In the early formation process of the complex, the complex-actin connection is reinforced. These structural changes of the nascent complex result in an increase in its mechanical integrity and overall stiffness, possibly leading to the maturation of the nascent complex by enhancing force transmission. In this study, we hypothesized that the complex component talin is a crucial factor in increasing the stiffness of the nascent complex. To test the hypothesis, we used atomic force microscopy (AFM) to measure the stiffness of the nascent complex using a probe coated with fibronectin. Stiffness measurements were conducted for intact and talin knocked-down cells. Our results demonstrated that talin was required to increase the stiffness of the nascent complex, which could be caused by the reinforced connection between the complex and actin filaments mediated by talin.

Functional Investigation of a Non-coding Variant Associated with Adolescent Idiopathic Scoliosis in Zebrafish: Elevated Expression of the Ladybird Homeobox Gene Causes Body Axis Deformation.

Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of embryonic and subsequent growth in zebrafish.

Nano-mechanical characterization of tension-sensitive helix bundles in talin rod.

Tension-induced exposure of a cryptic signaling binding site is one of the most fundamental mechanisms in molecular mechanotransduction. Helix bundles in rod domains of talin, a tension-sensing protein at focal adhesions, unfurl under tension to expose cryptic vinculin binding sites. Although the difference in their mechanical stabilities would determine which helix bundle is tension-sensitive, their respective mechanical behaviors under tension have not been characterized. In this study, we evaluated the mechanical behaviors of residues 486-654 and 754-889 of talin, which form helix bundles with low and high tension-sensitivity, by employing AFM nano-tensile testing. As a result, residues 754-889 exhibited lower unfolding energy for complete unfolding than residues 486-654. In addition, we found that residues 754-889 transition into intermediate conformations under lower tension than residues 486-654. Furthermore, residues 754-889 showed shorter persistence length in the intermediate conformation than residues 486-654, suggesting that residues 754-889 under tension exhibit separated α-helices, while residues 486-654 assume a compact conformation with inter-helix interactions. Therefore, we suggest that residues 754-889 of talin work as a tension-sensitive domain to recruit vinculin at the early stage of focal adhesion development, while residues 486-654 contribute to rather robust tension-sensitivity by recruiting vinculin under high tension.

Mechanical role of the spatial patterns of contractile cells in invagination of growing epithelial tissue.

Epithelial invagination is one of the fundamental deformation modes during morphogenesis, and is essential for deriving the three-dimensional shapes of organs from a flat epithelial sheet. Invagination occurs in an orderly manner according to the spatial pattern of the contractile cells; however, it remains elusive how tissue deformation can be caused by cellular activity in the patterned region. In this study, we investigated the mechanical role of the spatial patterns of the contractile cells in invagination of growing tissue using multicellular dynamics simulations. We found that cell proliferation and apical constriction were responsible for expanding the degree of tissue deformation and determining the location of the deformation, respectively. The direction of invagination depended on the spatial pattern of the contractile cells. Further, comparing the simulation results of surface and line contractions as possible modes of apical constriction, we found that the direction of invagination differed between these two modes even if the spatial pattern was the same. These results indicate that the buckling of the epithelial cell sheet caused by cell proliferation causes the invagination, with the direction and location determined by the configuration of the wedge-shaped cells given by the spatial pattern of the contractile cells.

In vitro tubulogenesis of Madin-Darby canine kidney (MDCK) spheroids occurs depending on constituent cell number and scaffold gel concentration.

In vitro tubulogenesis has been employed as an experimental model system to study tissue morphogenesis of internal organs. It has been previously shown that Madin-Darby canine kidney (MDCK) cells form tubes in the presence of hepatocyte growth factor in 3D cultures. Although these cells are expected to form tube structures in some microenvironments independent of chemical stimulation, little is known about the cellular mechanisms in organizing such an anisotropic multicellular structure. Here, we report 3D culture conditions that induce MDCK tubulogenesis without growth factor stimulation. We found that the cells spontaneously form elongated tube structures through aggregation processes in a specific range of both constituent cell number and scaffold gel concentration, while they form spherical aggregates in other conditions. We then examined cellular activities affecting tubulogenesis and showed that cell proliferation is not required for the tube elongation. Furthermore, we revealed that cells in the tube tips generate traction forces and pull the surrounding scaffold gel to migrate, resulting in the tube elongation. Our results suggest that the constituent cells during the aggregation process interact each other via mechanical forces transmitted in the scaffold gel, leading to the spontaneous tube formation.

Self-organizing optic-cup morphogenesis in three-dimensional culture.

Balanced organogenesis requires the orchestration of multiple cellular interactions to create the collective cell behaviours that progressively shape developing tissues. It is currently unclear how individual, localized parts are able to coordinate with each other to develop a whole organ shape. Here we report the dynamic, autonomous formation of the optic cup (retinal primordium) structure from a three-dimensional culture of mouse embryonic stem cell aggregates. Embryonic-stem-cell-derived retinal epithelium spontaneously formed hemispherical epithelial vesicles that became patterned along their proximal-distal axis. Whereas the proximal portion differentiated into mechanically rigid pigment epithelium, the flexible distal portion progressively folded inward to form a shape reminiscent of the embryonic optic cup, exhibited interkinetic nuclear migration and generated stratified neural retinal tissue, as seen in vivo. We demonstrate that optic-cup morphogenesis in this simple cell culture depends on an intrinsic self-organizing program involving stepwise and domain-specific regulation of local epithelial properties.

Spontaneous anterior arch fracture of the atlas following C1 laminectomy without fusion: A report of three cases and finite element analysis.

BACKGROUND: Only four cases of anterior arch fracture after C1 laminectomy without fusion have been previously reported. Although atlas fractures commonly occur in response to high-energy trauma, no obvious trauma that could cause the fracture was observed in these reported cases. The purpose of this study was to elucidate the biomechanical mechanism of anterior arch fracture of the atlas following C1 laminectomy and present three cases of this fracture. METHODS: Three cases of fracture of the anterior arch of the atlas following C1 laminectomy were retrospectively reviewed. Three atlas models (an intact model, a laminectomy model, and a transverse ligament-resected model) were created from computed tomography data of each case using a three-dimensional finite element method. Axial load was applied on the superior facet to mimic four conditions (neutral, flexion, extension, lateral bending). The distribution of von Mises stress in the anterior arch and the displacement of the posterior arch were compared among the three models. RESULTS: In all three cases, the anterior arch fracture clinically occurred after C1 laminectomy despite there being no obvious inciting trauma. During the finite element analysis, increased stress was observed in all postures of the laminectomy model as compared with the intact model. The stress-concentrated location observed in the finite element model was consistent with the fracture sites that were clinically observed. In terms of loading condition, much higher stress was observed in extension and lateral bending as compared with other postures. There were no significant differences in stress distribution between the laminectomy model and the transverse ligament-resected laminectomy model. CONCLUSIONS: Stress distribution concentrates in the anterior arch after C1 laminectomy, leading to fracture of the anterior arch despite no inciting trauma. There may be more frequent occult fractures observed after C1 laminectomy than has been reported. Therefore, surgeons should recognize anterior arch fracture as a possible complication of C1 laminectomy without fusion.

[Modeling and simulation of trabecular pattern formation in a cancellous bone defect under mechanical forces.]

Understanding regeneration of the trabecular structure in cancellous bone defects is an important issue in bone tissue engineering and regenerative medicine. Biochemical and biomechanical viewpoints are indispensable for understanding the fundamental mechanism that underlies the regeneration of the trabecular structure. In vitro observations of the Turing pattern-like bone differentiation into osteoblasts from human mesenchymal stem cells suggest that mathematical modeling and simulation based on a reaction-diffusion system model would help us to understand the mechanism of trabecular pattern formation during cancellous bone regeneration. In this article, we propose a mathematical model of trabecular morphogenesis based on the reaction-diffusion system in 3D, which comprises activators and inhibitors of bone formation by combining with mechanical factors. Based on the proposed model, we conduct computational simulation of trabecular regeneration in a cancellous bone defect using a voxel-based finite element method for stress analysis and a finite difference method for reaction-diffusion analysis. The proposed model could express the regeneration of the three-dimensional trabecular structure with mechanically adapted functions as a load-bearing structure. Based on these results, the proposed model and simulation framework are expected to facilitate the analysis of regeneration of the cancellous bone;this will help us to examine bone regeneration that involve complex biological factors.

A Novel Osteoblast/Osteocyte Selection Method in Primary Isolated Chick Bone Cells by Atomic Force Microscopy.

Selection of bone cells, particularly osteoblasts and osteocytes, for analysis of cellular processes and differentiation is a very important issue because bone remodeling is a highly complex and harmonized process, which includes molecular and cellular interactions and communications. In this study, we introduce a novel osteoblast and osteocyte selection method that uses atomic force microscopy and OB7.3, an antibody of Phex, which is a specific protein marker expressed on the surface of osteocytes. The elasticity and Phex expression levels were simultaneously detected by force spectroscopy using the OB7.3-modified atomic force microscopy probe on the bone cell surface. The elastic modulus was different between osteoblasts and osteocytes. Phex expression level was analyzed by the distribution of Phex-OB7.3 rupturing.