Mechanics of human embryo compaction

The shaping of the human embryo begins with compaction, during which cells come into close contact and form a tighter structure. Assisted reproductive technology (ART) studies suggest that human embryos fail compaction primarily because of defective adhesion. Based on our current understanding of animal morphogenesis, other morphogenetic engines, such as cell contractility, could be involved in shaping the human embryo. However, the molecular, cellular and physical mechanisms driving human embryo morphogenesis remain uncharacterized. Using micropipette aspiration on human embryos donated to research, we have mapped cell surface tensions during compaction. This reveals a 4-fold increase of tension at the cell-medium interface while cell-cell contacts keep a steady tension. Comparison between human and mouse reveals qualitatively similar but quantitively different mechanical strategies, with human embryos being mechanically least efficient. Inhibition of cell contractility and cell-cell adhesion in human embryos reveal that only contractility controls the surface tension responsible for compaction. Interestingly, if both cellular processes are required for compaction, they exhibit distinct mechanical signatures when faulty. Analyzing the mechanical signature of naturally failing embryos, we find evidence that non-compacting embryos or partially compacting embryos with excluded cells have defective contractility. Together, our study reveals that an evolutionarily conserved increase in cell contractility is required to generate the forces driving the first morphogenetic movement shaping the human body.

Hippo Pathway Effector Tead1 Induces Cardiac Fibroblast to Cardiomyocyte Reprogramming

Background The conversion of fibroblasts into induced cardiomyocytes may regenerate myocardial tissue from cardiac scar through in situ cell transdifferentiation. The efficiency transdifferentiation is low, especially for human cells. We explored the leveraging of Hippo pathway intermediates to enhance induced cardiomyocyte generation. Methods and Results We screened Hippo effectors Yap (yes‐associated protein), Taz (transcriptional activator binding domain), and Tead1 (TEA domain transcription factor 1; Td) for their reprogramming efficacy with cardio‐differentiating factors Gata4, Mef2C, and Tbx5 (GMT). Td induced nearly 3‐fold increased expression of cardiomyocyte marker cTnT (cardiac troponin T) by mouse embryonic and adult rat fibroblasts versus GMT administration alone ( P <0.0001), while Yap and Taz failed to enhance cTnT expression. Serial substitution demonstrated that Td replacement of TBX5 induced the greatest cTnT expression enhancement and sarcomere organization in rat fibroblasts treated with all GMT substitutions (GMTd versus GMT: 17±1.2% versus 5.4±0.3%, P <0.0001). Cell contractility (beating) was seen in 6% of GMTd‐treated cells by 4 weeks after treatment, whereas no beating GMT‐treated cells were observed. Human cardiac fibroblasts likewise demonstrated increased cTnT expression with GMTd versus GMT treatment (7.5±0.3% versus 3.0±0.3%, P <0.01). Mechanistically, GMTd administration increased expression of the trimethylated lysine 4 of histone 3 (H3K4me3) mark at the promoter regions of cardio‐differentiation genes and mitochondrial biogenesis regulator genes in rat and human fibroblast, compared with GMT. Conclusions These data suggest that the Hippo pathway intermediate Tead1 is an important regulator of cardiac reprogramming that increases the efficiency of maturate induced cardiomyocytes generation and may be a vital component of human cardiodifferentiation strategies.

NF-кB c-Rel modulates pre-fibrotic changes in human fibroblasts

Skin fibrosis is one central hallmark of the heterogeneous autoimmune disease systemic sclerosis. So far, there are hardly any standardized and effective treatment options. Pathogenic mechanisms underlying fibrosis comprise excessive and uncontrolled myofibroblast differentiation, increased extracellular matrix protein (ECM) synthesis and an intensification of the forces exerted by the cytoskeleton. A deeper understanding of fibroblast transformation could help to prevent or reverse fibrosis by specifically interfering with abnormally regulated signaling pathways. The transcription factor NF-κB has been implicated in the progression of fibrotic processes. However, the cellular processes regulated by NF-κB in fibrosis as well as the NF-κB isoforms preferentially involved are still completely unknown. In an in vitro model of fibrosis, we consistently observed the induction of the c-Rel subunit of NF-κB. Functional abrogation of c-Rel by siRNA resulted in diminished cell contractility of dermal fibroblasts in relaxed, but not in stressed 3D collagen matrices. Furthermore, directed migration was reduced after c-Rel silencing and total N-cadherin expression level was diminished, possibly mediating the observed cellular defects. Therefore, NF-кB c-Rel impacts central cellular adhesion markers and processes which negatively regulate fibrotic progression in SSc pathophysiology.

Non-coding function for mRNAs in Focal Adhesion Architecture and Mechanotransduction

Messenger RNA (mRNA) compartmentalization within the cytosol is well-recognized as a key mechanism of local translation-mediated regulation of protein levels, but whether such localization could be a means of exercising non-coding mRNA function is unknown. Here, we explore non-coding functions for mRNAs associated with focal adhesions (FAs), cellular structures responsible for mediating cell adhesion and response to changes in the extracellular matrix (ECM). Using high-throughput single molecule imaging and genomic profiling approaches, we find that mRNAs with distinct sequence characteristics localize to FAs in different human cell types. Notably, ∼85% of FA-mRNAs are not translationally active at steady state or under conditions of FA dissolution or activation. Untranslated mRNA sequences are anchored to FA based on their functional states by the RNA binding protein, G3BP1, forming biomolecular granules. Removing RNA or G3BP1, but not blocking new polypeptide synthesis, dramatically changes FA protein composition and organization, resulting in loss of cell contractility and cellular ability to adapt to changing ECM. We have therefor uncovered a novel, non-coding role for mRNAs as scaffolds to maintain FA structure and function, broadening our understating of noncanonical mRNA functions.

Electrospun fibre diameter and its effects on vascular smooth muscle cells

Bypass grafting is a technique used in the treatment of vascular disease, which is currently the leading cause of mortality worldwide. While technology has moved forward over the years, synthetic grafts still show significantly lower rates of patency in small diameter bypass operations compared to the gold standard (autologous vessel grafts). Scaffold morphology plays an important role in vascular smooth muscle cell (VSMC) performance, with studies showing how fibre alignment and surface roughness can modulate phenotypic and genotypic changes. Herein, this study has looked at how the fibre diameter of electrospun polymer scaffolds can affect the performance of seeded VSMCs. Four different scaffolds were electrospun with increasing fibre sizes ranging from 0.75 to 6 µm. Culturing VSMCs on the smallest fibre diameter (0.75 µm) lead to a significant increase in cell viability after 12 days of culture. Furthermore, interesting trends were noted in the expression of two key phenotypic genes associated with mature smooth muscle cell contractility (myocardin and smooth muscle alpha-actin 1), whereby reducing the fibre diameter lead to relative upregulations compared to the larger fibre diameters. These results showed that the smallest (0.75 µm) fibre diameter may be best suited for the culture of VSMCs with the aim of increasing cell proliferation and aiding cell maturity.

Computational model of 3D cell migration based on the molecular clutch mechanism

The external environment is a regulator of cell activity. Its stiffness and microstructure can either facilitate or prevent 3D cell migration in both physiology and disease. 3D cell migration results from force feedbacks between the cell and the extracellular matrix (ECM). Adhesions regulate these force feedbacks by working as molecular clutches that dynamically bind and unbind the ECM. Because of the interdependency between ECM properties, adhesion dynamics, and cell contractility, how exactly 3D cell migration occurs in different environments is not fully understood. In order to elucidate the effect of ECM on 3D cell migration through force-sensitive molecular clutches, we developed a computational model based on a lattice point approach. Results from the model show that increases in ECM pore size reduce cell migration speed. In contrast, matrix porosity increases it, given a sufficient number of ligands for cell adhesions and limited crowding of the matrix from cell replication. Importantly, these effects are maintained across a range of ECM stiffnesses’, demonstrating that mechanical factors are not responsible for how matrix microstructure regulates cell motility.

Condensation tendency and planar isotropic actin gradient induce radial alignment in confined monolayers

A monolayer of highly motile cells can establish long-range orientational order, which can be explained by hydrodynamic theory of active gels and fluids. However, it is less clear how cell shape changes and rearrangement are governed when the monolayer is in mechanical equilibrium states when cell motility diminishes. In this work, we report that rat embryonic fibroblasts (REF), when confined in circular mesoscale patterns on rigid substrates, can transition from the spindle shapes to more compact morphologies. Cells align radially only at the pattern boundary when they are in the mechanical equilibrium. This radial alignment disappears when cell contractility or cell-cell adhesion is reduced. Unlike monolayers of spindle-like cells such as NIH-3T3 fibroblasts with minimal intercellular interactions or epithelial cells like Madin-Darby canine kidney (MDCK) with strong cortical actin network, confined REF monolayers present an actin gradient with isotropic meshwork, suggesting the existence of a stiffness gradient. In addition, the REF cells tend to condense on soft substrates, a collective cell behavior we refer to as the 'condensation tendency'. This condensation tendency, together with geometrical confinement, induces tensile prestretch (i.e., an isotropic stretch that causes tissue to contract when released) to the confined monolayer. By developing a Voronoi-cell model, we demonstrate that the combined global tissue prestretch and cell stiffness differential between the inner and boundary cells can sufficiently define the cell radial alignment at the pattern boundary.

An Outside-In Switch in Integrin Signaling Caused by Chemical and Mechanical Signals in Reactive Astrocytes

Astrocyte reactivity is associated with poor repair capacity after injury to the brain, where chemical and physical changes occur in the damaged zone. Astrocyte surface proteins, such as integrins, are upregulated, and the release of pro-inflammatory molecules and extracellular matrix (ECM) proteins upon damage generate a stiffer matrix. Integrins play an important role in triggering a reactive phenotype in astrocytes, and we have reported that αVβ3 Integrin binds to the Thy-1 (CD90) neuronal glycoprotein, increasing astrocyte contractility and motility. Alternatively, αVβ3 Integrin senses mechanical forces generated by the increased ECM stiffness. Until now, the association between the αVβ3 Integrin mechanoreceptor response in astrocytes and changes in their reactive phenotype is unclear. To study the response to combined chemical and mechanical stress, astrocytes were stimulated with Thy-1-Protein A-coated magnetic beads and exposed to a magnetic field to generate mechanical tension. We evaluated the effect of such stimulation on cell adhesion and contraction. We also assessed traction forces and their effect on cell morphology, and integrin surface expression. Mechanical stress accelerated the response of astrocytes to Thy-1 engagement of integrin receptors, resulting in cell adhesion and contraction. Astrocyte contraction then exerted traction forces onto the ECM, inducing faster cell contractility and higher traction forces than Thy-1 alone. Therefore, cell-extrinsic chemical and mechanical signals regulate in an outside-in manner, astrocyte reactivity by inducing integrin upregulation, ligation, and signaling events that promote cell contraction. These changes in turn generate cell-intrinsic signals that increase traction forces exerted onto the ECM (inside-out). This study reveals αVβ3 Integrin mechanoreceptor as a novel target to regulate the harmful effects of reactive astrocytes in neuronal healing.

Repression of Smad4 by MicroRNA-1285 moderates TGF-β-induced epithelial–mesenchymal transition in proliferative vitreoretinopathy

The purpose of this study was to assess whether microRNA (miR)-1285 can suppress the epithelial–mesenchymal transition (EMT) in retinal pigment epithelial cells. Expression of miR-1285 was evaluated using quantitative real-time polymerase chain reaction (RT-qPCR). The features of EMT were assessed using Western blotting, immunocytochemical staining, scratch wound healing tests, modified Boyden chamber assay, and collagen gel contraction assay. A rabbit model of proliferative vitreoretinopathy (PVR) was used for in vivo testing, which involved the induction of PVR by injection of transfected ARPE cells into the vitreous chamber. Luciferase reporter assay was performed to identify the putative target of miR-1285. The expression of miR-1285 was downregulated in ARPE-19 cells treated with transforming growth factor (TGF)-β. Overexpression of miR-1285 led to upregulation of zonula occludens-1, downregulation of α-smooth muscle actin and vimentin, cell migration and cell contractility—all EMT features—in the TGF-β2-treated ARPE-19 cells. The reporter assay indicated that the 3′ untranslated region of Smad4 was the direct target of miR1285. PVR progression was alleviated in the miR-1285 transfected rabbits. In conclusion, overexpression of miR-1285 attenuates TGF-β2-induced EMT in a rabbit model of PVR, and the effect of miR-1285 in PVR is dependent on Smad4. Further research is warranted to develop a feasible therapeutic approach for the prevention and treatment of PVR.