bims-ecemfi 2024-10-13 papers

bims-ecemfi

Biomed News

on ECM and fibroblasts

Issue of 2024–10–13
29 papers selected by
Badri Narayanan Narasimhan, University of California, San Diego

An Optical System for Cellular Mechanostimulation in 3D Hydrogels.
Hydrogels with Independently Controlled Adhesion Ligand Mobility and Viscoelasticity Increase Cell Adhesion and Spreading.
YAP/TAZ drives Notch and angiogenesis mechanoregulation in silico.
Restoring mechanophenotype reverts malignant properties of ECM-enriched vocal fold cancer.
Force-mediated recruitment and reprogramming of healthy endothelial cells drive vascular lesion growth.
Strain-dependent glutathionylation of fibronectin fibers impacts mechano-chemical behavior and primes an integrin switch.
Mechanical Profiling of Biopolymer Condensates through Acoustic Trapping.
Gastric Cancer Actively Remodels Mechanical Microenvironment to Promote Chemotherapy Resistance via MSCs-Mediated Mitochondrial Transfer.
Ex vivo SIM-AFM measurements reveal the spatial correlation of stiffness and molecular distributions in 3D living tissue.
Jamming of nephron-forming niches in the developing mouse kidney creates cyclical mechanical stresses.
Myosin II tension sensors visualize force generation within the actin cytoskeleton in living cells.
Probing the Nanonewton Mitotic Cell Deformation Force by Ion-Resonance-Enhanced Photonics Force Microscopy.
Chemo-mechanical model of cell polarization initiated by structural polarity.
N-cadherin crosstalk with integrin weakens the molecular clutch in response to surface viscosity.
Transient Notch Activation Converts Pluripotent Stem Cell-Derived Cardiomyocytes Towards a Purkinje Fiber Fate.
Cell density couples tissue mechanics to control the elongation speed of the body axis.
Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells.
Controlled release of microorganisms from engineered living materials.
Microfluidic Modulation of Microvasculature in Microdissected Tumors.
The "weaken-fill-repair" model for cell budding: Linking cell wall biosynthesis with mechanics.
An engineered model of metastatic colonization of human bone marrow reveals breast cancer cell remodeling of the hematopoietic niche.
Microvascular Engineering for the Development of a Nonembedded Liver Sinusoid with a Lumen: When Endothelial Cells Do Not Lose Their Edge.
Stiffening of the Cytoplasm in Response to Intracellularly Applied Forces.
Tuning a Bioengineered Hydrogel for Studying Astrocyte Reactivity in Glioblastoma.
Obesity-driven changes in breast tissue exhibit a pro-angiogenic extracellular matrix signature.
Actin-driven nanotopography promotes stable integrin adhesion formation in developing tissue.
Functional heterogeneity of meniscal fibrochondrocytes and microtissue models is dependent on modality of fibrochondrocyte isolation.
In vitro vascularization of 3D cell aggregates in microwells with integrated vascular beds.
Diabetes induces modifications in costameric proteins and increases cardiomyocyte stiffness.

Acta Biomater. 2024 Oct 03. pii: S1742-7061(24)00578-6. [Epub ahead of print]

An Optical System for Cellular Mechanostimulation in 3D Hydrogels.

Rahul Sreedasyam, Bryce G Wilson, Patricia R Ferrandez, Elliot L Botvinick, Vasan Venugopalan.

  We introduce a method utilizing single laser-generated cavitation bubbles to stimulate cellular mechanotransduction in dermal fibroblasts embedded within 3D hydrogels. We demonstrate that fibroblasts embedded in either amorphous or fibrillar hydrogels engage in Ca2+ signaling following exposure to an impulsive mechanical stimulus provided by a single 250µm diameter laser-generated cavitation bubble. We find that the spatial extent of the cellular signaling is larger for cells embedded within a fibrous collagen hydrogel as compared to those embedded within an amorphous polyvinyl alcohol polymer (SLO-PVA) hydrogel. Additionally, for fibroblasts embedded in collagen, we find an increased range of cellular mechanosensitivity for cells that are polarized relative to the radial axis as compared to the circumferential axis. By contrast, fibroblasts embedded within SLO-PVA did not display orientation-dependent mechanosensitivity. Fibroblasts embedded in hydrogels and cultured in calcium-free media did not show cavitation-induced mechanotransduction; implicating calcium signaling based on transmembrane Ca2+ transport. This study demonstrates the utility of single laser-generated cavitation bubbles to provide local non-invasive impulsive mechanical stimuli within 3D hydrogel tissue models with concurrent imaging using optical microscopy. STATEMENT OF SIGNIFICANCE: : Currently, there are limited methods for the non-invasive real-time assessment of cellular sensitivity to mechanical stimuli within 3D tissue scaffolds. We describe an original approach that utilizes a pulsed laser microbeam within a standard laser scanning microscope system to generate single cavitation bubbles to provide impulsive mechanostimulation to cells within 3D fibrillar and amorphous hydrogels. Using this technique, we measure the cellular mechanosensitivity of primary human dermal fibroblasts embedded in amorphous and fibrillar hydrogels, thereby providing a useful method to examine cellular mechanotransduction in 3D biomaterials. Moreover, the implementation of our method within a standard optical microscope makes it suitable for broad adoption by cellular mechanotransduction researchers and opens the possibility of high-throughput evaluation of biomaterials with respect to cellular mechanosignaling.

Keywords:  3D hydrogels; cavitation bubble; cellular mechanosignaling; cellular mechanotransduction; collagen; laser microbeam; polyvinyl alcohol

DOI:  https://doi.org/10.1016/j.actbio.2024.09.050
bioRxiv. 2024 Sep 24. pii: 2024.09.23.614501. [Epub ahead of print]

Hydrogels with Independently Controlled Adhesion Ligand Mobility and Viscoelasticity Increase Cell Adhesion and Spreading.

Abolfazl Salehi Moghaddam, Katelyn Dunne, Wendy Breyer, Yingjie Wu, E Thomas Pashuck.

A primary objective in designing hydrogels for cell culture is recreating the cell-matrix interactions found within human tissues. Identifying the most important biomaterial features for these interactions is challenging because it is difficult to independently adjust variables such as matrix stiffness, stress relaxation, the mobility of adhesion ligands and the ability of these ligands to support cellular forces. In this work we designed a hydrogel platform consisting of interpenetrating polymer networks of covalently crosslinked poly(ethylene glycol) (PEG) and self-assembled peptide amphiphiles (PA). We can tailor the storage modulus of the hydrogel by altering the concentration and composition of each network, and we can tune the stress relaxation half-life through the non-covalent bonding in the PA network. Ligand mobility can be adjusted independently of the matrix mechanical properties by attaching the RGD cell adhesion ligand to either the covalent PEG network, the dynamic PA network, or both networks at once. Interestingly, our findings show that endothelial cell adhesion formation and spreading is maximized in soft, viscoelastic gels in which RGD adhesion ligands are present on both the covalent PEG and non-covalent PA networks. The dynamic nature of cell adhesion domains, coupled with their ability to exert substantial forces on the matrix, suggests that having different presentations of RGD ligands which are either mobile or are capable of withstanding significant forces are needed mimic different aspects of complex cell-matrix adhesions. By demonstrating how different presentations of RGD ligands affect cell behavior independently of viscoelastic properties, these results contribute to the rational design of hydrogels that facilitate desired cell-matrix interactions, with the potential of improving in vitro models and regenerative therapies.

DOI: https://doi.org/10.1101/2024.09.23.614501
NPJ Syst Biol Appl. 2024 Oct 05. 10(1): 116

YAP/TAZ drives Notch and angiogenesis mechanoregulation in silico.

Margot Passier, Katie Bentley, Sandra Loerakker, Tommaso Ristori.

Endothelial cells are key players in the cardiovascular system. Among other things, they are responsible for sprouting angiogenesis, the process of new blood vessel formation essential for both health and disease. Endothelial cells are strongly regulated by the juxtacrine signaling pathway Notch. Recent studies have shown that both Notch and angiogenesis are influenced by extracellular matrix stiffness; however, the underlying mechanisms are poorly understood. Here, we addressed this challenge by combining computational models of Notch signaling and YAP/TAZ, stiffness- and cytoskeleton-regulated mechanotransducers whose activity inhibits both Dll4 (Notch ligand) and LFng (Notch-Dll4 binding modulator). Our simulations successfully mimicked previous experiments, indicating that this YAP/TAZ-Notch crosstalk elucidates the Notch and angiogenesis mechanoresponse to stiffness. Additional simulations also identified possible strategies to control Notch activity and sprouting angiogenesis via cytoskeletal manipulations or spatial patterns of alternating stiffnesses. Our study thus inspires new experimental avenues and provides a promising modeling framework for further investigations into the role of Notch, YAP/TAZ, and mechanics in determining endothelial cell behavior during angiogenesis and similar processes.

DOI: https://doi.org/10.1038/s41540-024-00444-3
bioRxiv. 2024 Aug 23. pii: 2024.08.22.609159. [Epub ahead of print]

Restoring mechanophenotype reverts malignant properties of ECM-enriched vocal fold cancer.

Jasmin Kaivola, Karolina Punovuori, Megan R Chastney, Yekaterina A Miroshnikova, Hind Abdo, Fabien Bertillot, Fabian Krautgasser, Jasmin Di Franco, James R W Conway, Gautier Follain, Jaana Hagström, Antti Mäkitie, Heikki Irjala, Sami Ventelä, Hellyeh Hamidi, Giorgio Scita, Roberto Cerbino, Sara A Wickström, Johanna Ivaska.

Increased extracellular matrix (ECM) and matrix stiffness promote solid tumor progression. However, mechanotransduction in cancers arising in mechanically active tissues remains underexplored. Here, we report upregulation of multiple ECM components accompanied by tissue stiffening in vocal fold cancer (VFC). We compare non-cancerous (NC) and patient- derived VFC cells - from early (mobile, T1) to advanced-stage (immobile, T3) cancers - revealing an association between VFC progression and cell-surface receptor heterogeneity, reduced laminin-binding integrin cell-cell junction localization and a flocking mode of collective cell motility. Mimicking physiological movement of healthy vocal fold tissue (stretching/vibration), decreases oncogenic nuclear β-catenin and YAP levels in VFC. Multiplex immunohistochemistry of VFC tumors uncovered a correlation between ECM content, nuclear YAP and patient survival, concordant with VFC sensitivity to YAP-TEAD inhibitors in vitro. Our findings present evidence that VFC is a mechanically sensitive malignancy and restoration of tumor mechanophenotype or YAP/TAZ targeting, represents a tractable anti-oncogenic therapeutic avenue for VFC.

DOI: https://doi.org/10.1101/2024.08.22.609159
Nat Commun. 2024 Oct 06. 15(1): 8660

Force-mediated recruitment and reprogramming of healthy endothelial cells drive vascular lesion growth.

Apeksha Shapeti, Jorge Barrasa-Fano, Abdel Rahman Abdel Fattah, Janne de Jong, José Antonio Sanz-Herrera, Mylène Pezet, Said Assou, Emilie de Vet, Seyed Ali Elahi, Adrian Ranga, Eva Faurobert, Hans Van Oosterwyck.

Force-driven cellular interactions are crucial for cancer cell invasion but remain underexplored in vascular abnormalities. Cerebral cavernous malformations (CCM), a vascular abnormality characterized by leaky vessels, involves CCM mutant cells recruiting wild-type endothelial cells to form and expand mosaic lesions. The mechanisms behind this recruitment remain poorly understood. Here, we use an in-vitro model of angiogenic invasion with traction force microscopy to reveal that hyper-angiogenic Ccm2-silenced endothelial cells enhance angiogenic invasion of neighboring wild-type cells through force and extracellular matrix-guided mechanisms. We demonstrate that mechanically hyperactive CCM2-silenced tips guide wild-type cells by transmitting pulling forces and by creating paths in the matrix, in a ROCKs-dependent manner. This is associated with reinforcement of β1 integrin and actin cytoskeleton in wild-type cells. Further, wild-type cells are reprogrammed into stalk cells and activate matrisome and DNA replication programs, thereby initiating proliferation. Our findings reveal how CCM2 mutants hijack wild-type cell functions to fuel lesion growth, providing insight into the etiology of vascular malformations. By integrating biophysical and molecular techniques, we offer tools for studying cell mechanics in tissue heterogeneity and disease progression.

DOI: https://doi.org/10.1038/s41467-024-52866-6
Nat Commun. 2024 Oct 09. 15(1): 8751

Strain-dependent glutathionylation of fibronectin fibers impacts mechano-chemical behavior and primes an integrin switch.

Wei Li, Leandro Moretti, Xinya Su, Chiuan-Ren Yeh, Matthew P Torres, Thomas H Barker.

The extracellular matrix (ECM) is a protein polymer network that physically supports cells within a tissue. It acts as an important physical and biochemical stimulus directing cell behaviors. For fibronectin (Fn), a predominant component of the ECM, these physical and biochemical activities are inextricably linked as physical forces trigger conformational changes that impact its biochemical activity. Here, we analyze whether oxidative post-translational modifications, specifically glutathionylation, alter Fn's mechano-chemical characteristics through stretch-dependent protein modification. ECM post-translational modifications represent a potential for time- or stimulus-dependent changes in ECM structure-function relationships that could persist over time with potentially significant impacts on cell and tissue behaviors. In this study, we show evidence that glutathionylation of Fn ECM fibers is stretch-dependent and alters Fn fiber mechanical properties with implications on the selectivity of engaging integrin receptors. These data demonstrate the existence of multimodal post-translational modification mechanisms within the ECM with high relevance to the microenvironmental regulation of downstream cell behaviors.

DOI: https://doi.org/10.1038/s41467-024-52742-3
bioRxiv. 2024 Sep 19. pii: 2024.09.16.613217. [Epub ahead of print]

Mechanical Profiling of Biopolymer Condensates through Acoustic Trapping.

Kichitaro Nakajima, Tomas Sneideris, Lydia L Good, Nadia A Erkamp, Hirotsugu Ogi, Tuomas P J Knowles.

Characterizing the mechanical properties of single colloids is a central problem in soft matter physics. It also plays a key role in cell biology through biopolymer condensates, which function as membraneless compartments. Such systems can also malfunction, leading to the onset of a number of diseases, including many neurodegenerative diseases; the functional and pathological condensates are commonly differentiated by their mechanical signature. Probing the mechanical properties of biopolymer condensates at the single particle level has, however, remained challenging. In this study, we demonstrate that acoustic trapping can be used to profile the mechanical properties of single condensates in a contactless manner. We find that acoustic fields exert the acoustic radiation force on condensates, leading to their migration to a trapping point where acoustic potential energy is minimized. Furthermore, our results show that the Brownian motion fluctuation of condensates in an acoustic potential well is an accurate probe for their bulk modulus. We demonstrate that this framework can detect the change in the bulk modulus of polyadenylic acid condensates in response to changes in environmental conditions. Our results show that acoustic trapping opens up a novel path to profile the mechanical properties of soft colloids at the single particle level in a non-invasive manner with applications in biology, materials science, and beyond.

DOI: https://doi.org/10.1101/2024.09.16.613217
Adv Sci (Weinh). 2024 Oct 11. e2404994

Gastric Cancer Actively Remodels Mechanical Microenvironment to Promote Chemotherapy Resistance via MSCs-Mediated Mitochondrial Transfer.

Xin He, Li Zhong, Nan Wang, Baiwei Zhao, Yannan Wang, Xinxiang Wu, Changyu Zheng, Yueheng Ruan, Jianfeng Hou, Yusheng Luo, Yuehan Yin, Yulong He, Andy Peng Xiang, Jiancheng Wang.

  Chemotherapy resistance is the main reason of treatment failure in gastric cancer (GC). However, the mechanism of oxaliplatin (OXA) resistance remains unclear. Here, we demonstrate that extracellular mechanical signaling plays crucial roles in OXA resistance within GC. We selected OXA-resistant GC patients and analyzed tumor tissues by single-cell sequencing, and found that the mitochondrial content of GC cells increased in a biosynthesis-independent manner. Moreover, we found that the increased mitochondria of GC cells were mainly derived from mesenchymal stromal cells (MSCs), which could repair the mitochondrial function and reduce the levels of mitophagy in GC cells, thus leading to OXA resistance. Furthermore, we investigated the underlying mechanism and found that mitochondrial transfer was mediated by mechanical signals of the extracellular matrix (ECM). After OXA administration, GC cells actively secreted ECM in the tumor microenvironment (TEM), increasing matrix stiffness of the tumor tissues, which promoted mitochondria to transfer from MSCs to GC cells via microvesicles (MVs). Meanwhile, inhibiting the mechanical-related RhoA/ROCK1 pathway could alleviate OXA resistance in GC cells. In summary, these results indicate that matrix stiffness could be used as an indicator to identify chemotherapy resistance, and targeting mechanical-related pathway could effectively alleviate OXA resistance and improve therapeutic efficacy.

Keywords:  RhoA/ROCK1 signaling pathway; gastric cancer; matrix stiffness; oxaliplatin resistance

DOI:  https://doi.org/10.1002/advs.202404994
Acta Biomater. 2024 Sep 27. pii: S1742-7061(24)00539-7. [Epub ahead of print]

Ex vivo SIM-AFM measurements reveal the spatial correlation of stiffness and molecular distributions in 3D living tissue.

Itsuki Shioka, Ritsuko Morita, Rei Yagasaki, Duligengaowa Wuergezhen, Tadahiro Yamashita, Hironobu Fujiwara, Satoru Okuda.

  Living tissues each exhibit a distinct stiffness, which provides cells with key environmental cues that regulate their behaviors. Despite this significance, our understanding of the spatiotemporal dynamics and the biological roles of stiffness in three-dimensional tissues is currently limited due to a lack of appropriate measurement techniques. To address this issue, we propose a new method combining upright structured illumination microscopy (USIM) and atomic force microscopy (AFM) to obtain precisely coordinated stiffness maps and biomolecular fluorescence images of thick living tissue slices. Using mouse embryonic and adult skin as a representative tissue with mechanically heterogeneous structures inside, we validate the measurement principle of USIM-AFM. Live measurement of tissue stiffness distributions revealed the highly heterogeneous mechanical nature of skin, including nucleated/enucleated epithelium, mesenchyme, and hair follicle, as well as the role of collagens in maintaining its integrity. Furthermore, quantitative analysis comparing stiffness distributions in live tissue samples with those in preserved tissues, including formalin-fixed and cryopreserved tissue samples, unveiled the distinct impacts of preservation processes on tissue stiffness patterns. This series of experiments highlights the importance of live mechanical testing of tissue-scale samples to accurately capture the true spatiotemporal variations in mechanical properties. Our USIM-AFM technique provides a new methodology to reveal the dynamic nature of tissue stiffness and its correlation with biomolecular distributions in live tissues and thus could serve as a technical basis for exploring tissue-scale mechanobiology. STATEMENT OF SIGNIFICANCE: Stiffness, a simple mechanical parameter, has drawn attention in understanding the mechanobiological principles underlying the homeostasis and pathology of living tissues. To explore tissue-scale mechanobiology, we propose a technique integrating an upright structured illumination microscope and an atomic force microscope. This technique enables live measurements of stiffness distribution and fluorescent observation of thick living tissue slices. Experiments revealed the highly heterogeneous mechanical nature of mouse embryonic and adult skin in three dimensions and the previously unnoticed influences of preservation techniques on the mechanical properties of tissue at microscopic resolution. This study provides a new technical platform for live stiffness measurement and biomolecular observation of tissue-scale samples with micron-scale resolution, thus contributing to future studies of tissue- and organ-scale mechanobiology.

Keywords:  Atomic force microscopy; Elastic modulus; Embryonic skin; Ex vivo culture; Soft tissue mechanics

DOI:  https://doi.org/10.1016/j.actbio.2024.09.023
Nat Mater. 2024 Oct 09.

Jamming of nephron-forming niches in the developing mouse kidney creates cyclical mechanical stresses.

Louis S Prahl, Jiageng Liu, John M Viola, Aria Zheyuan Huang, Trevor J Chan, Gabriela Hayward-Lara, Catherine M Porter, Chenjun Shi, Jitao Zhang, Alex J Hughes.

Urinary collecting tubules form during kidney embryogenesis through the branching of the ureteric bud epithelium. A travelling mesenchyme niche of nephron progenitor cells caps each branching ureteric bud tip. These 'tip domain' niches pack more closely over developmental time and their number relates to nephron endowment at birth. Yet, how the crowded tissue environment impacts niche number and cell decision-making remains unclear. Here, through experiments and mathematical modelling, we show that niche packing conforms to physical limitations imposed by kidney curvature. We relate packing geometries to rigidity theory to predict a stiffening transition starting at embryonic day 15 in the mouse, validated by micromechanical analysis. Using a method to estimate tip domain 'ages' relative to their most recent branch events, we find that new niches overcome mechanical resistance as they branch and displace neighbours. This creates rhythmic mechanical stress in the niche. These findings expand our understanding of kidney development and inform engineering strategies for synthetic regenerative tissues.

DOI: https://doi.org/10.1038/s41563-024-02019-3
J Cell Sci. 2024 Oct 04. pii: jcs.262281. [Epub ahead of print]

Myosin II tension sensors visualize force generation within the actin cytoskeleton in living cells.

Ryan G Hart, Divya Kota, Fangjia Li, Mengdi Zhang, Diego Ramallo, Andrew J Price, Karla L Otterpohl, Steve J Smith, Alexander R Dunn, Mark O Huising, Jing Liu, Indra Chandrasekar.

  Nonmuscle myosin II generates cytoskeletal forces that drive cell division, embryogenesis, muscle contraction, and many other cellular functions. However, at present there is no method that can directly measure the forces generated by myosins in living cells. Here we describe a Förster resonance energy transfer (FRET)-based tension sensor that can detect myosin associated force along the filamentous actin network. Fluorescence lifetime imaging microscopy (FLIM)-FRET measurements indicate that the forces generated by NMIIB exhibit significant spatial and temporal heterogeneity as a function of donor lifetime and fluorophore energy exchange. These measurements provide a proxy for inferred forces that vary widely along the actin cytoskeleton. This initial report highlights the potential utility of myosin-based tension sensors in elucidating the roles of cytoskeletal contractility in a wide variety of contexts.

Keywords:  Actomyosin; Force in living cells; Tension Sensor

DOI:  https://doi.org/10.1242/jcs.262281
Nano Lett. 2024 Oct 08.

Probing the Nanonewton Mitotic Cell Deformation Force by Ion-Resonance-Enhanced Photonics Force Microscopy.

Xiangjun Di, Dejiang Wang, Xuchen Shan, Lei Ding, Zhaoxiang Zhong, Chaohao Chen, Dajing Wang, Zhiyong Song, Jianyun Wang, Qian Peter Su, Shuhua Yue, Min Zhang, Faliang Cheng, Fan Wang.

  Mechanical forces are essential for regulating dynamic changes in cellular activities. A comprehensive understanding of these forces is imperative for unraveling fundamental mechanisms. Here, we develop a microprobe capable of facilitating the measurement of biological forces up to nanonewton levels in living cells. This probe is designed by coating the core of anatase titania particles with amorphous titania and silica shells and an upconversion nanoparticles (UCNPs) layer. Leveraging both antireflection and ion resonance effects from the shells, the optically trapped probe attains a maximum lateral optical trap stiffness of 14.24 pN μm-1 mW-1, surpassing the best reported value by a factor of 3. Employing this advanced probe in a photonic force microscope, we determine the elasticity modulus of mitotic HeLa cells as 1.27 ± 0.3 kPa. Nanonewton probes offer the potential to explore 3D cellular mechanics with unparalleled precision and spatial resolution, fostering a deeper understanding of the underlying biomechanical mechanisms.

Keywords:  Cell stiffness; Nanonewton force; Titania particles; Upconversion nanoparticles (UCNPs)

DOI:  https://doi.org/10.1021/acs.nanolett.4c03610
Soft Matter. 2024 Oct 11.

Chemo-mechanical model of cell polarization initiated by structural polarity.

Hexiang Wang, Zhimeng Jia, Yuqiang Fang.

Cell polarization is crucial in most physiological functions. Living cells at the extracellular matrix (ECM) actively coordinate a polarized morphology to target the preferred signals. In particular, the initial heterogeneity of subcellular components, termed as structural polarity, has been discovered to mediate the early attachment and transmigration of cells in tumour metastasis. However, how heterogeneous cells initiate the early polarization remains incompletely discovered. Here, we establish a multiscale model of a cell to explore the chemo-mechanical mechanisms of cell polarization initiated by structural polarity. The two-dimensional vertex model of the cell is built with the main mechanical components of eukaryotic cells. The initial structural polarity of the modeled cell is introduced by seeding heterogeneous actin filaments at the cell cortex and quantified by the ratio of the filamentous forces at the vertices. Then, the structural polarity is integrated in the reaction-diffusion system of Rho GTPase (Cdc42) at the cell cortex to obtain the traction forces at the leading vertices. Finally, the modeled cell is actuated to spread under the traction forces and discovered to develop into a characteristic polarized morphology. The results indicate that the cell polarization is initiated and dynamically developed by structural polarity through the reaction-diffusion system of Cdc42. In addition, the bistability of Cdc42 activation at the cell cortex is defined and discovered to dominate the polarization status of the cell. Furthermore, biphasic (i.e., positive and negative) durotaxis of the cell is successfully modeled at an ECM with a stiffness gradient, and concluded to be codetermined by the chemo-mechanical coupling of the initial structural polarity and ECM stiffness gradient. The proposed multiscale model provides a quantitative way to probe cell polarization coupled with mechanical stimuli, biochemical reaction and cytoskeletal reorganization, and holds the potential to guide studies of cell polarization under multiple stimuli.

DOI: https://doi.org/10.1039/d4sm00800f
Nat Commun. 2024 Oct 12. 15(1): 8824

N-cadherin crosstalk with integrin weakens the molecular clutch in response to surface viscosity.

Eva Barcelona-Estaje, Mariana A G Oliva, Finlay Cunniffe, Aleixandre Rodrigo-Navarro, Paul Genever, Matthew J Dalby, Pere Roca-Cusachs, Marco Cantini, Manuel Salmeron-Sanchez.

Mesenchymal stem cells (MSCs) interact with their surroundings via integrins, which link to the actin cytoskeleton and translate physical cues into biochemical signals through mechanotransduction. N-cadherins enable cell-cell communication and are also linked to the cytoskeleton. This crosstalk between integrins and cadherins modulates MSC mechanotransduction and fate. Here we show the role of this crosstalk in the mechanosensing of viscosity using supported lipid bilayers as substrates of varying viscosity. We functionalize these lipid bilayers with adhesion peptides for integrins (RGD) and N-cadherins (HAVDI), to demonstrate that integrins and cadherins compete for the actin cytoskeleton, leading to an altered MSC mechanosensing response. This response is characterised by a weaker integrin adhesion to the environment when cadherin ligation occurs. We model this competition via a modified molecular clutch model, which drives the integrin/cadherin crosstalk in response to surface viscosity, ultimately controlling MSC lineage commitment.

DOI: https://doi.org/10.1038/s41467-024-53107-6
bioRxiv. 2024 Sep 28. pii: 2024.09.22.614353. [Epub ahead of print]

Transient Notch Activation Converts Pluripotent Stem Cell-Derived Cardiomyocytes Towards a Purkinje Fiber Fate.

David M Gonzalez, Rafael Dariolli, Julia Moyett, Stephanie Song, Bhavana Shewale, Jacqueline Bliley, Daniel Clarke, Avi Ma'ayan, Stacey Rentschler, Adam Feinberg, Eric Sobie, Nicole C Dubois.

Cardiac Purkinje fibers form the most distal part of the ventricular conduction system. They coordinate contraction and play a key role in ventricular arrhythmias. While many cardiac cell types can be generated from human pluripotent stem cells, methods to generate Purkinje fiber cells remain limited, hampering our understanding of Purkinje fiber biology and conduction system defects. To identify signaling pathways involved in Purkinje fiber formation, we analyzed single cell data from murine embryonic hearts and compared Purkinje fiber cells to trabecular cardiomyocytes. This identified several genes, processes, and signaling pathways putatively involved in cardiac conduction, including Notch signaling. We next tested whether Notch activation could convert human pluripotent stem cell-derived cardiomyocytes to Purkinje fiber cells. Following Notch activation, cardiomyocytes adopted an elongated morphology and displayed altered electrophysiological properties including increases in conduction velocity, spike slope, and action potential duration, all characteristic features of Purkinje fiber cells. RNA-sequencing demonstrated that Notch-activated cardiomyocytes undergo a sequential transcriptome shift, which included upregulation of key Purkinje fiber marker genes involved in fast conduction such as SCN5A, HCN4 and ID2, and downregulation of genes involved in contractile maturation. Correspondingly, we demonstrate that Notch-induced cardiomyocytes have decreased contractile force in bioengineered tissues compared to control cardiomyocytes. We next modified existing in silico models of human pluripotent stem cell-derived cardiomyocytes using our transcriptomic data and modeled the effect of several anti-arrhythmogenic drugs on action potential and calcium transient waveforms. Our models predicted that Purkinje fiber cells respond more strongly to dofetilide and amiodarone, while cardiomyocytes are more sensitive to treatment with nifedipine. We validated these findings in vitro , demonstrating that our new cell-specific in vitro model can be utilized to better understand human Purkinje fiber physiology and its relevance to disease.

DOI: https://doi.org/10.1101/2024.09.22.614353
iScience. 2024 Oct 18. 27(10): 110968

Cell density couples tissue mechanics to control the elongation speed of the body axis.

Changqing Lu, Joana M N Vidigueira, Christopher Chan Jin Jie, Alicja Maksymiuk, Fengzhu Xiong.

  The vertebrate body forms by the addition of new tissues at the posterior end. This elongates the body axis, allowing continued anterior segmentation to produce the stereotypic body plan. This balance requires the elongation speed to be constrained. Here we utilized modeling and tissue force microscopy on chicken embryos to show that cell density of the posterior presomitic mesoderm (pPSM) dynamically modulates elongation speed in a negative feedback loop. Elongation alters the cell density in the pPSM, which in turn controls progenitor cell influx through the mechanical coupling of body axis tissues. This enables responsive cell dynamics in over- and under-elongated axes that consequently self-adjust speed to achieve long-term robustness in axial length. Our simulations and experiments further suggest that cell density and FGF activity synergistically drive elongation. Our work supports a simple mechanism of morphogenetic speed control where the cell density relates negatively to progress, and positively to force generation.

Keywords:  Biophysics; Cell biology; Developmental biology

DOI:  https://doi.org/10.1016/j.isci.2024.110968
bioRxiv. 2024 Sep 23. pii: 2024.09.23.614499. [Epub ahead of print]

Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells.

Josh P Graham, Jose Gabriel Castro, Lisette C Werba, Luke C Fardone, Kevin P Francis, Anand Ramamurthi, Michael Layden, Helen O McCarthy, Tomas Gonzalez-Fernandez.

CRISPR gene editing offers unprecedented genomic and transcriptomic control for precise regulation of cell function and phenotype. However, delivering the necessary CRISPR components to therapeutically relevant cell types without cytotoxicity or unexpected side effects remains challenging. Viral vectors risk genomic integration and immunogenicity while non-viral delivery systems are challenging to adapt to different CRISPR cargos, and many are highly cytotoxic. The arginine-alanine-leucine-alanine (RALA) cell penetrating peptide is an amphiphilic peptide that self-assembles into nanoparticles through electrostatic interactions with negatively charged molecules before delivering them across the cell membrane. This system has been used to deliver DNAs, RNAs, and small anionic molecules to primary cells with lower cytotoxicity compared to alternative non-viral approaches. Given the low cytotoxicity, versatility, and competitive transfection rates of RALA, we aimed to establish this peptide as a new CRISPR delivery system in a wide range of molecular formats across different editing modalities. We report that RALA was able to effectively encapsulate and deliver CRISPR in DNA, RNA, and ribonucleic protein (RNP) formats to primary mesenchymal stem cells (MSCs). Comparisons between RALA and commercially available reagents revealed superior cell viability leading to higher numbers of transfected cells and the maintenance of cell proliferative capacity. We then used the RALA peptide for the knock-in and knock-out of reporter genes into the MSC genome as well as for the transcriptional activation of therapeutically relevant genes. In summary, we establish RALA as a powerful tool for safer and effective delivery of CRISPR machinery in multiple cargo formats for a wide range of gene editing strategies.

DOI: https://doi.org/10.1101/2024.09.23.614499
bioRxiv. 2024 Sep 27. pii: 2024.09.25.615042. [Epub ahead of print]

Controlled release of microorganisms from engineered living materials.

Manivannan Sivaperuman Kalairaj, Iris George, Sasha M George, Sofía E Farfán, Yoo Jin Lee, Laura K Rivera-Tarazona, Suitu Wang, Mustafa K Abdelrahman, Seelay Tasmim, Asaf Dana, Philippe E Zimmern, Sargurunathan Subashchandrabose, Taylor H Ware.

Probiotics offer therapeutic benefits by modulating the local microbiome, the host immune response, and the proliferation of pathogens. Probiotics have the potential to treat complex diseases, but their persistence or colonization is required at the target site for effective treatment. Although probiotic persistence can be achieved by repeated delivery, no biomaterial that releases clinically relevant doses of metabolically active probiotics in a sustained manner has been previously described. Here, we encapsulate stiff probiotic microorganisms within relatively less stiff hydrogels and show a generic mechanism where these microorganisms proliferate and induce hydrogel fracture, resulting in microbial release. Importantly, this fracture-based mechanism leads to microorganism release with zero-order release kinetics. Using this mechanism, small (∼1 μL) engineered living materials (ELMs) release >10 8 colony-forming-units (CFUs) of E. coli in 2 h. This release is sustained for at least 10 days. Cell release can be varied by more than three orders of magnitude by varying initial cell loading and modulating the mechanical properties of encapsulating matrix. As the governing mechanism of microbial release is entirely mechanical, we demonstrate controlled release of model Gram-negative, Gram-positive, and fungal probiotics from multiple hydrogel matrices.
SIGNIFICANCE: Probiotics offer therapeutic benefits and have the potential to treat complex diseases, but their persistence at the target site is often required for effective treatment. Although probiotic persistence can be achieved by repeated delivery, no biomaterial that releases metabolically active probiotics in a sustained manner has been developed yet. This work demonstrates a generic mechanism where stiff probiotics encapsulated within relatively less stiff hydrogels proliferate and induce hydrogel fracture. This allows a zero-order release of probiotics which can be easily controlled by adjusting the properties of the encapsulating matrices. This generic mechanism is applicable for a wide range of probiotics with different synthetic matrices and has the potential to be used in the treatment of a broad range of diseases.

DOI: https://doi.org/10.1101/2024.09.25.615042
bioRxiv. 2024 Oct 07. pii: 2024.09.26.615278. [Epub ahead of print]

Microfluidic Modulation of Microvasculature in Microdissected Tumors.

Tran N H Nguyen, Lisa F Horowitz, Brandon Nguyen, Ethan Lockhart, Songli Zhu, Taranjit S Gujral, Albert Folch.

The microvasculature within the tumor microenvironment (TME) plays an essential role in cancer signaling beyond nutrient delivery. However, it has been challenging to control the generation and/or maintenance of microvasculature in ex vivo systems, a critical step for establishing cancer models of high clinical biomimicry. There have been great successes in engineering tissues incorporating microvasculature de novo (e.g., organoids and organs-on-chip), but these reconstituted tissues are formed with non-native cellular and molecular components that can skew certain outcomes such as drug efficacy. Microdissected tumors, on the other hand, show promise in preserving the TME, which is key for creating cancer models that can bridge the gap between bench and bedside. However, microdissected tumors are challenging to perfuse. Here, we developed a microfluidic platform that allows for perfusing the microvasculature of microdissected tumors. We demonstrate that, compared to diffusive transport, microfluidically perfused tissues feature larger and longer microvascular structures, with a better expression of CD31, a marker for endothelial cells, as analyzed by 3D imaging. This study also explores the effects of nitric oxide pathway-related drugs on endothelial cells, which are sensitive to shear stress and can activate endothelial nitric oxide synthase, producing nitric oxide. Our findings highlight the critical role of controlled perfusion and biochemical modulation in preserving tumor microvasculature, offering valuable insights for developing more effective cancer treatments.

DOI: https://doi.org/10.1101/2024.09.26.615278
iScience. 2024 Oct 18. 27(10): 110981

The "weaken-fill-repair" model for cell budding: Linking cell wall biosynthesis with mechanics.

Yu Liu, Chunxiuzi Liu, Shaohua Tang, Hui Xiao, Xinlin Wu, Yunru Peng, Xianyi Wang, Linjie Que, Zengru Di, Da Zhou, Matthias Heinemann.

  The interplay between cellular mechanics and biochemical processes in the cell cycle is not well understood. We propose a quantitative model of cell budding in Saccharomyces cerevisiae as a "weaken-fill-repair" process, linking Newtonian mechanics of the cell wall with biochemical changes that affect its properties. Our model reveals that (1) oscillations in mother cell size during budding are an inevitable outcome of the process; (2) asymmetric division is necessary for the daughter cell to maintain mechanical stiffness; and (3) although various aspects of the cell are constrained and interconnected, the budding process is governed by a single reduced parameter, ψ, which balances osmolyte accumulation with enzymatic wall-weakening to ensure homeostasis. This model provides insights into the evolution of cell walls and their role in cell division, offering a system-level perspective on cell morphology.

Keywords:  Biophysics; Cell biology; Systems biology

DOI:  https://doi.org/10.1016/j.isci.2024.110981
Proc Natl Acad Sci U S A. 2024 Oct 15. 121(42): e2405257121

An engineered model of metastatic colonization of human bone marrow reveals breast cancer cell remodeling of the hematopoietic niche.

Ilaria Baldassarri, Daniel Naveed Tavakol, Pamela L Graney, Alan G Chramiec, Hanina Hibshoosh, Gordana Vunjak-Novakovic.

  Incomplete understanding of metastatic disease mechanisms continues to hinder effective treatment of cancer. Despite remarkable advancements toward the identification of druggable targets, treatment options for patients in remission following primary tumor resection remain limited. Bioengineered human tissue models of metastatic sites capable of recreating the physiologically relevant milieu of metastatic colonization may strengthen our grasp of cancer progression and contribute to the development of effective therapeutic strategies. We report the use of an engineered tissue model of human bone marrow (eBM) to identify microenvironmental cues regulating cancer cell proliferation and to investigate how triple-negative breast cancer (TNBC) cell lines influence hematopoiesis. Notably, individual stromal components of the bone marrow niche (osteoblasts, endothelial cells, and mesenchymal stem/stromal cells) were each critical for regulating tumor cell quiescence and proliferation in the three-dimensional eBM niche. We found that hematopoietic stem and progenitor cells (HSPCs) impacted TNBC cell growth and responded to cancer cell presence with a shift of HSPCs (CD34+CD38-) to downstream myeloid lineages (CD11b+CD14+). To account for tumor heterogeneity and show proof-of-concept ability for patient-specific studies, we demonstrate that patient-derived tumor organoids survive and proliferate in the eBM, resulting in distinct shifts in myelopoiesis that are similar to those observed for aggressively metastatic cell lines. We envision that this human tissue model will facilitate studies of niche-specific metastatic progression and individualized responses to treatment.

Keywords:  cancer; hematopoiesis; metastasis; organoids; tissue engineering

DOI:  https://doi.org/10.1073/pnas.2405257121
ACS Biomater Sci Eng. 2024 Oct 10.

Microvascular Engineering for the Development of a Nonembedded Liver Sinusoid with a Lumen: When Endothelial Cells Do Not Lose Their Edge.

Ana Ximena Monroy-Romero, Brenda Nieto-Rivera, Wenjin Xiao, Mathieu Hautefeuille.

  Microvascular engineering seeks to exploit known cell-cell and cell-matrix interactions in the context of vasculogenesis to restore homeostasis or disease development of reliable capillary models in vitro. However, current systems generally focus on recapitulating microvessels embedded in thick gels of extracellular matrix, overlooking the significance of discontinuous capillaries, which play a vital role in tissue-blood exchanges particularly in organs like the liver. In this work, we introduce a novel method to stimulate the spontaneous organization of endothelial cells into nonembedded microvessels. By creating an anisotropic micropattern at the edge of a development-like matrix dome using Marangoni flow, we achieved a long, nonrandom orientation of endothelial cells, laying a premise for stable lumenized microvessels. Our findings revealed a distinctive morphogenetic process leading to mature lumenized capillaries, demonstrated with both murine and human immortalized liver sinusoidal endothelial cell lines (LSECs). The progression of cell migration, proliferation, and polarization was clearly guided by the pattern, initiating the formation of a multicellular cord that caused a deformation spanning extensive regions and generated a wave-like folding of the gel, hinged at a laminin-depleted zone, enveloping the cord with gel proteins. This event marked the onset of lumenogenesis, regulated by the gradual apico-basal polarization of the wrapped cells, leading to the maturation of vessel tight junctions, matrix remodeling, and ultimately the formation of a lumen─recapitulating the development of vessels in vivo. Furthermore, we demonstrate that the process strongly relies on the initial gel edge topography, while the geometry of the vessels can be tuned from a curved to a straight structure. We believe that our facile engineering method, guiding an autonomous self-organization of vessels without the need for supporting cells or complex prefabricated scaffolds, holds promise for future integration into microphysiological systems featuring discontinuous, fenestrated capillaries.

Keywords:  endothelium; folding; micropatterning; morphogenesis; self-organization

DOI:  https://doi.org/10.1021/acsbiomaterials.4c00939
Nano Lett. 2024 Oct 08.

Stiffening of the Cytoplasm in Response to Intracellularly Applied Forces.

Wentian Tang, Jintian Wang, Aojun Jiang, Yu Sun.

  Cells constantly encounter mechanical forces that regulate various cellular functions, such as migration, division, and differentiation. Understanding how cells respond to forces at the intracellular level is essential for elucidating the mechanical adaptability of living cells. This study investigates how the cytoplasm alters its mechanical properties in response to forces applied inside a cell. The mechanical properties were measured through in situ characterization using magnetic tweezers to apply mechanical forces on magnetic beads internalized into cells. The findings reveal that the cytoplasm stiffens within seconds when force is applied to the cytoplasm. Macromolecular crowding and cytoskeletal structures, particularly F-actin, were found to significantly contribute to cytoplasm stiffening. The stiffening response was also observed across multiple length scales by using magnetic beads of varying diameters. These results highlight the rapid adaptation of the cytoplasm to mechanical forces applied to the inside of a cell.

Keywords:  cell mechanics; cytoplasm; intracellular measurement; stiffening; viscoelasticity

DOI:  https://doi.org/10.1021/acs.nanolett.4c03979
Acta Biomater. 2024 Oct 04. pii: S1742-7061(24)00576-2. [Epub ahead of print]

Tuning a Bioengineered Hydrogel for Studying Astrocyte Reactivity in Glioblastoma.

Thomas DePalma, Colin L Hisey, Kennedy Hughes, David Fraas, Marie Tawfik, Jason Scharenberg, Sydney Wiggins, Kim Truc Nguyen, Derek Hansford, Eduardo Reátegui, Aleksander Skardal.

  Astrocytes play many essential roles in the central nervous system (CNS) and are altered significantly in disease. These reactive astrocytes contribute to neuroinflammation and disease progression in many pathologies, including glioblastoma (GB), an aggressive form of brain cancer. Current in vitro platforms do not allow for accurate modeling of reactive astrocytes. In this study, we sought to engineer a simple bioengineered hydrogel platform that would support the growth of primary human astrocytes and allow for accurate analysis of various reactive states. After validating this platform using morphological analysis and qPCR, we then used the platform to begin investigating how astrocytes respond to GB derived extracellular vesicles (EVs) and soluble factors (SF). These studies reveal that EVs and SFs induce distinct astrocytic states. In future studies, this platform can be used to study how astrocytes transform the tumor microenvironment in GB and other diseases of the CNS. STATEMENT OF SIGNIFICANCE: Recent work has shown that astrocytes help maintain brain homeostasis and may contribute to disease progression in diseases such as glioblastoma (GB), a deadly primary brain cancer. In vitro models allow researchers to study basic mechanisms of astrocyte biology in healthy and diseased conditions, however current in vitro systems do not accurately mimic the native brain microenvironment. In this study, we shown that our hydrogel system supports primary human astrocyte culture with an accurate phenotype and allows us to study how astrocytes change in response to a variety of inflammatory signals in GB. This platform could be used further investigate astrocyte behavior and possible therapeutics that target reactive astrocytes in GB and other brain diseases.

Keywords:  Astrocyte; Extracellular Vesicles; Glioblastoma; Hydrogel; Quiescence; Reactivity

DOI:  https://doi.org/10.1016/j.actbio.2024.09.048
Matrix Biol Plus. 2024 Dec;24 100162

Obesity-driven changes in breast tissue exhibit a pro-angiogenic extracellular matrix signature.

Ellen E Bamberg, Mark Maslanka, Kiran Vinod-Paul, Sharon Sams, Erica Pollack, Matthew Conklin, Peter Kabos, Kirk C Hansen.

  Obesity has reached epidemic proportions in the United States, emerging as a risk factor for the onset of breast cancer and a harbinger of unfavorable outcomes [1], [2], [3]. Despite limited understanding of the precise mechanisms, both obesity and breast cancer are associated with extracellular matrix (ECM) rewiring [4], [5], [6]. Utilizing total breast tissue proteomics, we analyzed normal-weight (18.5 to < 25 kg/m2), overweight (25 to < 30 kg/m2), and obese (≥30 kg/m2) individuals to identify potential ECM modifying proteins for cancer development and acceleration. Obese individuals exhibited substantial ECM alterations, marked by increased basement membrane deposition, angiogenic signatures, and ECM-modifying proteins. Notably, the collagen IV crosslinking enzyme peroxidasin (PXDN) emerged as a potential mediator of the ECM changes in individuals with an elevated body mass index (BMI), strongly correlating with angiogenic and basement membrane signatures. Furthermore, glycan-binding proteins galectin-1 (LGALS1) and galectin-3 (LGALS3), which play crucial roles in matrix interactions and angiogenesis, also strongly correlate with ECM modifications. In breast cancer, elevated PXDN, LGALS1, and LGALS3 correlate with reduced relapse-free and distant-metastatic-free survival. These proteins were significantly associated with mesenchymal stromal cell markers, indicating adipocytes and fibroblasts may be the primary contributors of the obesity-related ECM changes. Our findings unveil a pro-angiogenic ECM signature in obese breast tissue, offering potential targets to inhibit breast cancer development and progression.

Keywords:  Adipocytes; Body Mass Index; Breast; Extracellular matrix; Fibroblasts; Galectin; LGALS1; LGALS3; Obesity; PXDN; Peroxidasin; Stromal cells; Tissue Proteomics; Tumor Microenvironment

DOI:  https://doi.org/10.1016/j.mbplus.2024.100162
Nat Commun. 2024 Oct 07. 15(1): 8691

Actin-driven nanotopography promotes stable integrin adhesion formation in developing tissue.

Tianchi Chen, Cecilia H Fernández-Espartero, Abigail Illand, Ching-Ting Tsai, Yang Yang, Benjamin Klapholz, Pierre Jouchet, Mélanie Fabre, Olivier Rossier, Bianxiao Cui, Sandrine Lévêque-Fort, Nicholas H Brown, Grégory Giannone.

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerization-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MASs). Super-resolution microscopy reveals that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking shows that, during MAS development, integrins become immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also display restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscle cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerization-driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis.

DOI: https://doi.org/10.1038/s41467-024-52899-x
Cell Prolif. 2024 Oct 08. e13735

Functional heterogeneity of meniscal fibrochondrocytes and microtissue models is dependent on modality of fibrochondrocyte isolation.

Zhiyao Ma, Shikha Chawla, Xiaoyi Lan, Eva Zhou, Aillette Mulet-Sierra, Melanie Kunze, Mark Sommerfeldt, Adetola B Adesida.

Collagenase digestion (d) and cellular outgrowth (og) are the current modalities of meniscus fibrochondrocytes (MFC) isolation for bioengineering and mechanobiology-related studies. However, the impact of these modalities on study outcomes is unknown. Here, we show that og- and d-isolated MFC have distinct proliferative capacities, transcriptomic profiles via RNA sequencing (RNAseq), extracellular matrix (ECM)-forming, and migratory capacities. Our data indicate that microtissue pellet models developed from og-isolated MFC display a contractile phenotype with higher expressions of alpha-smooth muscle actin (ACTA2) and transgelin (TAGLN) and are mechanically stiffer than their counterparts from d-MFC. Moreover, we introduce a novel method of MFC isolation designated digestion-after-outgrowth (dog). The transcriptomic profile of dog-MFC is distinct from d- and og-MFC, including a higher expression of mechanosensing caveolae-associated caveolin-1 (CAV1). Additionally, dog-MFC were superior chondrogenically and generated larger-size microtissue pellet models containing a higher frequency of smaller collagen fibre diameters. Thus, we demonstrate that the modalities of MFC isolation influence the downstream outcomes of bioengineering and mechanobiology-related studies.

DOI: https://doi.org/10.1111/cpr.13735
Mater Today Bio. 2024 Dec;29 101260

In vitro vascularization of 3D cell aggregates in microwells with integrated vascular beds.

Maria G Fois, Zeinab N Tahmasebi Birgani, Carmen López-Iglesias, Kèvin Knoops, Clemens van Blitterswijk, Stefan Giselbrecht, Pamela Habibović, Roman K Truckenmüller.

  Most human tissues possess vascular networks supplying oxygen and nutrients. Engineering of functional tissue and organ models or equivalents often require the integration of artificial vascular networks. Several approaches, such as organs on chips and three-dimensional (3D) bioprinting, have been pursued to obtain vasculature and vascularized tissues in vitro. This technical feasibility study proposes a new approach for the in vitro vascularization of 3D microtissues. For this, we thermoform arrays of round-bottom microwells into thin non-porous and porous polymer films/membranes and culture vascular beds on them from which endothelial sprouting occurs in a Matrigel-based 3D extra cellular matrix. We present two possible culture configurations for the microwell-integrated vascular beds. In the first configuration, human umbilical vein endothelial cells (HUVECs) grow on and sprout from the inner wall of the non-porous microwells. In the second one, HUVECs grow on the outer surface of the porous microwells and sprout through the pores toward the inside. These approaches are extended to lymphatic endothelial cells. As a proof of concept, we demonstrate the in vitro vascularization of spheroids from human mesenchymal stem cells and MG-63 human osteosarcoma cells. Our results show the potential of this approach to provide the spheroids with an abundant outer vascular network and the indication of an inner vasculature.

Keywords:  3D cell culture; Endothelial cells; Microthermoforming; Microwells; Spheroids; Vascularization

DOI:  https://doi.org/10.1016/j.mtbio.2024.101260
Am J Physiol Cell Physiol. 2024 Oct 07.

Diabetes induces modifications in costameric proteins and increases cardiomyocyte stiffness.

Gerardo Romanelli, Lihuén Villarreal, Camila Espasandín, Juan Claudio Benech.

  Several studies have demonstrated that Diabetes mellitus can increase the risk of cardiovascular disease and remains the principal cause of death in these patients. Costameres connect the sarcolemma with the cytoskeleton and extracellular matrix, facilitating the transmission of mechanical forces and cell signaling. They are related to cardiac physiology because individual cardiac cells are connected by intercalated discs that synchronize muscle contraction. Diabetes impacts the nano-mechanical properties of cardiomyocytes, resulting in increased cellular and left ventricular stiffness, as evidenced in clinical studies of these patients. The question of whether costameric proteins are affected by diabetes in the heart has not been studied. This work analyzes whether T1DM modifies the costameric proteins and coincidentally changes the cellular mechanics in the same cardiomyocytes. The samples were analyzed by immunotechniques using laser confocal microscopy. Significant statistical differences were found in the spatial arrangement of the costameric proteins. However, these differences are not due to their expression. Atomic force microscopy was used to compare intrinsic cellular stiffness between diabetic and normal cardiomyocytes and obtain the first elasticity map sections of diabetic living cardiomyocytes. Data obtained demonstrated that diabetic cardiomyocytes had higher stiffness than control. The present work shows experimental evidence that intracellular changes related to cell-cell and cell-extracellular matrix communication occur, which could be related to cardiac pathogenic mechanisms. These changes could contribute to alterations in the mechanical and electrical properties of cardiomyocytes and consequently, to diabetic cardiomyopathy.

Keywords:  Costamere; Diabetes mellitus; cardiomyocytes; cardiomyopathy; nanomechanics

DOI:  https://doi.org/10.1152/ajpcell.00273.2024