bims-ecemfi 2024-11-10 papers

bims-ecemfi

Biomed News

on ECM and fibroblasts

Issue of 2024–11–10
twenty papers selected by
Badri Narayanan Narasimhan, University of California, San Diego

Interpenetrating Polymer Network Hydrogels with Tunable Viscoelasticity and Proteolytic Cleavability to Direct Stem Cells In Vitro.
Talin and vinculin combine their activities to trigger actin assembly.
Concurrently Probing the Mechanical and Electrical Characteristics of Living Cells via an Integrated Microdevice.
Cell tumbling enhances stem cell differentiation in hydrogels via nuclear mechanotransduction.
Scale-dependent interactions enable emergent microrheological stress response of actin-vimentin composites.
Synthetic hydrogel substrate for human induced pluripotent stem cell definitive endoderm differentiation.
Characterising Cancer Cell Responses to Cyclic Hypoxia Using Mathematical Modelling.
Extracellular Matrix Limits Nanoparticle Diffusion and Cellular Uptake in a Tissue-Specific Manner.
Exploring heterogeneous cell population dynamics in different microenvironments by novel analytical strategy based on images.
PIEZO1 mediates matrix stiffness-induced tumor progression in kidney renal clear cell carcinoma by activating the Ca2+/Calpain/YAP pathway.
A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow.
Biomimetic Structural Hydrogels Reinforced by Gradient Twisted Plywood Architectures.
Assessing mechanical agency during apical apoptotic cell extrusion.
Developing a soft micropatterned substrate to enhance maturation of human induced pluripotent stem cell-derived cardiomyocytes.
Foreign Body Immune Response to Zwitterionic and Hyaluronic Acid Granular Hydrogels Made with Mechanical Fragmentation.
Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion.
Magnetic 3D bioprinting of skeletal muscle spheroid for a spheroid-based screening assay.
Liquid crystalline collagen assemblies as substrates for directed alignment of human Schwann cells.
Piezo2 interacts with E-cadherin in specialized gastrointestinal epithelial mechanoreceptors.
Optogenetic patterning generates multi-strain biofilms with spatially distributed antibiotic resistance.

Adv Healthc Mater. 2024 Nov 06. e2402656

Interpenetrating Polymer Network Hydrogels with Tunable Viscoelasticity and Proteolytic Cleavability to Direct Stem Cells In Vitro.

Prannoy Seth, Jens Friedrichs, Yanuar Dwi Putra Limasale, Nicole Fertala, Uwe Freudenberg, Yixin Zhang, Ayala Lampel, Carsten Werner.

  The dynamic nature of cellular microenvironments, regulated by the viscoelasticity and enzymatic cleavage of the extracellular matrix, remains challenging to emulate in engineered synthetic biomaterials. To address this, a novel platform of cell-instructive hydrogels is introduced, composed of two concurrently forming interpenetrating polymer networks (IPNs). These IPNs consist of the same basic building blocks - four-armed poly(ethylene glycol) and the sulfated glycosaminoglycan (sGAG) heparin - are cross-linked through either chemical or physical interactions, allowing for precise and selective tuning of the hydrogel's stiffness, viscoelasticity, and proteolytic cleavability. The studies of the individual and combined effects of these parameters on stem cell behavior revealed that human mesenchymal stem cells exhibited increased spreading and Yes-associated protein transcriptional activity in more viscoelastic and cleavable sGAG-IPN hydrogels. Furthermore, human induced pluripotent stem cell (iPSC) cysts displayed enhanced lumen formation, growth, and pluripotency maintenance when cultured in sGAG-IPN hydrogels with higher viscoelasticity. Inhibition studies emphasized the pivotal roles of actin dynamics and matrix metalloproteinase activity in iPSC cyst morphology, which varied with the viscoelastic properties of the hydrogels. Thus, the introduced sGAG-IPN hydrogel platform offers a powerful methodology for exogenous stem cell fate control.

Keywords:  dynamic matrix restructuring; interpenetrating sGAG hydrogels; proteolytic cleavability; stem cell morphogenesis; viscoelasticity

DOI:  https://doi.org/10.1002/adhm.202402656
Nat Commun. 2024 Nov 03. 15(1): 9497

Talin and vinculin combine their activities to trigger actin assembly.

Hong Wang, Rayan Said, Clémence Nguyen-Vigouroux, Véronique Henriot, Peter Gebhardt, Julien Pernier, Robert Grosse, Christophe Le Clainche.

Focal adhesions (FAs) strengthen their link with the actin cytoskeleton to resist force. Talin-vinculin association could reinforce actin anchoring to FAs by controlling actin polymerization. However, the actin polymerization activity of the talin-vinculin complex is not known because it requires the reconstitution of the mechanical and biochemical activation steps that control the association of talin and vinculin. By combining kinetic and binding assays with single actin filament observations in TIRF microscopy, we show that the association of talin and vinculin mutants, mimicking mechanically stretched talin and activated vinculin, triggers a sequential mechanism in which filaments are nucleated, capped and released to elongate. In agreement with these observations, FRAP experiments in cells co-expressing the same constitutive mutants of talin and vinculin revealed accelerated growth of stress fibers. Our findings suggest a versatile mechanism for the regulation of actin assembly in FAs subjected to various combinations of biochemical and mechanical cues.

DOI: https://doi.org/10.1038/s41467-024-53859-1
Nano Lett. 2024 Nov 04.

Concurrently Probing the Mechanical and Electrical Characteristics of Living Cells via an Integrated Microdevice.

Johnson Q Cui, Ye Tian, Zhihao Wu, Lu Zhang, William C Cho, Shuhuai Yao, Yuan Lin.

  The mechanical and electrical properties of cells serve as critical indicators of their physiological and pathological state. Currently, distinct setups are required to measure the electrical and mechanical responses of cells. In addition, most existing methods such as optical trapping (OT) and atomic force microscopy (AFM) are labor-intensive, expensive, and low-throughput. Here, we developed a microdevice that integrates automated cell trapping, deformation, and electric impedance spectroscopy to overcome these limitations. Our device enables parallel aspiration of tens of trapped cells in a highly scalable manner by simply adjusting the applied pressures, allowing for rapid probing of the dynamic viscoelastic properties of cells. Furthermore, embedded microelectrodes enable concurrent investigations of the electrical impedance of the cells. Through testing on different cell types, our platform demonstrated superior capabilities in comprehensive cell characterization and phenotyping, highlighting its great potential as a versatile tool for single cell analysis, drug screening, and disease detection.

Keywords:  algorithm-assisted; cell trapping; cell viscoelasticity; cellular impedance; microfluidics; single cell characterization

DOI:  https://doi.org/10.1021/acs.nanolett.4c05005
Nat Mater. 2024 Nov 01.

Cell tumbling enhances stem cell differentiation in hydrogels via nuclear mechanotransduction.

Manish Ayushman, Georgios Mikos, Xinming Tong, Sauradeep Sinha, Eunice Lopez-Fuentes, Sarah Jones, Pamela C Cai, Hung-Pang Lee, Ashby J Morrison, Andrew Spakowitz, Sarah C Heilshorn, Alejandro Sweet-Cordero, Fan Yang.

Cells can deform their local niche in three dimensions via whole-cell movements such as spreading, migration or volume expansion. These behaviours, occurring over hours to days, influence long-term cell fates including differentiation. Here we report a whole-cell movement that occurs in sliding hydrogels at the minutes timescale, termed cell tumbling, characterized by three-dimensional cell dynamics and hydrogel deformation elicited by heightened seconds-to-minutes-scale cytoskeletal and nuclear activity. Studies inhibiting or promoting the cell tumbling of mesenchymal stem cells show that this behaviour enhances differentiation into chondrocytes. Further, it is associated with a decrease in global chromatin accessibility, which is required for enhanced differentiation. Cell tumbling also occurs during differentiation into other lineages and its differentiation-enhancing effects are validated in various hydrogel platforms. Our results establish that cell tumbling is an additional regulator of stem cell differentiation, mediated by rapid niche deformation and nuclear mechanotransduction.

DOI: https://doi.org/10.1038/s41563-024-02038-0
Soft Matter. 2024 Nov 04.

Scale-dependent interactions enable emergent microrheological stress response of actin-vimentin composites.

Julie Pinchiaroli, Renita Saldanha, Alison E Patteson, Rae M Robertson-Anderson, Bekele J Gurmessa.

The mechanical properties of the mammalian cell regulate many cellular functions and are largely dictated by the cytoskeleton, a composite network of protein filaments, including actin, microtubules, and intermediate filaments. Interactions between these distinct filaments give rise to emergent mechanical properties that are difficult to generate synthetically, and recent studies have made great strides in advancing our understanding of the mechanical interplay between actin and microtubule filaments. While intermediate filaments play critical roles in the stress response of cells, their effect on the rheological properties of the composite cytoskeleton remains poorly understood. Here, we use optical tweezers microrheology to measure the linear viscoelastic properties and nonlinear stress response of composites of actin and vimentin with varying molar ratios of actin to vimentin. We reveal a surprising, nearly opposite effect of actin-vimentin network mechanics compared to single-component networks in the linear versus nonlinear regimes. Namely, the linear elastic plateau modulus and zero-shear viscosity are markedly reduced in composites compared to single-component networks of actin or vimentin, whereas the initial response force and stiffness are maximized in composites versus single-component networks in the nonlinear regime. While these emergent trends are indicative of distinct interactions between actin and vimentin, nonlinear stiffening and long-time stress response appear to both be dictated primarily by actin, at odds with previous bulk rheology studies. We demonstrate that these complex, scale-dependent effects arise from the varied contributions of network density, filament stiffness, non-specific interactions, and poroelasticity to the mechanical response at different spatiotemporal scales. Cells may harness this complex behavior to facilitate distinct stress responses at different scales and in response to different stimuli to allow for their hallmark multifunctionality.

DOI: https://doi.org/10.1039/d4sm00988f
Biomaterials. 2024 Oct 25. pii: S0142-9612(24)00454-X. [Epub ahead of print]315 122920

Synthetic hydrogel substrate for human induced pluripotent stem cell definitive endoderm differentiation.

Adriana Mulero-Russe, Ana Mora-Boza, Elijah N Marquez, Morgan Ziegelski, Michael Helmrath, Andrés J García.

  Human induced pluripotent stem cells (hiPSCs) can give rise to multiple lineages derived from three germ layers, endoderm, mesoderm and ectoderm. Definitive endoderm (DE) cell types and tissues have great potential for regenerative medicine applications. Current hiPSC differentiation protocols focus on the addition of soluble factors; however, extracellular matrix properties are known to also play a role in dictating cell fate. Matrigel™ is the gold standard for DE differentiation, but this xenogeneic, poorly defined basement membrane extract limits the clinical translatability of DE-derived tissues. Here we present a fully defined PEG-based hydrogel substrate to support hiPSC-derived DE differentiation. We screened hydrogel formulations presenting different adhesive peptides and matrix stiffness. Our results demonstrate that presenting a short peptide, cyclic RGD, on the engineered PEG hydrogel supports the transition from undifferentiated hiPSCs to DE using a serum-free, commercially available kit. We show that increasing substrate stiffness (G' = 1.0-4.0 kPa) results in an increased linear response in DE differentiation efficiency. We also include a temporal analysis of the expression of integrin and syndecan receptors as the hiPSCs undergo specification towards DE lineage. Finally, we show that focal adhesion kinase activity regulates hiPSC growth and DE differentiation efficiency. Overall, we present a fully defined matrix as a synthetic alternative for Matrigel™ supporting DE differentiation.

Keywords:  FAK; Matrix stiffness; PEG hydrogel; RGD; hiPSCs

DOI:  https://doi.org/10.1016/j.biomaterials.2024.122920
Bull Math Biol. 2024 Nov 06. 86(12): 145

Characterising Cancer Cell Responses to Cyclic Hypoxia Using Mathematical Modelling.

Giulia L Celora, Ruby Nixson, Joe M Pitt-Francis, Philip K Maini, Helen M Byrne.

  In vivo observations show that oxygen levels in tumours can fluctuate on fast and slow timescales. As a result, cancer cells can be periodically exposed to pathologically low oxygen levels; a phenomenon known as cyclic hypoxia. Yet, little is known about the response and adaptation of cancer cells to cyclic, rather than, constant hypoxia. Further, existing in vitro models of cyclic hypoxia fail to capture the complex and heterogeneous oxygen dynamics of tumours growing in vivo. Mathematical models can help to overcome current experimental limitations and, in so doing, offer new insights into the biology of tumour cyclic hypoxia by predicting cell responses to a wide range of cyclic dynamics. We develop an individual-based model to investigate how cell cycle progression and cell fate determination of cancer cells are altered following exposure to cyclic hypoxia. Our model can simulate standard in vitro experiments, such as clonogenic assays and cell cycle experiments, allowing for efficient screening of cell responses under a wide range of cyclic hypoxia conditions. Simulation results show that the same cell line can exhibit markedly different responses to cyclic hypoxia depending on the dynamics of the oxygen fluctuations. We also use our model to investigate the impact of changes to cell cycle checkpoint activation and damage repair on cell responses to cyclic hypoxia. Our simulations suggest that cyclic hypoxia can promote heterogeneity in cellular damage repair activity within vascular tumours.

Keywords:  Cell cycle; Damage repair; Fluctuating oxygen levels; Individual-based modelling; Mathematical oncology; Tumour Hypoxia

DOI:  https://doi.org/10.1007/s11538-024-01359-0
ACS Nano. 2024 Nov 05.

Extracellular Matrix Limits Nanoparticle Diffusion and Cellular Uptake in a Tissue-Specific Manner.

Devorah Cahn, Alexa Stern, Michael Buckenmeyer, Matthew Wolf, Gregg A Duncan.

  Overexpression and remodeling of the extracellular matrix (ECM) in cancer and other diseases may significantly reduce the ability of nanoparticles to reach target sites, preventing the effective delivery of therapeutic cargo. Here, we evaluate how tissue-specific properties of the ECM affect nanoparticle diffusion using fluorescence video microscopy and cellular uptake via flow cytometry. In addition, we determined how poly(ethylene glycol) (PEG) chain length and branching influence the ability of PEGylated nanoparticles to overcome the ECM barrier from different tissues. We found that purified collagen, in the absence of other ECM proteins and polysaccharides, presented a greater barrier to nanoparticle diffusion compared to the decellularized ECM from the liver, lung, and small intestine submucosa. Nanoparticles with dense PEG coatings achieved up to ∼2000-fold enhancements in diffusion rate and cellular uptake up to ∼5-fold greater than non-PEGylated nanoparticles in the presence of the ECM. We also found nanoparticle mobility in the ECM varied significantly between tissue types, and the optimal nanoparticle PEGylation strategy to enhance ECM penetration was strongly dependent on ECM concentration. Overall, our data support the use of low molecular weight PEG coatings which provide an optimal balance of nanoparticle penetration through the ECM and uptake in target cells. However, tissue-specific enhancements in ECM penetration and cellular uptake were observed for nanoparticles bearing a branched PEG coating. These studies provide insights into tissue specific ECM barrier functions, which can facilitate the design of nanoparticles that effectively transport through target tissues, improving their therapeutic efficacy.

Keywords:  PEGylation; decellularized ECM; extracellular matrix; multiple particle tracking; nanoparticle drug delivery

DOI:  https://doi.org/10.1021/acsnano.4c10381
NPJ Syst Biol Appl. 2024 Nov 06. 10(1): 129

Exploring heterogeneous cell population dynamics in different microenvironments by novel analytical strategy based on images.

Yihong Huang, Zidong Zhou, Tianqi Liu, Shengnan Tang, Xuegang Xin.

Understanding the dynamic states and transitions of heterogeneous cell populations is crucial for addressing fundamental biological questions. High-content imaging provides rich datasets, but it remains increasingly difficult to integrate and annotate high-dimensional and time-resolved datasets to profile heterogeneous cell population dynamics in different microenvironments. Using hepatic stellate cells (HSCs) LX-2 as model, we proposed a novel analytical strategy for image-based integration and annotation to profile dynamics of heterogeneous cell populations in 2D/3D microenvironments. High-dimensional features were extracted from extensive image datasets, and cellular states were identified based on feature profiles. Time-series clustering revealed distinct temporal patterns of cell shape and actin cytoskeleton reorganization. We found LX-2 showed more complex membrane dynamics and contractile systems with an M-shaped actin compactness trend in 3D culture, while they displayed rapid spreading in early 2D culture. This image-based integration and annotation strategy enhances our understanding of HSCs heterogeneity and dynamics in complex extracellular microenvironments.

DOI: https://doi.org/10.1038/s41540-024-00459-w
Biochim Biophys Acta Mol Cell Res. 2024 Oct 26. pii: S0167-4889(24)00214-3. [Epub ahead of print] 119871

PIEZO1 mediates matrix stiffness-induced tumor progression in kidney renal clear cell carcinoma by activating the Ca2+/Calpain/YAP pathway.

Biqiang Zhu, Fan Li, Jiajun Yu, Zhulin Liang, Xinwen Ke, Yong Wang, Zhengshuai Song, Zhongyuan Li, Guohao Li, Yonglian Guo.

   OBJECTIVE: The significance of physical factors in the onset and progression of tumors has been increasingly substantiated by a multitude of studies. The extracellular matrix, a pivotal component of the tumor microenvironment, has been the subject of extensive investigation in connection with the advancement of KIRC (Kidney Renal Clear Cell Carcinoma) in recent years. PIEZO1, a mechanosensitive ion channel, has been recognized as a modulator of diverse physiological processes. Nonetheless, the precise function of PIEZO1 as a transducer of mechanical stimuli in KIRC remains poorly elucidated.
METHODS: A bioinformatics analysis was conducted using data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) to explore the correlation between matrix stiffness indicators, such as COL1A1 and LOX mRNA levels, and KIRC prognosis. Expression patterns of mechanosensitive ion channels, particularly PIEZO1, were examined. Collagen-coated polyacrylamide hydrogel models were utilized to simulate varying stiffness environments and study their effects on KIRC cell behavior in vitro. Functional experiments, including PIEZO1 knockdown and overexpression, were performed to investigate the molecular mechanisms underlying matrix stiffness-induced cellular changes. Interventions in the Ca2+/Calpain/YAP Pathway were conducted to evaluate their effects on cell growth, EMT, and stemness characteristics.
RESULTS: Our findings indicate a significant correlation between matrix stiffness and the prognosis of KIRC patients. It is observed that higher mechanical stiffness can facilitate the growth and metastasis of KIRC cells. Notably, we have also observed that the deficiency of PIEZO1 hinders the proliferation, EMT, and stemness characteristics of KIRC cells induced by a stiff matrix. Our study suggests that PIEZO1 plays a crucial role in mediating KIRC growth and metastasis through the activation of the Ca2+/Calpain/YAP Pathway.
CONCLUSION: This study elucidates a novel mechanism through which the activation of PIEZO1 leads to calcium influx, subsequent calpain activation, and YAP nuclear translocation, thereby contributing to the progression of KIRC driven by matrix stiffness.

Keywords:  Calpain; Kidney renal clear cell carcinoma; Matrix stiffness; PIEZO1; YAP

DOI:  https://doi.org/10.1016/j.bbamcr.2024.119871
J Vis Exp. 2024 Oct 18.

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow.

Samantha Howerton, Yanping Liang, Jennifer Hammel, Benjamin Purow, Jennifer Munson.

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

DOI: https://doi.org/10.3791/66604
Adv Mater. 2024 Nov 01. e2411372

Biomimetic Structural Hydrogels Reinforced by Gradient Twisted Plywood Architectures.

Yulu Tang, Bentao Wu, Jie Li, Canhui Lu, Jianing Wu, Rui Xiong.

  Naturally structural hydrogels such as crustacean exoskeletons possess a remarkable combination of seemingly contradictory properties: high strength, modulus, and toughness coupled with exceptional fatigue resistance, owing to their hierarchical structures across multiple length scales. However, replicating these unique mechanical properties in synthetic hydrogels remains a significant challenge. This work presents a synergistic approach for constructing hierarchical structural hydrogels by employing cholesteric liquid crystal self-assembly followed by nanocrystalline engineering. The resulting hydrogels exhibit a long-range ordered gradient twisted plywood structure with high crystallinity to mimic the design of crustacean exoskeletons. Consequently, the structural hydrogels achieve an unprecedented combination of ultrahigh strength (46 ± 3 MPa), modulus (496 ± 25 MPa), and toughness (170 ± 14 MJ m-3), together with recorded high fatigue threshold (32.5 kJ m-2) and superior impact resistance (48 ± 2 kJ m-1). Additionally, through controlling geometry and compositional gradients of the hierarchical structures, a programmable shape morphing process allows for the fabrication of complex 3D hydrogels. This study not only offers valuable insights into advanced design strategies applicable to a broad range of promising hierarchical materials, but also pave the ways for load-bearing applications in tissue engineering, wearable devices, and soft robotics.

Keywords:  fatigue resistance; gradient structure; mechanical properties; structural hydrogels; twisted plywood structure

DOI:  https://doi.org/10.1002/adma.202411372
iScience. 2024 Nov 15. 27(11): 111017

Assessing mechanical agency during apical apoptotic cell extrusion.

Sommer Anjum, Llaran Turner, Youmna Atieh, George T Eisenhoffer, Lance A Davidson.

  Homeostasis is necessary for epithelia to maintain barrier function and prevent the accumulation of defective cells. Unfit, excess, and dying cells in the larval zebrafish tail fin epidermis are removed via controlled cell death and extrusion. Extrusion coincides with oscillations of cell area, both in the extruding cell and its neighbors. Here, we develop a biophysical model of this process to explore the role of autonomous and non-autonomous mechanics. We vary biophysical properties and oscillatory behaviors of extruding cells and their neighbors along with tissue-wide cell density and viscosity. We find that cell autonomous processes are major contributors to the dynamics of extrusion, with the mechanical microenvironment providing a less pronounced contribution. We also find that some cells initially resist extrusion, influencing the duration of the expulsion process. Our model provides insights into the cellular dynamics and mechanics that promote elimination of unwanted cells from epithelia during homeostatic tissue maintenance.

Keywords:  Bioinformatics; Biophysics; Cell biology

DOI:  https://doi.org/10.1016/j.isci.2024.111017
Acta Biomater. 2024 Oct 26. pii: S1742-7061(24)00621-4. [Epub ahead of print]

Developing a soft micropatterned substrate to enhance maturation of human induced pluripotent stem cell-derived cardiomyocytes.

Yasaman Maaref, Shayan Jannati, Farah Jayousi, Philipp Lange, Mohsen Akbari, Mu Chiao, Glen F Tibbits.

  Human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) offer numerous advantages as a biological model, yet their inherent immaturity compared to adult cardiomyocytes poses significant limitations. This study addresses hiPSCCM immaturity by introducing a physiologically relevant micropatterned substrate for long-term culture and maturation. An innovative microfabrication methodology combining laser etching and casting creates a micropatterned polydimethylsiloxane (PDMS) substrate with varying stiffness, from 2 to 50 kPa, mimicking healthy and fibrotic cardiac tissue. Platinum electrodes were integrated into the cell culture chamber enable pacing of cells at various frequencies. Subsequently, cells were transferred to the incubator for time-course analysis, ensuring contamination-free conditions. Cell contractility, cytosolic Ca2+ transient, sarcomere orientation, and nucleus aspect ratio were analyzed in a 2D hiPSCCM monolayer up to 90 days post-replating in relation to substrate micropattern dimensions. Culturing hiPSCCMs for three weeks on a micropatterned PDMS substrate (2.5-5 µm deep, 20 µm center-to-center spacing of grooves, 2-5 kPa stiffness) emerges as optimal for cardiomyocyte alignment, contractility, and cytosolic Ca2+ transient. The study provides insights into substrate stiffness effects on hiPSCCM contractility and Ca2+ transient at immature and mature states. Maximum contractility and fastest Ca2+transient kinetics occur in mature hiPSCCMs cultured for two to four weeks, with the optimum at three weeks, on a soft micropatterned PDMS substrate. MS proteomic analysis further revealed that hiPSCCMs cultured on soft micropatterned substrates exhibit advanced maturation, marked by significant upregulation of key structural, electrophysiological, and metabolic proteins. This new substrate offers a promising platform for disease modeling and therapeutic interventions. STATEMENT OF SIGNIFICANCE: Human induced pluripotent stem cell derived cardiomyocytes (hiPSCCMs) have been transformative to disease-in-a-dish modeling, drug discovery and testing, and autologous regeneration for human hearts and their role will continue to expand dramatically. However, one of the major limitations of hiPSCCMs is that without intervention, the cells are immature and represent those in the fetal heart. We developed protocols for the fabrication of the PDMS matrices that includes variations in its stiffness and micropatterning. Growing our hiPSCCMs on matrices of comparable stiffness to a healthy heart (5 kPa) and grooves of 20 μm, generate heart cells typical of the healthy adult human heart.

Keywords:  Contractility; HiPSC-CMs; Long-term culture; Maturation; Micropatterned substrate; PDMS

DOI:  https://doi.org/10.1016/j.actbio.2024.10.029
Adv Healthc Mater. 2024 Nov 05. e2402890

Foreign Body Immune Response to Zwitterionic and Hyaluronic Acid Granular Hydrogels Made with Mechanical Fragmentation.

Maryam Asadikorayerm, Patrick Weber, František Surman, Anna Puiggalí-Jou, Marcy Zenobi-Wong.

  Granular hydrogels have recently attracted the attention for diverse tissue engineering applications due to their versatility and modularity. Despite previous studies showing enhanced viability and metabolism of cells encapsulated in these hydrogels, the in vitro immune response and long-term fibrotic response of these scaffolds have not been well characterized. Here, bulk and granular hydrogels are studied based on synthetic zwitterionic (ZI) and natural polysaccharide hyaluronic acid (HA) made with mechanical fragmentation. In vitro, immunomodulatory studies show an increased stimulatory effect of HA granular hydrogels compared to bulk, while both bulk and granular ZI hydrogels do not induce an inflammatory response. Subcutaneous implantation in mice shows that both ZI and HA granular hydrogels resulted in less collagen capsule deposition around implants compared to bulk HA hydrogels 10 weeks after implantation. Moreover, the HA granular hydrogels are infiltrated by host cells, including macrophages and mature blood vessels, in a porosity-dependent manner. However, a large number of cells, including multinucleated giant cells as well as blood vessels, surround bulk and granular ZI hydrogels and are not able to infiltrate. Overall, this study provides new insights on the long-term stability and fibrotic response of granular hydrogels, paving the way for future studies and applications.

Keywords:  fragmentation; granular; hyaluronic acid; hydrogels; immune response; microgels; zwitterionic

DOI:  https://doi.org/10.1002/adhm.202402890
Nat Cell Biol. 2024 Nov 01.

Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion.

Yue Lu, Tezin Walji, Benjamin Ravaux, Pratima Pandey, Changsong Yang, Bing Li, Delgermaa Luvsanjav, Kevin H Lam, Ruihui Zhang, Zhou Luo, Chuanli Zhou, Christa W Habela, Scott B Snapper, Rong Li, David J Goldhamer, David W Schmidtke, Duojia Pan, Tatyana M Svitkina, Elizabeth H Chen.

Invasive membrane protrusions play a central role in a variety of cellular processes. Unlike filopodia, invasive protrusions are mechanically stiff and propelled by branched actin polymerization. However, how branched actin filaments are organized to create finger-like invasive protrusions is unclear. Here, by examining the mammalian fusogenic synapse, where invasive protrusions are generated to promote cell membrane juxtaposition and fusion, we have uncovered the mechanism underlying invasive protrusion formation. We show that two nucleation-promoting factors for the Arp2/3 complex, WAVE and N-WASP, exhibit different localization patterns in the protrusions. Whereas WAVE is closely associated with the plasma membrane at the leading edge of the protrusive structures, N-WASP is enriched with WIP along the actin bundles in the shafts of the protrusions. During protrusion initiation and growth, the Arp2/3 complex nucleates branched actin filaments to generate low-density actin clouds in which the large GTPase dynamin organizes the new branched actin filaments into bundles, followed by actin-bundle stabilization by WIP, the latter functioning as an actin-bundling protein. Disruption of any of these components results in defective protrusions and failed myoblast fusion in cultured cells and mouse embryos. Together, our study has revealed the intricate spatiotemporal coordination between two nucleation-promoting factors and two actin-bundling proteins in building invasive protrusions at the mammalian fusogenic synapse and has general implications in understanding invasive protrusion formation in cellular processes beyond cell-cell fusion.

DOI: https://doi.org/10.1038/s41556-024-01541-5
SLAS Discov. 2024 Oct 26. pii: S2472-5552(24)00052-2. [Epub ahead of print] 100190

Magnetic 3D bioprinting of skeletal muscle spheroid for a spheroid-based screening assay.

Chayanit Chaweewannakorn, Khin The Nu Aye, Joao N Ferreira.

  Over the past decade, there has been a rapid development in the use of magnetic three dimensional (3D) based cell culture systems. Concerning the skeletal muscle, 3D culture systems can provide biological insights for translational clinical research in the fields of muscle physiology and metabolism. These systems can enhance the cell culture environment by improving spatially-oriented cellular assemblies and morphological features closely mimicking the in vivo tissues/organs, since they promote strong interactions between cells and the extracellular matrix (ECM). However, the time-consuming and complex nature of 3D traditional culture techniques pose a challenge to the widespread adoption of 3D systems. Herein, a bench protocol is presented for creating an innovative, promptly assembled and user-friendly culture platform for the magnetic 3D bioprinting of skeletal muscle spheroids. Our protocol findings revealed consistent morphological outcomes and the functional development of skeletal muscle tissue, as evidenced by the expression of muscle-specific contractile proteins and myotubes and the responsiveness to stimulation with cholinergic neurotransmitters. This proof-of-concept protocol confirmed the future potential for further validation and application of spheroid-based assays in human skeletal muscle research.

Keywords:  3D cell culture; bioprinting; magnetic; nanoparticles; skeletal muscle

DOI:  https://doi.org/10.1016/j.slasd.2024.100190
Soft Matter. 2024 Nov 04.

Liquid crystalline collagen assemblies as substrates for directed alignment of human Schwann cells.

Homa Ghaiedi, Luis Carlos Pinzon Herrera, Saja Alshafeay, Leonard Harris, Jorge Almodovar, Karthik Nayani.

Collagen is a key component of the extracellular matrix (ECM) and well-oriented domains of collagen are important for mimicking the local cell environment in vitro. While there has been significant attention directed towards the alignment of collagen, formation of large-scale oriented domains remains a key challenge. Type I collagen self-assembles to form liquid crystalline (LC) mesophases in acidic conditions at concentrations above 100 mg mL-1. The LC mesophase provides an efficient platform for large-scale alignment and patterning of collagen coated substrates. However, there still exist challenges related to solubilizing and processing of collagen at such high concentrations in order to replicate the native ECM. In this contribution, we report on centimeter-scale alignment in collagen-coated glass substrates using solutions that are well below the LC-forming concentrations. Importantly, we are also able to extend this method to macroscopic 3-D LC-collagen hydrogels with programmed anisotropy within them to create a mimic of the native ECM. We show that the orientation and aspect ratio of human Schwann cells are strongly coupled with the alignment of the collagen substrate/hydrogel. We use a simple model to estimate the critical magnetic field strength needed for a given concentration of collagen to permit macroscopic alignment-enabling guidance for future studies on alignment of collagen at high concentrations.

DOI: https://doi.org/10.1039/d4sm00534a
J Gen Physiol. 2024 Dec 02. pii: e202213324. [Epub ahead of print]156(12):

Piezo2 interacts with E-cadherin in specialized gastrointestinal epithelial mechanoreceptors.

Arnaldo Mercado-Perez, Jeric P Hernandez, Yaroslav Fedyshyn, Anthony J Treichel, Vikram Joshi, Kimberlee Kossick, Kalpana R Betageri, Gianrico Farrugia, Brooke Druliner, Arthur Beyder.

Piezo2 is a mechanically gated ion channel most commonly expressed by specialized mechanoreceptors, such as the enteroendocrine cells (EECs) of the gastrointestinal epithelium. A subpopulation of EECs expresses Piezo2 and functionally resembles the skin's touch sensors, called Merkel cells. Low-magnitude mechanical stimuli delivered to the mucosal layer are primarily sensed by mechanosensitive EECs in a process we term "gut touch." Piezo2 transduces cellular forces into ionic currents, a process that is sensitive to bilayer tension and cytoskeletal depolymerization. E-cadherin is a widely expressed protein that mediates cell-cell adhesion in epithelia and interacts with scaffold proteins that anchor it to actin fibers. E-cadherin was shown to interact with Piezo2 in immortalized cell models. We hypothesized that the Piezo2-E-cadherin interaction is important for EEC mechanosensitivity. To test this, we used super-resolution imaging, co-immunoprecipitation, and functional assays in primary tissues from mice and gut organoids. In tissue EECs and intestinal organoids, we observed multiple Piezo2 cellular pools, including one that overlaps with actin and E-cadherin at the cells' lateral walls. Further, E-cadherin co-immunoprecipitated with Piezo2 in the primary colonic epithelium. We found that E-cadherin knockdown decreases mechanosensitive calcium responses in mechanically stimulated primary EECs. In all, our results demonstrate that Piezo2 localizes to the lateral wall of EECs, where it physically interacts with E-cadherin and actin. They suggest that the Piezo2-E-cadherin-actin interaction is important for mechanosensitivity in the gut epithelium and possibly in tissues where E-cadherin and Piezo2 are co-expressed in epithelial mechanoreceptors, such as skin, lung, and bladder.

DOI: https://doi.org/10.1085/jgp.202213324
Nat Commun. 2024 Nov 01. 15(1): 9443

Optogenetic patterning generates multi-strain biofilms with spatially distributed antibiotic resistance.

Xiaofan Jin, Ingmar H Riedel-Kruse.

Spatial organization of microbes in biofilms enables crucial community function such as division of labor. However, quantitative understanding of such emergent community properties remains limited due to a scarcity of tools for patterning heterogeneous biofilms. Here we develop a synthetic optogenetic toolkit 'Multipattern Biofilm Lithography' for rational engineering and orthogonal patterning of multi-strain biofilms, inspired by successive adhesion and phenotypic differentiation in natural biofilms. We apply this toolkit to profile the growth dynamics of heterogeneous biofilm communities, and observe the emergence of spatially modulated commensal relationships due to shared antibiotic protection against the beta-lactam ampicillin. Supported by biophysical modeling, these results yield in-vivo measurements of key parameters, e.g., molecular beta-lactamase production per cell and length scale of antibiotic zone of protection. Our toolbox and associated findings provide quantitative insights into the spatial organization and distributed antibiotic protection within biofilms, with direct implications for future biofilm research and engineering.

DOI: https://doi.org/10.1038/s41467-024-53546-1