Biology subjects

Generali, M.

Publications and source records attributed to Generali, M..

9 recordsLinked to original sources

bioRxiv2026

3D Human Neuromuscular Architecture Encoded by Compliant Patterned Hydrogel Scaffolds

Engineered human neuromuscular models are limited by a trade-off between biological fidelity and architectural control: self-assembled organoids organize stochastically, while engineered 3D tissues impose anisotropy through passive tension at the edges without internal guidance. Here we encode architectural guidance into human neuromuscular tissues by coupling a three-dimensional, cell-laden biomatrix to a xolographically printed hydrogel substrate featuring sub-millimeter surface topography. We demonstrate that grooved substrates increase human primary myotube alignment and maturation in the overlying 3D tissue, while guiding directional axon extension from human iPSC-derived motor neuron spheroids. We show that combining both cell types along a shared alignment axis establishes functional neuromuscular connectivity, evidenced by acetylcholine receptor clustering, glutamate-evoked contraction and its blockade by d-tubocurarine (curare), and yields innervation coverage of centimeter scale tissues. Overall, this platform combines compliant substrates, mesoscale tissue architectural guidance, and human-relevant cell biology in a single untethered construct for studies of neuromuscular tissue development and function.

bioengineering↗

bioRxiv2026

Stimulatrix: An open-source automated platform for high-throughput functional characterization of engineered contractile tissues

Engineered cardiac and skeletal muscle tissues suspended between flexible posts support disease modeling and pharmacology, yet their stimulation, longitudinal imaging and quantitative analysis often remain fragmented and labor-intensive. Here we present Stimulatrix, an open-source platform integrating automated video acquisition within a cell culture incubator, synchronized electrical stimulation and deep learning for longitudinal assessment of contractile tissues in multiwell plates. The platform quantifies tissue compaction, force generation and contraction kinetics with optional cloud processing reducing dependence on local GPU hardware. We demonstrate the workflow in cardiac tissues comprising human induced pluripotent stem cell-derived cardiomyocytes and cardiac fibroblasts, and in primary human skeletal muscle constructs. Force and kinetic measurements were benchmarked against manual annotations. Longitudinal profiling resolved responses to matrix composition and pacing regimens and tracked doxorubicin-associated loss of cardiac contractile force. Stimulatrix provides an accessible workflow for automated functional phenotyping of engineered muscle tissues.

bioengineering↗

bioRxiv2026

Neural graft elimination via dual safety switch compromises therapeutic recovery after stroke

Safety switch systems are increasingly incorporated into stem cell therapies to enable selective graft elimination in the event of adverse outcomes. Yet how removing transplanted cells affects the underlying pathology remains largely unexplored. Here, we show that iPSC-derived neural stem cells (NSCs) co-transduced with herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase-9 (iC9) are more efficiently ablated by combined ganciclovir (GCV) and chemical inducer of dimerization (CID) treatment than either single switch alone, under both proliferating and differentiating conditions. Using bioluminescence imaging to track graft survival longitudinally, we demonstrate that dual safety switch NSC (DS-NSC) grafts are selectively eliminated by GCV and CID administration in immunodeficient mice following photothrombotic stroke. The combination treatment initiated on day 8 post-transplantation resulted in reduced graft volume, lower NSC proliferation, and persistently reduced bioluminescence signal compared to mice receiving only solvent. Histological analysis revealed that combination treatment was associated with larger stroke lesions, higher IBA1 fluorescence intensity, and reduced vascular density in the ischemic border zone. Mice receiving GCV and CID showed persistently elevated contralateral hindlimb error rates on a ladder walk task from day 28 post-stroke onward compared to solvent-treated mice. These findings demonstrate that while DS-NSC elimination is feasible, the pharmacological activation of the safety switch system is associated with impaired functional stroke recovery.

neuroscience↗

bioRxiv2026

Targeting integrin αvβ3 by chimeric antigen receptor neural stem cell (CAR-NSC) therapy for stroke

Stroke remains a leading cause of adult disability due to the brains limited regenerative capacity. Although stem cell therapies show favorable safety and feasibility profiles in early clinical trials, poor spatial retention and limited engagement with peri-infarct salvageable tissue constrain efficacy. Here, we engineered human induced pluripotent stem cell-derived neural stem cells (NSC) with a chimeric antigen receptor (CAR)-like architecture to enable targeted recognition of injury-associated cues. Specifically, cells were modified to express a membrane-anchored single chain variable fragment (scFv) targeting integrin v{beta}3, a receptor selectively upregulated in peri-infarct vasculature after stroke. Engineered CAR-NSC retained progenitor identity and selectively bound recombinant integrin v{beta}3 in vitro. Following focal transplantation into a photothrombotic stroke mouse model, CAR-NSC displayed broader dispersion within peri-infarct tissue and covered a greater proportion of the ischemic lesion compared to non-binding control-CAR-NSC. CAR-NSC grafts extended longer neurites that aligned more closely with the lesion border. In addition, CAR-NSC transplantation reduced microglial activation and was associated with increased vascular density and blood-brain barrier integrity in the peri-infarct zone. Together, these findings establish a CAR-like NSC strategy for stroke to direct the spatial distribution and tissue engagement of transplanted cells. Molecular targeting of injury-associated cues may improve the precision and regenerative efficacy of cell-based therapies for stroke and related neurological disorders.

neuroscience↗

bioRxiv2025

Hypoxia preconditioned neural xenografts promote repair of brain tissue after stroke

Stroke is a leading cause of long-term disability, yet no effective regenerative therapies exist. While cell-based therapies have shown promise in preclinical animal models, their clinical application remains limited due to poor survival of transplanted cells in the ischemic stroke environment. Hypoxic preconditioning has emerged as a strategy to potentially enhance graft survival, but the cellular mechanisms and translational relevance in human iPSC-derived neural progenitor cells (NPCs) are not fully understood. Here, we tested whether hypoxic preconditioning of NPCs affects their molecular and functional properties including proliferation and survival in vitro and after transplantation into a stroke mouse model. Hypoxic preconditioning enhanced proliferation and glial differentiation in vitro, improved cell survival post-transplantation, and enhanced regeneration-associated tissue responses such as vascular remodeling in the peri-infarct brain. These findings suggest that hypoxic preconditioning is a clinically translatable approach to increase the NPC graft survival in the post-stroke brain.

neuroscience↗

bioRxiv2025

Generation of Valvular Interstitial Cells from Human Pluripotent Stem Cells

Heart valves are living structures whose sophisticated functions are mediated by a specialized population of mesenchymal cells known as valvular interstitial cells (VICs). Given their central role in valve homeostasis, VICs represent a promising cell population for studying heart valve diseases and developing novel therapies to treat them. Here, we describe a strategy for generating VICs from human pluripotent stem cells (hPSCs) by stage-specific manipulation of developmental signalling pathways. Our results demonstrate that hPSC-derived VICs show a high transcriptional similarity to primary human fetal VICs and can secrete key proteins of the valve extracellular matrix. We further investigate the heterogeneity of hPSC-derived VICs and identify two major subpopulations with distinct molecular and functional properties, mirroring the cellular diversity observed in vivo. Finally, we utilize an in vitro model of Noonan syndrome to demonstrate that hPSC-derived VICs can accurately recapitulate key aspects of valve disease. Collectively, these findings provide a reproducible method for the scaled generation of bona fide hPSC-derived VICs and establish their utility in disease modelling and tissue engineering applications. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- We established a robust platform to generate bona fide valvular interstitial cells (VICs) from human pluripotent stem cells (hPSCs), recapitulating native VIC identity and function. - We delineated signaling pathways that promote the development of two distinct VIC subsets and identified a surface marker to distinguish between them. What are the clinical implications?- A renewable, human-specific source of VICs enables precise mechanistic studies of valve development and disease that are not possible with limited surgical specimens or animal models. - This platform creates opportunities for therapeutic applications, including drug discovery and tissue engineering approaches for valve repair.

cell biology↗

bioRxiv2025

Volumetric 3D Printing and Melt-Electrowriting to Fabricate Implantable Reinforced Cardiac Tissue Patches

Cardiac patches for repairing myocardial defects require mechanically stable materials that prevent bleeding and can be implanted via suturing. The current clinical standard, bovine pericardial patches (BPPs), serve this purpose but do not degrade or integrate with the myocardium, limiting their long-term effectiveness. Therefore, we have developed the Reinforced engineered Cardiac tissue Patch (RCPatch). This multimaterial patch consists of a stiffness-tuned, cardiomyocyte-infiltrated 3D metamaterial and a suturable, hydrogel-infiltrated mesh to reduce permeability and bleeding. We first designed and computationally optimized anisotropic metamaterials using a generative modelling approach and fabricated them from biodegradable poly({varepsilon}-caprolactone) (PCL) via volumetric 3D printing (VP). The metamaterial supported the infiltration of cardiomyocytes, which maintained cell viability and contractility in vitro. In a second step, we enhanced implantability and reduced blood permeability through the patch by combining a melt-electrowritten (MEW) mesh with a fibrin hydrogel. Finally, in an acute large animal trial, the RCPatch was used on an induced myocardial defect, where it withstood intraventricular blood pressure and enabled partial hemodynamic recovery. Our findings establish a scalable framework for fabricating cardiac tissue patches that integrate mechanical reinforcement with biological function, offering a surgically implantable, and potentially regenerative solution for intraventricular myocardial repair. Table of Contents (ToC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/642643v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@66293dorg.highwire.dtl.DTLVardef@11d9122org.highwire.dtl.DTLVardef@235d16org.highwire.dtl.DTLVardef@1b46911_HPS_FORMAT_FIGEXP M_FIG C_FIG This study presents an implantable intraventricular cardiac patch (RCPatch) combining volumetric 3D-printed metamaterials with melt-electrowritten (MEW) meshes. The design integrates tunable stiff structures with soft, cell-laden hydrogels. The RCPatch withstood suturing, intraventricular pressure, and cardiac contraction in an acute large animal myocardial defect model. The patch prevented bleeding and enabled partial hemodynamic recovery, demonstrating its potential for myocardial repair.

bioengineering↗

bioRxiv2024

Human iPSC-derived cell grafts promote functional recovery by molecular interaction with stroke-injured brain

Stroke is a leading cause of disability and death due to the brains limited ability to regenerate damaged neural circuits. To date, stroke patients have only few therapeutic options and are often left with considerable disabilities. Induced pluripotent stem cell (iPSC)-based therapies are emerging as a promising therapeutic approach for stroke recovery. In this study, we demonstrate that local transplantation of iPSC-derived neural progenitor cells (NPCs) improves long-term brain tissue repair responses and reduces neurological deficits after cerebral ischemia in mice. Using in vivo bioluminescence imaging and post-mortem histology, we show long-term graft survival over the course of five weeks and preferential graft differentiation into mature neurons without signs of pluripotent residuals. Transplantation of NPCs led to a set of brain tissue repair responses including increased vascular sprouting and repair, improved blood-brain barrier integrity, reduced microglial activation, and increased neurogenesis compared to littermate control animals receiving sham transplantation. Employing deep learning-assisted behavior analysis, we found that NPC-treated mice displayed improved gait performance and complete fine-motor recovery in the horizontal ladder rung walk, five weeks post-injury. To dissect the molecular graft composition and identify graft-host interactions, single nucleus profiling of the cell transplants and host stroke tissue was performed. We identified graft differentiation preferentially towards neurons with GABAergic and glutamatergic phenotypes in similar proportions, with the remaining cells acquiring astrocyte and NPC-like phenotypes. Interaction between graft and host transcriptome indicated that GABAergic cell grafts were primarily involved in graft-host communication through the regeneration-associated neurexin (NRXN), neuregulin (NRG), neural cell adhesion molecule (NCAM) and SLIT signalling pathways. In conclusion, our study reveals that transplanted iPSC-derived NPCs primarily differentiate into GABAergic neurons contributing to long-term recovery and further delineates the regenerative interactions between the graft and the stroke-injured host tissue.

neuroscience↗

bioRxiv2022

Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications

Cell-based therapies are a promising treatment paradigm for neurodegenerative diseases and other brain injuries. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. Here, we generate good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. The produced NPCs have a stable gene-expression over at least 15 passages and can be scaled for up to 1018 cells per initially seeded 106 cells. To ensure a pure NPC population for in vivo applications, we reduce risks of iPSC contamination using micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted cells have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month.

neuroscience↗