Advanced Science

AbstactThe conservation of genetic resources from aquaculture species and endangered fish is increasingly challenged by large body size, long reproductive cycles, and limited opportunities for timely intervention after death. Here, we establish and validate an ultra-fast genetic platform based on germline stem cell transplantation to enable postmortem genetic recovery in fish. Using grass carp (Ctenopharyngodon idella) as a representative warm-water species, we systematically quantified the relationships among postmortem tissue freshness, germline stem cell viability, and transplantation efficiency, and demonstrated that low-temperature preservation plays a decisive role in maintaining germline activity after death. Germline stem cells isolated from deceased grass carp were transplanted into germ-cell-depleted zebrafish recipients, where they rapidly colonized recipient gonads, underwent proliferation and differentiation, and generated functional donor-derived gametes within three months. These gametes supported successful fertilization and normal embryonic development, ultimately yielding viable grass carp offspring. Our results reveal an intrinsic postmortem resilience of germline stem cells and demonstrate that cross-species transplantation into small, fast-maturing hosts can dramatically accelerate genetic recovery. This strategy overcomes key biological and logistical constraints associated with conventional breeding-based rescue approaches and provides a rapid, scalable, and broadly applicable framework for postmortem genetic resource conservation in aquaculture and endangered fish species.

Cervical spinal cord injury (SCI) causes profound and persistent loss of hand function, and effective neuromodulation strategies remain limited. We report the first-in-human implantation of a 32-contact cervical epidural paddle array in two individuals with severe chronic SCI. Individualized motor pool recruitment maps, derived from systematic bipolar and multipolar configurations, enabled person-specific stimulation parameters. Optimized stimulation restored volitional hand opening, closing and coordinated upper-limb movements that were previously unattainable. This approach achieved a >91% success rate in complex reach-grasp-lift-release sequences, supported by substantial gains in range of motion, grip, and pinch strength. Electrophysiological and kinematic analyses demonstrated parameter-dependent, selective recruitment of flexor and extensor motor pools. Personalized stimulation programs integrated with goal-directed activities enabled functional hand use in home and community settings, sustained over several months of continued autonomous use. These findings establish a mechanistically grounded and translational framework for restoring upper-limb function after chronic severe SCI.

The blood-brain barrier (BBB) is a highly specialized system that is critical for regulating transport between the blood and the central nervous system. In brain tumors, the vasculature system is compromised, and is referred to as the blood-tumor barrier (BTB). The ability to precisely model the unique physiological properties of the BTB is essential to decipher its role in tumor pathophysiology and for the rational design of efficacious therapeutics. Here, we introduce a robust and high-throughput in vitro 3D human BTB organoid model that recapitulates various key features of the BTB observed in vivo and in clinical GBM samples. The organoids are composed of patient-derived glioblastoma stem cells (GSCs), human brain endothelial cells (EC), astrocytes and pericytes, which are formed through self-assembly. Transcriptomic and functional analyses reveal that the GSCs in the BTB organoids exhibit enhanced level of stemness, mesenchymal signature, invasiveness and angiogenesis, and this is further confirmed in in vivo studies. We demonstrate the ability of the BTB organoids to model therapeutic delivery and drug efficacy on brain tumor cells. Collectively, our findings show that the BTB organoid model has broad utility as a clinically representative system for studying the BTB and evaluating brain tumor therapies.

The Neuron-as-a-Sensor (NaaS) methodology is a human-relevant platform designed to detect compound-induced effects by capturing functional changes in neuronal activity. This is achieved by integrating hiPSC-derived neuronal cultures, compartmentalized MEA microfluidic devices, a detailed electrophysiology paradigm and a standardized analysis pipeline. Applied to chemotherapy-induced peripheral neuropathy (CIPN), NaaS leverages electrophysiological profiling to capture alterations in neuronal excitability beyond cytotoxicity. Using paclitaxel and oxaliplatin as reference compounds, we demonstrated drug-specific, time-dependent changes in spontaneous and thermally evoked activity that align with their distinct clinical neuropathic phenotypes. Dimensionality reduction of electrophysiological metrics enabled construction of a functional discrimination map, allowing robust separation of compound signatures from vehicle controls. These findings highlight the ability of NaaS to model clinically relevant neurotoxic effects in a scalable manner, supporting its application in both adverse effect prediction and therapeutic screening.

Glaucoma is characterized by progressive stiffening of the trabecular meshwork (TM), which elevates intraocular pressure and contributes to tissue dysfunction. Although substrate stiffness and mechanical stimulation both regulate TM homeostasis, their combined effects remain poorly understood. Here, a hydrogel-integrated microfluidic platform is presented that enables simultaneous control of substrate stiffness via tunable gelatin methacryloyl (GelMA) hydrogels and equi-biaxial quasi-static stretch via hydraulic actuation. Finite element analysis validates the applied strain field, and optimized crosslinking ensures structural stability. Primary normal TM (nTM) and glaucomatous TM (gTM) cells cultured under coupled conditions exhibit selective mechanotransduction dysregulation rather than global mechanosensory impairment. While nTM cells dynamically regulate -smooth muscle actin (-SMA), myocilin (MYOC), matrix metalloproteinase-2 (MMP2), and collagen type I (COL1), gTM cells display constitutively elevated -SMA, loss of mechanical regulation of MMP2, and impaired stretch-mediated COL1 suppression, while retaining stiffness-dependent focal adhesion kinase and MYOC sensitivity. Key differences between normal and glaucomatous cells emerge only under combined stiff and stretched conditions, underscoring the importance of coupled mechanical cues in revealing disease-relevant phenotypes. These findings implicate tissue stiffening in selective pathway dysregulation and highlight mechanotransduction-targeted therapeutic strategies.

While extracellular matrix (ECM) mechanics strongly regulate stem cell commitment, the fields mechanistic understanding of this phenomenon largely derives from simplified two-dimensional (2D) culture substrates. Here we found a three-dimensional (3D) matrix-specific mechanoresponsive mechanism for neural stem cell (NSC) differentiation. NSC lineage commitment in 3D is maximally stiffness-sensitive in the range of 0.1-1.2 kPa, a narrower and more brain-mimetic range than we had previously identified in 2D (0.75 - 75 kPa). Transcriptomics revealed stiffness-dependent upregulation of early growth response 1 (Egr1) in 3D but not in 2D. Egr1 knockdown enhanced neurogenesis in stiff ECMs by driving {beta}-catenin nuclear localization and activity in 3D, but not in 2D. Mechanical modeling and experimental studies under osmotic pressure indicate that stiff 3D ECMs are likely to stimulate Egr1 via increases in confining stress during cell volumetric growth. To our knowledge, Egr1 represents the first 3D-specific stem cell mechanoregulatory factor.

Bacterial endocarditis is a fatal cardiovascular disease exacerbated by weakened heart contraction, yet the direct impact of cardiac contractility on bacterial adhesion remains elusive. Here, we present a novel quantitative physics model integrating finite element analysis and live-cell imaging to uncover their strong correlation. Using this model, we quantified the real-time force magnitude generated by organoid-type cardiac microtissue derived from healthy donors and dilated cardiomyopathy patients - mimicking normal and suppressed heart contractility, respectively - to the approaching bacteria in a real fluidic system. The data revealed that weakened cardiac contractility facilitated bacterial invasion of the myocardium. Verifying this finding in a mouse transverse aortic constriction model demonstrated that increasing heart contraction efficiently mitigated bacterial invasion, with a 25% increase in heart contractility reducing endocarditis risk by 80%. Our findings demonstrate that patient-derived cardiac organoids provide a physiologically relevant platform for studying bacterial infections in vitro, offering high clinical fidelity. This platform establishes a valuable tool for drug screening and the development of novel therapeutic strategies.

Growth plate (GP) is the critical cartilaginous structure for longitudinal bone growth. Herein, employing high-resolution analytical techniques, we explore the intricate mechanisms that govern the polarized mineralization patterns within GP. The GP-epiphysis interface displays a sharp transition in tissue modulus, acting as a "protective shell" for the underlying GP, whereas the GP-metaphysis interface exhibits a gradual modulus increase, enabling efficient load redistribution to metaphysis. The unique mechanical environments at these two interfaces contribute to polarized CaP mineralization patterns, which are regulated by a complex protein-based molecular machinery. The mineralization inhibitors SPP1 and AHSG enriched at the GP-epiphysis interface could act as a line of defence against mineralization. When these two proteins coexisted with the mineralization-promoting ENPP1 and ALPL at the GP-metaphysis interface, a sequential event of precise nucleation and programmable assembly of CaP minerals occur, forming "mineralization waves" to guide bone elongation. By replicating such specific macromolecular environment at GP-metaphysis interface, a hypertable amorphous calcium phosphate (ACP) phase is well-retained in vitro, demonstrating the possibility for precise and gentle control of ACP-hydroxyapatite (HAp) transformation under physiological conditions. Our study defines a novel concept of "mineralization waves" that govern the velocity and amplitude of GP-guided mineralization process.

The heterogeneity of dermal mesenchymal cells, including perivascular mesenchymal cells and papillary and reticular fibroblasts, plays critical roles in skin homeostasis. Herein, we present human skin equivalents (HSEs), in which pericytes, papillary fibroblasts, and reticular fibroblasts are spatially organized through autonomous three-cell interactions among epidermal keratinocytes, dermal fibroblasts, and vascular endothelial cells. The replication of dermal mesenchymal cell heterogeneity enhances skin functions, including epithelialization, epidermal barrier formation, and dermal elasticity, enabling in vitro evaluation of drug efficacy using methodologies that are identical to those used in human clinical studies. Furthermore, ascorbic acid-induced epidermal turnover and synthesis of well-aligned extracellular matrix via perivascular niche cells play crucial roles in improving skin barrier function and elasticity. Therefore, HSEs with heterogeneous dermal mesenchymal cells may improve our understanding of the mechanisms underlying skin homeostasis through cell-to-cell communication and serve as a model to animal experiments for developing precision medicine.

Artificial intelligence (AI)-driven discovery of antimicrobial peptides (AMPs) is yet to fully utilise their three-dimensional (3D) structural characteristics, microbial specie-specific antimicrobial activities and mechanisms. Here, we constructed a QLAPD database comprising the sequence, structures and antimicrobial properties of 12,914 AMPs. QLAPD underlies a multimodal, multitask, multilabel, and conditionally controlled AMP discovery (M3-CAD) pipeline, which is proposed for the de novo design of multi-mechanism AMPs to combat multidrug-resistant organisms (MDROs). This pipeline integrates the generation, regression, and classification modules, using a innovative 3D voxel coloring method to capture the nuanced physicochemical context of amino acids, significantly enhancing structural characterizations. QL-AMP-1, discovered by M3-CAD, which possesses four antimicrobial mechanisms, exhibited low toxicity and significant activity against MDROs. The skin wound infection model demonstrates its considerable antimicrobial effects and negligible toxicity. Altogether, integrating 3D features, specie-specific antimicrobial activities and mechanisms enhanced AI-driven AMP discovery, making the M3-CAD pipeline a viable tool for de novo AMP design.

Lightning has been proposed as a pivotal energy source driving prebiotic chemical evolution and early life processes. Similarly, external physical stimuli such as sound and light have been shown to modulate neurophysiological activity and mitigate Alzheimers disease (AD) pathology by promoting amyloid-{beta} (A{beta}) clearance. Here, we demonstrate that cold atmospheric plasma (CAP) acts as a joule-level analog of lightning, integrating light, sound, and reactive species to modulate AD progression in a mouse model. Using multimodal analyses, we show that CAP treatment enhances key neuroimmune signaling pathways in an AD murine model, including microglial activation, without inducing pathological alterations at the functional, transcriptomic, or proteomic levels in healthy mice. These findings highlight CAP as a safe and efficacious modality for modulating neurodegenerative processes, establishing a foundation for its therapeutic translation in AD and related disorders.

Persistent hyperglycemia impairs wound healing in diabetic patients, and severe cases may even lead to disability or death. Glycemic control alone cannot effectively prevent the occurrence of diabetic foot ulcers, a serious complication of diabetes. However, safe, efficient, and cost-effective therapies remain unavailable and are urgently needed. Using a novel sports medicine paradigm, we predicted, based on reverse-transcriptomics, that exercise-induced sweat has the potential to promote would healing in diabetic foot ulcers. Subsequent animal experiments demonstrated that sweat can indeed promote re-epithelialization and collagen deposition, upregulate the expression of the proliferation marker Ki-67, the angiogenesis marker CD31, and -SMA, and significantly accelerate wound healing in a mouse model of diabetic foot ulcers. This study provides a new direction for sports medicine and offers a novel therapeutic strategy for patients with diabetic foot ulcers.

The kidney epitheliums pivotal role in molecular filtration, metabolism, and excretion highlights the crucial importance of understanding kidney physiology in drug development. However, our knowledge is largely derived from non-human or non-physiological models, potentially limiting its applicability to humans. To address this significant gap, we have pioneered a human kidney epithelial microphysiological analysis platform (Epi-MAP) designed to establish, mature, and monitor renal functions of the human collecting epithelium within a physiologically relevant microenvironment. We first demonstrate the highly mature collecting duct physiology derived from human stem cells, enabled by the Epi-MAPs microenvironments that recapitulate in vivo asymmetries in fluidic and biochemical conditions. The integrated biosensors of the Epi-MAP provide long-term, time-resolved epithelial maturation trajectories, revealing advanced integrity and functional maturity with transepithelial metrics. Furthermore, Epi-MAPs electrophysiological analytics for measuring water flux, in conjunction with transepithelial potential and resistance, allow for real-time decoding of intricate epithelial responses to substance stimulation, showcasing its effectiveness as a robust pharmacological test model. This human cell-derived, physiologically advanced model on a chip stands as a robust in vitro tool, offering comprehensive insights into human kidney biology and significantly enhancing drug discovery process based on human physiology.

Peripheral neuropathy (PN) is the most common complication of diabetes. It is characterized by sensory loss which often causes major health consequences including foot ulceration, chronic pain, poor mobility and increased risk of falls. However, present treatments do not counteract the cause of the disease, namely lack of sensory feedback, but rather aim at partial and temporal symptoms relief (e.g. analgesics for pain or creams for ulcers healing). Electrical stimulation is a promising solution for sensory restoration, but it is yet unknown if it can elicit perceivable sensations in PN damaged nerves and whether it could lead to any health or functional benefits. To this aim, we designed a wearable sensory neuroprosthesis providing targeted neurostimulation at the ankle level (NeuroStep) restoring feet lost sensations. We tested it in 14 participants with PN, evaluating its effects on functional outcomes and pain, and the cortical activation related to the restored sensations. Our system was able to restore lost sensations in all participants. The nerves of PN participants resulted significantly less excitable and sensitive than healthy individuals (N=22). Thanks to the neurostimulation, participants improved cadence and functional gait, with even stronger improvements in individuals with higher risk of falls. A full day of NeuroStep use led to a clinically significant reduction of 30.4% {+/-} 9.2% in neuropathic pain. Restored sensations activated cortical patterns, as measured via fMRI, similar to the naturally located foot sensations, thus not requiring training by the user. NeuroStep restores intuitive sensations in PN participants, improving mobility and decreasing pain, possibly replacing multiple inefficient treatments. It holds potential to drastically improve patients quality of life thanks to functional and health benefits, while paving the way to new effective neuromodulation treatments.

Fibrotic scarring is a pervasive and unresolved challenge in medicine, leading to permanent disfigurement, impaired mobility, and severe disruption of basic skin functions including elasticity, barrier protection, and thermoregulation. Despite its far-reaching personal, clinical, and economic impact, affecting hundreds of millions worldwide after surgery, trauma, and burns, no effective treatments exist to halt or reverse pathological scar formation. Scarring results from uncontrolled TGF-b1 signaling, which drives excessive deposition of extracellular matrix (ECM) proteins such as collagen-I/III and accumulation of alpha-smooth muscle actin (alpha-SMA), producing rigid, dysfunctional tissue. Here, we present a mechanistically guided approach targeting this unmet clinical need, leveraging the natural antifibrotic peptide hormone relaxin-2 (RLX-2) to actively remodel dermal architecture. RLX-2 signals via its G-protein coupled receptor RXFP1, upregulating matrix metalloproteinases (MMPs) and inhibiting aberrant ECM production. In TGF-b1-activated dermal fibroblasts across 2D and 3D in vitro models, ex vivo healthy and scarred human skin samples - cultured under physiological and pathological tension - and in an in vivo murine burn wound model, RLX-2 robustly suppresses fibrosis, restores regenerative tissue features, and rescues dermal architecture. Importantly, RLX-2 achieves this result without compromising the normal wound healing process, highlighting its potential as a transformative therapy for both prevention and reversal of pathological scarring.

The gut-brain axis has emerged as a crucial factor in neurodegeneration, with growing evidence linking gut dysbiosis and metabolic dysfunction to Alzheimers disease (AD) progression. Unfortunately, the lack of human-relevant in vitro models limits our ability to effectively explore the mechanism of this axis. To address this gap, we have developed a human induced pluripotent stem cell (iPSC)-derived gut-blood-brain barrier (BBB)-brain microphysiological system that enables systematic investigation of gut-brain interaction in the context of AD under controlled conditions. Our findings reveal that the interaction between gut and brain organoids can promote the maturation of brain organoids, making them more similar to their physiological characteristics in vivo. Additionally, co-culture gut and brain organoids better recapitulates the pathological features of AD. We also discovered that gut organoids of AD can trigger neurodegenerative disease manifestations in healthy brain organoids. In summary, our microphysiological system provides a novel and versatile in vitro platform for studying the interaction between the gut and brain in neurodegenerative diseases.

Clostridioides difficile infection (CDI) is a leading healthcare-associated diarrhea with high recurrence rates, partially due to antibiotic-induced dysbiosis and dysregulated host inflammation. Specialized pro-resolving mediators (SPMs), such as Lipoxin A4 (LXA4), offer promise in controlling excessive inflammation and promoting tissue repair, yet their role in CDI remains unexplored. Here, we developed a compact, gas-tight gut-on-a-chip (GOC) system that reconciles the anaerobic requirements of C. difficile with the oxic needs of human intestinal epithelium, enabling physiologically relevant co-culture within a standard incubator. A CDI in vitro model was established based on this GOC system. Using the model, we demonstrated that prophylactic administration of LXA4 significantly preserved epithelial barrier integrity, attenuated pro-inflammatory cytokine secretion (IL-8 and IFN-{gamma}), and reduced bacterial colonization. Transcriptomic analysis revealed that LXA4 pretreatment upregulated genes involved in cell junction organization while downregulated immune activation pathways. These protective effects were validated in a murine CDI model, where LXA4 pretreatment reduced weight loss, pathological damage, and fecal bacterial burden. Furthermore, prophylactic administration of LXA4 synergized with vancomycin treatment further enhanced antibiotic efficacy while allowing a 50% dose reduction without compromising therapeutic outcomes. Our study establishes a robust approach for CDI research and highlights the prophylactic and adjuvant potential of inflammation-resolving strategies, offering a novel approach to mitigate CDI incidence and improve treatment outcomes.

The lymphatic system plays various crucial but underappreciated roles in fluid transport and immune response in numerous organs and tissue types. Consequently, generation of lymphatic vessels has been postulated as an innovative therapeutic strategy for various diseases. However, there is a lack of efficient and reliable method to differentiate human pluripotent stem cells into lymphatic endothelial cells (LEC) for lymphatic regeneration. Current differentiation methods suffer from poor yield and low lymphatic marker expression, while also having limited clinical applicability due to its reliance on either the embryoid body intermediates or xenogenic supporting cells. Given that LECs exclusively rely on anaerobic and fatty acid metabolism due to the hypoxic environment of the lymph, here we report that the unique lymphatic-specific metabolic pathways can be exploited to promote lymphatic identity in differentiated LECs (dLECs). We show that dLECs express elevated levels of lymphatic markers compared to native endothelial cells, which is up to 15 times higher than the current leading standard of dLECs. Moreover, dLECs can form lymphatic vascular networks in both 2D and 3D, as well as secrete important lymphangiocrine for organ maturation. Upon implantation into double-ligation tail lymphedema and mammary fat pad models, dLECs were able to integrate with the host lymphatic vessels, restore fluid flow, and reduce swelling. Collectively, we show that metabolite supplementation can drive stem cell differentiation into dLECs, which can be incorporated into new alternative methods for personalized therapies and disease modeling, as well as provide a direct therapeutic option for lymphedema and lymphatic disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/686405v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1c4caa5org.highwire.dtl.DTLVardef@d42b08org.highwire.dtl.DTLVardef@1554ffcorg.highwire.dtl.DTLVardef@1f64ae6_HPS_FORMAT_FIGEXP M_FIG C_FIG A xeno-free and stepwise differentiation protocol with VEGF-C and sodium acetate induces lymphatic identity in iPSC-derived endothelial cells. Differentiated LECs (dLECs) express key lymphatic markers as verified using bulk RNA-sequencing, qPCR, FACS, and immunostaining. These dLECs secrete important cytokines for lympangiocrine signaling, able to form robust 2D and 3D lymphatic networks, as well as demonstrate in vivo functionality and host integration in murine models.

Diffuse axonal injury (DAI) is the most severe pathological feature of traumatic brain injury. However, how primary axonal injury is induced by mechanical stress and whether it could be mitigated remain unknown, largely due to the resolution limits of medical imaging approaches. Here we established an Axon-on-a-Chip (AoC) model for mimicking DAI and investigating its early cellular responses. By integrating computational fluid dynamics and microfluidic techniques, DAI was observed for the first time during mechanical stress, and a clear correlation between stress intensity and severity of DAI was elucidated. This AoC was further used to investigate the dynamic intracellular changes occurring simultaneously with stress, and identified delayed local Ca2+ surges escorted rapid disruption of periodic axonal cytoskeleton during the early stage of DAI. Compatible with high-resolution live-microscopy, this model hereby provides a versatile system to identify early mechanisms underlying DAI, offering a platform for screening effective treatments to alleviate brain injuries.

Mechanical forces, including flow shear stress, regulate fundamental cellular process by modulating the nucleocytoplasmic transport of transcription factors, such as Yes-associated Protein (YAP). However, the mechanical mechanism how flow induces the nucleocytoplasmic transport remains largely unclear. Here we found that unidirectional flow applied to endothelial cells induces biphasic YAP nucleocytoplasmic transport with initial nuclear import, followed by nuclear export as perinuclear actin cap forms and nuclear stiffening in a dose and timing-dependent manner. In contrast, pathological oscillatory flow induces slight actin cap formation and nuclear softening, sustaining YAP nuclear localization. To explain the disparately spatiotemporal distribution of YAP, we developed a three-dimensional mechanochemical model considering coupling processes of flow sensing, cytoskeleton organization, nucleus mechanotransduction, and YAP spatiotemporal transport. We discovered that actin cap formation and nuclear stiffness alteration under flow synergically regulate nuclear deformation, hence governing YAP transport. Furthermore, we expanded our single cell model to a collective vertex framework and found that actin cap irregularities in individual cells under flow shear stress potentially induce topological defects and spatially heterogeneous YAP distribution in cellular monolayers. Our work unveils the unified mechanism of flow-induced nucleocytoplasmic transport, offering a universal linkage between transcriptional regulation and mechanical stimulation.