Biology subjects

Li, A.

Publications and source records attributed to Li, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

A single-cell transcriptomic atlas of the pigtail macaque placenta in late gestation

The placenta is a complex organ with multiple immune and non-immune cell types that promote fetal tolerance and facilitate the transfer of nutrients and oxygen. The nonhuman primate (NHP) is a key experimental model for studying human pregnancy complications, in part due to similarities in placental structure, which makes it essential to understand how single-cell populations compare across the human and NHP maternal-fetal interface. We constructed a single-cell RNA-Seq (scRNA-Seq) atlas of the placenta from the pigtail macaque (Macaca nemestrina) in the third trimester, comprising three different tissues at the maternal-fetal interface: the chorionic villi (placental disc), chorioamniotic membranes, and the maternal decidua. Each tissue was separately dissociated into single cells and processed through the 10X Genomics and Seurat pipeline, followed by aggregation, unsupervised clustering, and cluster annotation. Next, we determined the maternal-fetal origins of cell populations and analyzed single-cell RNA trajectory, Gene Ontology enrichment, and cell-cell communication. Single-cell populations in the pigtail macaque were strikingly similar in their identity and frequency to those found in the human placenta, including cells from trophoblast, stromal cell, immune, and macrophage lineages. An advantage of our approach was the deep sequencing of three tissues at the maternal-fetal interface, which yielded a rich diversity of common and rare single-cell populations. The third-trimester pigtail macaque single-cell atlas enables the identification of cellular subclusters analogous to those in humans and provides a powerful resource for understanding experimental perturbations on the NHP placenta.

genomics↗

bioRxiv2025

Global and local origins of trial-to-trial spike count variability in visual cortex

Sensory neuron spiking responses vary across repeated presentations of the same stimuli, but whether this trial-to-trial variability represents noise versus unidentified signals remains unresolved. Some of the variability can be attributed to correlations between neural activity and arousal, locomotion, and other overt movements. We hypothesized that correlations with global activity factors, i.e., patterns of neural activity observable in other brain regions, may explain additional variability in spike count responses of visual cortical neurons. To test this, we used Neuropixels 2.0 probes to record neural activity in mouse primary visual cortex (V1) while subjects passively viewed images. We recorded videos of behavior alongside neural activity from other brain regions, either spiking activity of neural populations in anterior cingulate area (ACA) or widefield calcium signals from across the dorsal cortex. We then used a model based on reduced rank regression to partition the explainable variability of visual cortical responses by source. Some of the trial-to-trial variability in V1 spike counts was attributable to locally shared patterns of activity uncorrelated with either behavior or global activity patterns. Locally shared activity patterns explained trial-to-trial variability that was in excess of Poisson spike generation. Of the parts of variability attributable to non-local sources, global cortical activity predicted significantly more V1 spike count variability than behavioral factors. Additionally, behavioral factors explained little variability uniquely and comprised a geometric subspace of the globally predictable V1 activity. Finally, optogenetically perturbing ACA directly impacted V1 activity, and ACA activity patterns predicted V1 spike count variability even on trials without overt behaviors. Our data indicate that globally shared factors from other cortical areas contribute substantially to shared spike count variability in V1, with only a minority of shared variability confined to local V1 circuits.

neuroscience↗

bioRxiv2025

Distinguishing cortical indexes of arousal and awareness in sleeping patients with unresponsive wakefulness syndrome

Consciousness is assumed to be defined by two components: awareness and arousal. However, compared with awareness, the cortical index for arousal remains lacking in clues. Since patients with unresponsive wakefulness syndrome (UWS) are supposed to have no awareness but arousal, sleep-wake cycle in patients with UWS may reflect pure arousal fluctuations, and then could highlight the cortical indexes for arousal. This study recorded nighttime polysomnography in patients with UWS, patients with minimally conscious state and healthy controls. The EEG signal showed that spectral slope could index arousal, fluctuating among sleep stages in all three groups. Both spectral entropy and Lempel-Ziv complexity indexed awareness, showing a significant difference between conscious and unconscious states. These findings provide fundamental evidence for the two-component hypothesis of consciousness.

neuroscience↗

bioRxiv2025

Profiling and Functional Analysis of Urinary Exosomal MicroRNAs in Pregnant Women with Systemic Lupus Erythematosus

BackgroundPregnancy in Systemic Lupus Erythematosus (pSLE) is high-risk, necessitating non-invasive biomarkers for monitoring and predicting complications. Urinary exosomes, containing miRNAs, offer a promising source reflecting systemic and renal states, yet their profile in late gestation pSLE is less studied. ObjectiveThis study aimed to investigate the profile of urinary exosomal miRNAs in pregnant women with SLE during late gestation compared to healthy pregnant controls and to explore their potential biological roles and pathways. MethodsUrinary exosomes were isolated from 6 pSLE patients and 5 controls. Exosomes were characterized, and miRNAs were sequenced. Bioinformatics analyses, including target gene prediction and functional enrichment (GO, KEGG), were performed. ResultsExosomes were successfully isolated. Sequencing identified 29 significantly downregulated miRNAs in pSLE exosomes. Enrichment analysis of these miRNAs and their target genes indicated involvement in metabolic and immune pathways, virus infection, and cellular senescence. Target genes also enriched in protein stability and transport, and high-level analysis showed broader roles in metabolic, immune, and cellular maintenance/stress processes. Targets of top differentially expressed miRNAs linked to mRNA regulation, AMPK, and senescence pathways. ConclusionOur findings demonstrate that urinary exosomal miRNAs are significantly altered in pregnant women with SLE during late gestation. The functional enrichment analysis suggests their target genes are involved in critical biological processes relevant to pSLE pathophysiology and pregnancy complications, including metabolism, immunity, viral response, senescence, and cellular maintenance. This highlights the potential of urinary exosomal miRNAs as non-invasive biomarkers for monitoring and predicting risks in this high-risk population.

immunology↗

bioRxiv2025

Screening of FDA-approved small molecules to discover inhibitors of the Pseudomonas aeruginosa quorum-sensing enzyme, PqsE

Pseudomonas aeruginosa is a notorious pathogen that is a leading cause of hospital-acquired infections. Due to its heightened resistance to a broad range of antibiotics, there is great need for new antimicrobial agents that are effective in treating P. aeruginosa infections. One attractive option for exploring new antibiotic targets is the quorum sensing (QS) pathway, as it governs many of the pathogenic behaviors that allow P. aeruginosa to stage and maintain infections. Within the QS pathway, there is a key protein-protein interaction between an enzyme, PqsE, and one of the master QS regulators, RhlR. Although its catalytic function is dispensable for its interaction with RhlR, previous mutagenic work characterizing the active site of PqsE identified active site mutations that induce a conformational change in PqsE, preventing it from forming a complex with RhlR. These active site mutations, when introduced stably into the genome of P. aeruginosa, also lead to a significant decrease in production of a key toxin, pyocyanin, and prevent colonization in the lungs of a murine host. These findings encourage drug discovery efforts to identify small molecules that bind in the active site of PqsE and induce a conformational change similar to the ones induced by particular amino acid substitutions in the active site. Here, we performed a fluorescence polarization screen to identify molecules that bind in the active site of PqsE. We screened a library of FDA-approved drugs, with the intention of potentially repurposing a known molecule for the treatment of P. aeruginosa infections. Three molecules were identified, two of which showed inhibitory activity that was consistent with a competitive mode of inhibition. One hit molecule, Apomorphine, had a distinctly different inhibitory profile, and is potentially binding outside of the active site to allosterically inhibit enzyme activity of PqsE. All three hit molecules were tested in an in vivo enzyme activity assay, and one of the competitive inhibitors, Vorinostat, was found to inhibit intracellular PqsE. Vorinostat is now being explored as a candidate for synthetic derivatization to inhibit the PqsE-RhlR protein-protein interaction via binding in the PqsE active site.

biochemistry↗

bioRxiv2025

Motor Cortex Modulates Ipsilateral Limb Movement Through a Direct Cortico-Cerebellar Circuit

Motor cortex is traditionally associated with control of contralateral limb movements via corticospinal and cortico-ponto-cerebellar pathways. However, the contribution of ipsilateral motor cortical outputs on motor control remains unclear. Here, we identify and characterize a distinct population of cortico-cerebellar (C-C) neurons in the motor cortex that form monosynaptic projections to the ipsilateral cerebellar nuclei. The C-C neurons receive preferential local motor cortical inputs and exhibit projection patterns distinct from cortico-pontine projecting neurons. Using in vivo imaging and optogenetic perturbations, we show that these neurons are active during locomotion and transitions of volitional movements. Disruption of the C-C projection severely affects the locomotion and balancing. Interestingly, the C-C pathway is selectively involved in the initiation and coordination of ipsilateral forelimb movements, without affecting contralateral movement kinematics. These findings shed light on a non-canonical cortico-cerebellar pathway that supports ipsilateral motor control, complementing the traditional control mechanisms of the cerebral cortex over the contralateral motor domains.

neuroscience↗

bioRxiv2025

Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

Enhancers are gene-distal cis-regulatory elements that drive cell type-specific gene expression. While significant progress has been made in identifying enhancers and characterizing their epigenomic features, much less effort has been devoted to elucidating mechanistic interactions among clusters of functionally linked regulatory elements within their endogenous chromatin contexts. Here, we developed a novel recombinase-mediated genome rewriting platform and applied our divergent transcription architectural model to understand how a long-range human enhancer confers a remarkable 10,000-fold activation to its target gene, NMU, at its native locus. Our systematic dissection reveals transcription factor synergy at this enhancer and highlights the interplay between a divergently transcribed core enhancer unit and emerging new types of cis-regulatory elements--notably, intrinsically inactive facilitators that augment and buffer core enhancer activity, and an adjacent retroviral long terminal repeat promoter that represses enhancer activity. We discuss the broader implications of our focused study on enhancer mechanisms and regulation genome-wide.

genomics↗

bioRxiv2025

LCK-targeting molecular glues overcome resistance to inhibitor-based therapy in T-cell acute lymphoblastic leukemia

Drug resistance is a major challenge in cancer therapy, especially in the context of kinase inhibitors. While targeted protein degradation (TPD) was a distinct mode of action compared to inhibition-based therapeutic targeting, the potential value of TPD in drug-resistant cancer remains unclear. Here, we report the discovery of cereblon-recruiting molecular glue degraders (MGDs) targeting LCK, an oncogenic kinase in T-cell acute lymphoblastic leukemia (T-ALL). By high-throughput screening and medicinal chemistry optimization, we developed a series of MGDs that induced CRBN-dependent degradation of LCK as well as potent cytotoxicity in T-ALL in vitro. Structure-activity relationship analysis and ternary complex modeling revealed a non-canonical degron at the LCK-CRBN interface involving the G-loop, whose mutation disrupts this interaction. Unlike inhibitors and inhibitor-based PROTACs, these MGDs engage LCK in regions distal to the ATP binding site and thus their activities in T-ALL are not affected by gate-keeper LCK mutations that drive resistance to inhibitor-based therapeutics. Taken together, our data underscore the potential of LCK-targeting MGDs as a strategy to overcome kinase inhibitor resistance in T-ALL, highlighting a potentially generalizable strategy in cancer therapy.

cancer biology↗

bioRxiv2025

Multiparametric Correlative Topographical and Volumetric Fluorescence Microscopy

Live-cell imaging of cell surface topography and intracellular architecture is essential for understanding cellular function. However, conventional approaches often involve trade-offs between resolution, invasiveness, and volumetric coverage. Here, we present an integrated Scanning Ion Conductance Microscope and single-objective Oblique Plane Microscope (SICM-OPM) system that enables simultaneous non-contact topographical imaging and volumetric fluorescence imaging within the same live cell. Beyond correlative live imaging, the platform supports nanomechanical mapping with tens-of-nanometres resolution, fluorescence-guided localised molecular delivery via the SICM, and benefits from reduced photobleaching due to light-sheet excitation. We demonstrate this platforms capabilities by visualising imipramine-induced T-tubule remodelling in live cardiomyocytes, revealing subsurface detubulation while surface morphology remains preserved. Additionally, we show precision delivery of fluorescent cargos--including dextrans and -synuclein--into diatom and mammalian cells, alongside localised stiffness mapping to evaluate mechanical responses. We believe this technique opens new avenues for correlative structural, functional, and biophysical studies in live cells, with broad relevance to cell biology, neurodegeneration, and mechanobiology.

biophysics↗

bioRxiv2025

Red Blood Cell Transfusion is a Non-Canonical Immune Stimulus Characterized by the Suboptimal Induction of CD4+ T Cell Help

Red blood cell (RBC) alloimmunization to non-ABO antigens is a major clinical complication for chronically transfused patients. When exposed to transfused RBCs carrying foreign antigens, some patients generate IgG antibodies that target these antigens, creating potential barriers to future transfusions. Interestingly, other patients produce only IgM antibodies against the same non-ABO antigens, which generally have fewer clinical consequences. Despite the stark differences in their impact, the factors regulating IgM versus IgG production in response to transfused RBCs remain poorly understood. This study explores the balance between IgM and IgG production following transfusion, comparing it to the well-characterized antibody response induced by vaccination in mouse models. By directly assessing antibody levels following RBC transfusion versus Alum-adjuvanted vaccination, we demonstrate that transfusion of RBCs expressing a model antigen is a relatively weak inducer of IgG class switching. Additionally, loss-of-function experiments using CD40L blockade and CD4 depletion confirmed that T cell help is essential for class switching after transfusion but has no effect on IgM production. Most notably, providing supra-physiological levels of T cell help enhanced class switching in a dose-dependent manner after transfusion, whereas vaccination-induced class switching remained unaffected. These findings support a model in which the limited IgG class switching following transfusion stems from suboptimal T cell help compared to vaccination. Furthermore, they suggest that transfusion activates T cells through a non-canonical pathway, distinct from the mechanisms driving immune responses to standard Alum vaccination.

immunology↗

bioRxiv2025

Tracking biological hallucinations in single-cell perturbation predictions using scArchon, a comprehensive benchmarking platform

The accurate prediction of cellular responses to perturbations, such as drug treatments, remains a pivotal challenge in single-cell transcriptomics. While numerous deep learning tools have been developed for this task, their systematic benchmarking across diverse datasets and performance metrics has been limited. Here, we present scArchon, a reproducible, modular benchmarking platform built on Snakemake, designed to evaluate perturbation response prediction tools in an unbiased and extensible manner. Employing four representative single-cell RNA-seq datasets, we compare leading methods such as scGen, CPA, trVAE, scPRAM, scVIDR, scDisInFact, SCREEN, scPreGAN, and CellOT against baselines. We assess model performance using a composite of statistical and biological metrics. Our analysis reveals heterogeneous performance: while methods like trVAE, scGen, scPRAM, and scVIDR achieve robust results across multiple datasets, other tools occasionally underperform even compared to linear or control baselines. Notably, models with favorable quantitative scores may fail to retain key biological perturbation signatures, underscoring the need for gene-level evaluation. scArchon provides a unified, extensible foundation for large-scale, standardized benchmarking of perturbation prediction tools, facilitating methodological transparency and accelerating development in this rapidly evolving field. We encourage adoption of scArchon and sharing of containerized tools to drive progress in single-cell perturbation modeling.

bioinformatics↗

bioRxiv2025

Extracellular Ca2+-Sensing Receptor (CaSR) Regulates Hypothalamic Function to Control Energy and Skeletal Metabolism in Mice

Introductory paragraphThe extracellular Calcium-sensing receptor (CaSR) regulates cellular responsiveness to physiological changes in ionized calcium (Ca2+) concentrations. The CaSR is expressed in the brain, including in hypothalamic growth hormone stimulating GHRH and anorectic POMC neurons that control growth and energy homeostasis. We embryonically deleted the Casr gene in neurons to create NeuronCaSR-/- mice to delineate the role of this receptor in regulating growth, skeletal development, and energy metabolism. NeuronCaSR-/- mice had reduced size, weight and bone mass compared to littermate controls, with a dysregulated growth hormone axis. They also showed increased adiposity and circulating leptin levels, leptin resistance, and decreased glucose tolerance, along with reduced expression of the anorectic precursor peptide POMC and secondary increases in the expression of the anorectic peptide AgRP in the hypothalamus of NeuronCaSR-/- mice. Knockdown of CaSR in adult mice specifically in the hypothalamic arcuate nucleus, where GHRH, POMC and AgRP neurons reside, also resulted in increased body weight, adiposity, leptin resistance, and glucose intolerance, and reduced bone mass. Together these data suggest that neuronal CaSR critically regulates energy and skeletal metabolism and body growth by modulating hypothalamic function, representing a new paradigm for central integration of calcaemic activities with body function.

physiology↗

bioRxiv2025

Feeding induces c-Fos in hepatocytes contributing to hepatocellular carcinoma in obesity

The transcription factor c-Fos plays an important role in hepatic metabolism; however, its role in metabolic dysfunction-associated steatotic liver disease (MASLD) and hepatocellular carcinoma (HCC) is unclear. Here, we show that hepatic c-Fos is induced by insulin after feeding and suppressed by glucagon during fasting in chow-fed mice. In lean mice, adenovirus-mediated c-Fos ectopic expression in the liver is sufficient to cause insulin resistance. In diet-induced obesity or after ectopic expression in hepatocytes, c-Fos promotes MASLD progression by altering PPAR signaling and fatty acid metabolism pathways. Mechanistically, c-Fos drives glycolysis, stress-associated MAPK, and insulin-related PI3K-Akt signaling, exacerbating metabolic dysregulation. In HCC, c-Fos expression correlates with PI3K-Akt, MAPK, and calcium signaling pathways activation. Moreover, c-Fos siRNA knockdown in human liver cancer cells reduces proliferation and increases apoptosis under lipotoxic or ER stress conditions. These findings identify c-Fos as a critical mediator of liver steatosis progression, linking hepatocyte signaling and metabolic reprogramming to liver dysfunction and tumorigenesis.

cancer biology↗

bioRxiv2025

Transcriptional activity generates chromatin motion that drives nuclear blebbing

Abnormal nuclear morphology is a hallmark of human diseases, including cancers and age-related disorders. Previously, maintenance of nuclear morphology and integrity was thought to be solely dependent on a force balance between nuclear mechanical resistance and actin antagonism. However, our recent work revealed that inhibiting RNA polymerase II suppresses nuclear blebbing independent of altering force balance, but the mechanism remains unknown. Through removing cell culture media serum and then adding it back, we can decrease and then restore transcriptional activity. Decreasing transcriptional activity decreases nuclear bleb formation, stability, and rupture while returning transcriptional activity restores nuclear blebbing. These modulations of transcriptional activity did not alter nuclear or actin mechanics. The mean square displacement (MSD) of chromatin domains labeled via transfected Cy3-dNTPs revealed that transcription activity regulates chromatin motion. To determine if increasing chromatin motion is a mechanism to increase nuclear blebbing, we used an established RAD51 inhibitor BO2. We verified BO2 increases chromatin domain motion which resulted in increased nuclear blebbing. We reveal the mechanism by which transcriptional activity drives nuclear blebbing is through chromatin motion. Thus, two hallmarks of human disease are directly linked via transcriptional activity and abnormal nuclear shape. Statement of SignificanceO_LINuclear blebs are hallmarks of disease progression that cause dysfunction, but how they are formed remains unanswered. C_LIO_LIWe find that chromatin motion generated by transcriptional activity is essential for both nuclear bleb formation and stability. This was independent of changes in nuclear stiffness or actin antagonism. C_LIO_LIThis finding provides a key advancement in our understanding of nuclear bleb formation. Furthermore, it reveals transcriptional activity as a novel contributor to nuclear blebbing in addition to the paradigm of nuclear shape determined as a force balance between nuclear resistance and actin antagonism. C_LI

cell biology↗

bioRxiv2025

Transcription Factor (TF) validation using Dam-IT simultaneously captures genome-wide TF-DNA binding, direct gene regulation, and chromatin accessibility in plant cells

Transcription Factors (TFs) govern vast networks of gene regulation. However, TF-DNA binding and TF-gene regulation datasets are typically measured separately due to experimental constraints, making it challenging to disentangle true biological relationships from batch effects. To fill this gap, we developed DamID-seq Incorporating Transcriptomics (Dam-IT), which simultaneously captures TF-DNA binding, direct TF-gene regulation, and chromatin accessibility in the same batch of cells. Dam-IT uses a transient cell-based TF-target validation system that is scalable and flexible to many experimental designs. As proof of concept, we used Dam-IT to reveal that bZIP1 directly regulates genes by binding to DNA regions of relatively low chromatin accessibility, supporting a "Hit-and-Run" mechanism of transcription.

systems biology↗

bioRxiv2025

Integrated Classification of Cortical Cells and Quantitative Projectomic Mapping Unveil Organizational Principles of Brain-Wide Connectomes at Single Cell Level

Molecularly defined cortical cell types have recently been linked to whole neuronal morphology (WNM), particularly those characterized by whole-brain-wide projections, such as intratelencephalic (IT), extratelencephalic (ET), and corticothalamic (CT) neurons. In contrast, classical morphological classifications (e.g., tufted TPC, small tufted SPC, and stellate SSC) are based primarily on local dendrosomatic and axonal structures, especially apical dendrites. This study bridges these perspectives by establishing a new neuronal taxonomy, analyzing the connectomes of defined cortical cell types, and comparing them with those obtained from bulk anterograde injections. Neurons were sparsely labeled via tamoxifen-inducible Cre lines with GFP reporters, and 1,419 WNM cells were comprehensively reconstructed with Vaa3D-TeraVR from [~]15 areas across six functional regions of molecularly labeled brains imaged with 2p-fMOST. These cells were newly classified by integrating current molecular-WNM and classical morphological perspectives, with sample size augmented by 1,455 publicly available WNM cells reconstructed from the Mouse-Light project and CEBSIT. This effort defined ten combined molecular-WNM-classical morphological cell types: L5ET_TPC, L6CT_NPC, L6b_HPC, and seven IT types--L2/3IT_TPC, L4IT_SSC, L4IT_UPC, L4IT_TPC, L5IT_SPC, L6IT_IPC, and L6IT_car3PC. Clustering, quantitative analyses and random Forest classifier objectively validated these types and revealed their distinct connectomes, along with convergent, topographic, and hierarchical organizations across their projection brain regions. At the single-cell level, multiple organizational principles governing cortico-cortical (C-C) and cortico-subcortical (C-subC) connectomes emerged with unprecedented detail, offering a precise GPS-like tool for in vivo recordings and robust datasets for neuronal network modeling. Comparisons with bulk anterograde injection data underscored the limitations of traditional methods in identifying projection targets. Overall, our approach provides significant insights into cortical circuitry and elucidates the complex interplay between neuronal molecular identity, whole morphology, and classical morphological classification.

neuroscience↗

bioRxiv2025

Cell type-specific contributions to impaired blood-brain barrier and cerebral metabolism in presymptomatic 5XFAD mice

Altered cerebral metabolism and blood-brain barrier (BBB) dysfunction are emerging as critical contributors to the preclinical phase of Alzheimers disease (AD), underscoring their role in early pathogenesis. To identify sensitive biomarkers before irreversible neuronal loss and cognitive decline, we examined 5XFAD mice at 3 months of age by applying multiple advanced MRI techniques. Arterial spin tagging based MRI revealed increased BBB permeability and water extraction fraction, indicating compromised BBB integrity at the early stage of pathogenesis in 5xFAD mice. Despite preserved cerebral blood flow, a decreased unit mass cerebral metabolic rate of oxygen (CMRO2) was evident in the same cohorts of 5XFAD mice. Interestingly, a region-specific decrease of tissue pH values was detected in the hippocampus of these 5XFAD mice by creatine chemical exchange saturation transfer MRI. Elevated neuronal H4K12 lactylation in the hippocampus supports the reduced pH values. To further dissect the cellular and molecular mechanisms underlying these MRI-detectable changes in 5XFAD mice, we conducted single-nucleus RNA sequencing (snRNA-Seq) with optimized blood vessel enrichment protocols. Our results revealed cell type-specific transcriptomic changes in the hippocampus of 3-month-old 5XFAD mice, including downregulation of synaptogenesis and synaptic transmission genes in the CA1 and dentate gyrus excitatory neurons, impaired endothelial gene expression linked to brain barrier function and angiogenesis, altered innate immune response genes in astrocytes, as well as upregulation of cholesterol biosynthesis and metabolism genes in the CA1 excitatory neurons. These findings underlie the intricate interplay between BBB disruption and metabolic dysregulation before the onset of cognitive decline in AD. Our study demonstrates that BBB dysfunction and cerebral metabolic alterations preceded brain hypoperfusion and cognitive decline, emphasizing potential molecular pathways for early intervention. These findings, once validated in human studies, could significantly enhance early diagnosis and inform novel therapeutic strategies targeting early AD pathogenesis.

neuroscience↗

bioRxiv2025

A Continuously Oxygenated Macroencapsulation System Enables High-Density Packing and Delivery of Insulin-Secreting Cells

The encapsulation of insulin-secreting cells within immuno-protective systems holds significant promise for curative treatment of type 1 diabetes without immunosuppression. A major challenge, however, remains the inadequate oxygen tension within the encapsulation systems, which compromises the survival and function of encapsulated cells and necessitates low packing density and impractically large systems to deliver a curative cell mass. In this study, we present a novel cell encapsulation system capable of generating oxygen via the electrolysis of tissue moisture to provide a continuous oxygen supply to densely packed insulin-secreting cells. Our system comprises a miniaturized implantable electrochemical oxygen generator (iEOG) and a scalable cylindrical cell encapsulation pouch, designed in a linear configuration to facilitate minimally invasive implantation and retrieval. The oxygen generation from the system was shown to be precisely controlled, stable, and capable of supporting clinically relevant doses of pancreatic islets. In vitro studies demonstrated that the oxygenated system effectively maintained the viability and function of insulinoma cell aggregates and human pancreatic islets at densities of 60,000 islet equivalents per mL (IEQ/mL) or 4,200 IEQ/cm{superscript 2} under a hypoxic cell culture condition (1% O ). In an allogeneic rat model, the oxygenated systems containing pancreatic islets implanted into the poorly vascularized but clinically attractive subcutaneous space at a density of 60,000 IEQ/mL successfully reversed diabetes for up to about 3 months without the need for immunosuppression, while animals implanted with non-oxygenated systems remained diabetic. Most ([~] 90%) of the pancreatic islets encapsulated in the continuously oxygenated systems were found viable and functional upon retrieval. These findings suggest the feasibility of using continuous oxygenation to support insulin-secreting cells at high loading densities in subcutaneous space, enabling the development of an encapsulation system with clinically practical dimensions.

bioengineering↗