Cell Proliferation

BackgroundIt has been demonstrated that stem cell transplantation promotes healing of the infarcted heart through paracrine effects. However, the therapeutic potential of exosomes secreted by hiPSC-derived epicardial cells (hEP-Exos) for treating infarcted hearts remains unclear. Myocardial infarction (MI) can trigger EP activation, increasing EP paracrine function. Therefore, this study aims to determine and compare the cardioprotective effects of exosomes secreted by hEPs under normoxic (Exo-N) and hypoxic (Exo-H) conditions in MI mice and to explore the underlying mechanisms. MethodsTwo types of exosomes were collected by ultracentrifugation and delivered via intramyocardial injection in a murine MI model. The protective effects of Exo-N and Exo-H on the infarcted heart were assessed using echocardiography, histological examination, and immunofluorescence analysis. Additionally, microRNA sequencing, luciferase activity assays, and miRNA gain-and loss-of-function experiments were performed to identify enriched miRNAs and investigate their roles in different exosome populations. ResultsIn vitro, both Exo-N and Exo-H enhanced the migration and tube-formation capacities in human umbilical vein endothelial cells (HUVECs) and reduced the apoptosis in hiPSC-derived cardiomyocytes (hCMs) under oxygen-glucose deprivation (OGD), with Exo-H exhibiting a stronger effect. In vivo, both Exo-N and Exo-H significantly improved contractile function, reduced infarct size, and mitigated adverse remodeling in mouse hearts with MI, accompanied by increased cardiomyocyte survival and angiogenesis, with Exo-H showing superior efficacy. Mechanistically, miRNA sequencing revealed distinct cargo profiles between Exo-N and Exo-H. miR-214-3p was identified as a key mediator of the enhanced therapeutic potency of Exo-H. miR-214-3p promoted EC angiogenesis by suppressing vasohibin-1 and attenuated cardiomyocyte mitochondrial fission and apoptosis by suppressing mitochondrial elongation factor 2 (MIEF2). ConclusionsThis study demonstrates that administration of hEP-Exos, particularly Exo-H, provides robust cardioprotection by enhancing cardiomyocyte survival and angiogenesis, potentially mediated by miR-214-3p. These findings suggest that conditioned hEP-Exos could be a promising and effective acellular therapeutic option for treating MI.

The ability to revascularize target tissues and organs through cell-based therapy would provide a novel approach for the treatment of a range of ischemic disorders including cardiovascular diseases, stroke and peripheral artery disease. Towards this goal, we have identified a human pluripotent stem cell (hPSC)-derived vascular progenitor (VP) population generated via an epicardial intermediate with functional engraftment properties. VP cells efficiently engraft the mammary fat pad and hind limb skeletal muscle of NSG recipient mice and form vessel-like structures that integrate with the host vasculature. In an ischemic hind limb mouse model, VPs generate extensive vascular grafts that improve perfusion, restore some function and preserve muscle integrity over a three-month period post-transplant. Single-cell transcriptomic and flow cytometric analyses show that the VP population, initially identified by the co-expression of CD140b, CD13 and KDR, displays an epicardial lineage signature and expresses a spectrum of genes and proteins indicative of vascular progenitor stage cells. Together, these findings demonstrate that it is possible to revascularize both normal and ischemic tissue through the transplantation of an appropriate hPSC-derived progenitor and in doing so, lay the foundation for developing cell-based therapy approaches to treat ischemic diseases. Graphical Abstract LegendHuman pluripotent stem cells are differentiated through an epicardial intermediate to generate vascular progenitor (VP) cells characterized by expression of CD140b, CD13 and KDR. These VP cells demonstrate the capacity to engraft both mammary fat pad and skeletal muscle tissue where they form stable perfused vascular networks. In a hindlimb ischemia model, VP cell transplantation restores blood flow and improves functional outcomes. eTOC BlurbFernandes et al. develop a protocol to generate engraftable vascular progenitors from human pluripotent stem cells through an epicardial intermediate. These cells form functional vessels in vivo, restore perfusion in ischemic tissue, and demonstrate tissue-specific adaptation while maintaining endothelial identity, providing a foundation for therapeutic revascularization. HighlightsO_LIA staged differentiation protocol generates vascular progenitors (VPs) from hPSCs via an epicardial intermediate. C_LIO_LIVP cells form stable, perfused vascular networks following transplantation into multiple tissue sites. C_LIO_LIVP cell therapy with or without VEGF nanoparticles restores perfusion and improves functional outcomes in hindlimb ischemia. C_LIO_LISingle-cell analysis reveals tissue-specific adaptation while maintaining endothelial identity. C_LI

Mesenchymal stem cells (MSCs) cultured in hypoxic conditions have been suggested to have more therapeutic efficacy than those cultured under normoxic conditions, and there is growing interest in using hypoxic MSCs for clinical treatment, particularly human umbilical cord (hUC)-MSCs. We investigated how hUC-MSCs and human bone marrow (hBM)-MSCs change from normoxia to hypoxia (1% O2) for 2 weeks of culture. In the growth speed and population doubling time, hUC-MSCs cultured under hypoxia exhibited a significantly higher proliferation rate beyond cancerous cells, such as human glioblastoma and breast cancer cells, while hBM-MSCs did not show a significant difference between normoxia and hypoxia, and were statistically slower than these cancerous cells. Notably, hypoxic hUC-MSCs showed upregulation of genes related to metabolic reprogramming (cholesterol biosynthesis and fatty acid metabolism pathways) and cancer stem cell-like phenotype (factors related to Wnt and Hedgehog signaling pathways, cell proliferation drivers, and apoptosis-resistance), and lesser migration and homing to the traumatic brain injury than normoxic hUC-MSCs after intravenous injection. Thus, whether hUC-MSCs cultured under hypoxia offer clinical benefits and use are safe, given their extremely accelerated proliferation rate and partial cancer stem cell-like traits, requires comprehensive and careful investigation.

This study investigates the therapeutic potential of secretomes derived from Adipose-derived Mesenchymal Stem Cells (ADMSC-CM) and Limbal-derived Mesenchymal Stem Cells (LMSC-CM) against oxidative stress-induced damage in Retinal Pigment Epithelium (RPE-1) cells. RPE dysfunction, often triggered by oxidative stress, is a hallmark of various retinal degenerations. Here, we induced RPE-1 injury using H2O2 and evaluated the restorative effects of both MSC-conditioned media (CM). Our results demonstrated that both ADMSC-CM and LMSC-CM significantly enhanced cell viability and successfully reversed H2O2-induced G2/M phase cell cycle arrest. While oxidative stress triggered a pro-inflammatory response characterized by elevated IL-1{beta}, IL-6, and IL-10 expression, MSC-CM treatment, particularly ADMSC-CM, effectively modulated these levels and suppressed the p38 MAPK signaling pathway. Furthermore, MSC-CM reduced the Bax/Bcl-2 ratio, indicating an anti-apoptotic effect, and appeared to stabilize autophagic flux. To investigate the impact of oxidative-stress induced alterations in retinal pigment epithelial cells on angiogenesis, the effects of RPE-derived secreted factors on endothelial cell function were evaluated. Crucially, in terms of safety and secondary complications, neither secretome exhibited pro-angiogenic tendencies; instead, they significantly inhibited HUVEC migration and invasion compared to the H2O2 damaged group. These findings suggest that both ADMSC and LMSC secretomes provide a potent multi-targeted therapeutic effect, making them promising candidates for cell-free therapies in retinal diseases.

Unlike the conventional mature neutrophils, immature neutrophils have been investigated for their regenerative properties; however, their limited availability necessitates alternative generation strategies. Here, we used a combination of dimethylsulfoxide (DMSO) and 1,25-dihydroxyvitamin D3 (D3) to differentiate myeloid leukemia (HL-60) cells into immature neutrophil-like cells. Differentiated cells exhibited reduced cell size, loss of uniformity, decreased nuclear-to-cytoplasmic ratio, band-shaped nuclei, increased proportion of CD11b+CD14+ cells (indicative of immature neutrophils), decreased proportion of CD11b+CD16+ cells (indicative of mature neutrophils), higher levels of arginase 1, TGF{beta}1 (markers of immature neutrophils), and no expression of CD16, MRC1 (markers of mature neutrophils and M2 macrophages, respectively). Proteomic analysis revealed enrichment of proteins associated with immature neutrophils and wound healing. Functionally, these cells supported limbal stem cell growth and wound closure in vitro, indicating relevance for corneal regeneration. Administration of these cells to ex-vivo and in-vivo alkali-injured corneas, resulted in significant effect on promotion of wound healing, with epithelial regeneration and decreased fibrotic markers, proving that such cells hold promise for clinical translation as a therapeutic tool for tissue repair.

Subretinal fibrosis underlies the end-stage pathogenesis of retinal diseases including age- related macular degeneration (AMD). It can disrupt retinal structure and eventually lead to legal blindness by generating contractile force, fibrotic scarring, subretinal hemorrhage, and retinal detachment. Myofibroblasts are the predominant cells critically involved in subretinal fibrosis, however, the cellular contribution to myofibroblasts remains unclear. Here we demonstrate that multiple cell lineages, including macrophages, endothelial cells (EC), retinal pigment epithelial (RPE) cells and pericytes, significantly contribute to myofibroblasts in a laser-induced subretinal fibrosis model. We found microRNA miR-24 is significantly downregulated in the plasma of wet AMD patients. Overexpression of miR-24 represses epithelial-mesenchymal transition (EMT), endothelial-mesenchymal transition (EndMT), and the resulting fibrosis by regulating TGF- {beta}/SMAD3 and PAK4/LIMK2/MRTF pathways. Consistently, a combination of SMAD3 and MRTF inhibitors show superior efficacy to individual inhibitors in repressing fibrosis in vitro and laser-induced subretinal fibrosis in vivo. Together, these suggest the contribution of multiple cell-types in myofibroblast transformation in subretinal fibrosis, and repression of miR-24-regulated TGF-{beta}/SMAD3 and PAK4/LIMK2/MRTF pathways in multiple cell types holds therapeutic potential for treating subretinal fibrosis in AMD and other fibrotic disorders.

Porcine organotypic retinal explant cultures are widely used to study retinal neurodegeneration under controlled conditions, but the biological process that occurs in the retinal explant over time due to preparation-induced injury and culture are not well understood. Here, we generated a time-resolved transcriptomic reference for porcine neural retinal explants-maintained ex vivo for 10 days. Global expression profiles are strongly separated by culture time, with Day 0 clearly distinct from cultured samples and at Day 7 and Day 10 showing the highest similarity, indicating a transition toward a later stabilized state. Across the time course, 3,187 genes were differentially expressed relative to Day 0, with the largest shifts occurring at an early stage of culture (Day 1-Day 3). Pathway-level analyses revealed coordinated remodeling involving inflammatory signaling, and metabolic/bioenergetic changes, including reduced mitochondrial and oxidative phosphorylation-related programs at later time points. Here, we provide a time-resolved transcriptomics reference dataset for cultured porcine retinal explants. These data can build a foundation to interpret data generated in this model, differentiate changes inherent to the explant culture from treatment-specific effects and to select appropriate experimental windows for mechanistic studies of retinal degeneration.

BackgroundGains of chromosome 20q11.21 are among the most common culture-acquired abnormalities in human pluripotent stem cells (hPSC), conferring a well-defined survival advantage while altering differentiation capacity. However, it remains unclear whether this advantage persists during differentiation, how the aneuploidy alters ectodermal and retinal pigment epithelium (RPE) lineage specification, and which genes within the minimal amplicon drive these effects. MethodsWe used three isogenic human embryonic stem cell line pairs (wild-type and 20q11.21 gain) and assessed their behaviour in two neuroectoderm differentiation systems: directed neuroectoderm induction (dual SMAD inhibition) and long-term spontaneous RPE differentiation. Competitive dynamics were measured in mixed cultures, and lineage outcomes were analysed using immunostaining, gene expression profiling and single-cell RNA sequencing. To identify driver genes, we generated BCL2L1 and ID1 overexpression lines and tested their effects under both directed and spontaneous differentiation conditions. ResultsAcross all lines and conditions, 20q cells expanded from a minor fraction to dominate mixed cultures, indicating that their competitive advantage persists beyond the undifferentiated state. Despite this dominance, pure 20q cells failed to specify to neuroectoderm or RPE. Single-cell transcriptomics revealed consistent diversion toward non-neural ectodermal and extraembryonic fates. Mechanistically, overexpression of BCL2L1 and ID1 alone or in combination impaired neuroectoderm specification, while synergistic effect of both genes promoted non-neural ectodermal outcomes under directed differentiation conditions. In spontaneous differentiation, both genes could disrupt differentiation. ConclusionsThe 20q11.21 gain couples a persistent survival advantage with a disruption of neural and RPE lineage competence, redirecting cells toward alternative ectodermal and extraembryonic fates. These effects arise from the combined action of two dosage-sensitive genes BCL2L1 and ID1 within the amplicon, illustrating how regional gene dosage can reshape developmental signalling responses in hPSC.

Angiogenesis, the process by which new blood vessels form from existing vasculature, is fundamental to tissue repair and regeneration but also underlies pathological conditions such as cancer progression. Targeting angiogenesis has thus become a promising approach for developing novel cancer therapeutics. While various phytochemicals have demonstrated anti-angiogenic effects, the role of 2-5(H)-Furanone, a naturally occurring lactone found in various plants and marine sources with diverse biological activities, remains insufficiently explored. In this study, we systematically evaluate the anti-angiogenic potential of 2-5(H)-Furanone using Human Umbilical Vein Endothelial Cells (HUVECs) as an in vitro model and zebrafish embryos as an in vivo model. Experimental findings demonstrated that treatment of HUVECs with increasing concentrations of 2-5(H)-Furanone led to significant, dose-dependent reductions in proliferation, invasion, migration, and tube formation. Analyses of gene expression revealed marked downregulation of key pro-angiogenic mediators, VEGF, and HIF-1. Complementing these in vitro results, in vivo studies in zebrafish embryos showed robust, dose-dependent inhibition of intersegmental vessel (ISV) formation, accompanied by suppression of critical angiogenesis-related genes. Molecular docking further supported these observations by indicating stable binding of 2-5(H)-Furanone to major angiogenic targets, including VEGFR2, MMP2, HIF-1, and PIK3CA. Collectively, our data demonstrate that 2-5(H)-Furanone potently inhibits angiogenesis, as evidenced in both HUVEC and zebrafish models, through functional and molecular mechanisms. These findings support the further development of 2-5(H)-Furanone as a promising anti-angiogenic therapy candidate.

The role of DNA topoisomerase II beta (TOP2B) in cardiomyocyte differentiation is poorly understood. To address this, Human induced pluripotent stem cells (hiPSC) were differentiated into cardiomyocytes (CM) that are wildtype or contain a genomic deletion of Topoisomerase 2B (BKO). Both WT and BKO hiPSC could be induced to differentiate into sheets of beating cardiomyocytes. BKO hiPSC take slightly longer to differentiate into sheets of beating CM than WT iPSC. RNA was prepared from both undifferentiated and differentiated WT and BKO hiPSC. RNA seq was used to examine gene expression changes when the WT and BKO hiPSC were differentiated into CM. Gene expression changes following differentiation of BKO cells were largely similar to those in WT cells. In addition, the differentiated WT CM were treated with dexrazoxane (ICRF-187), a TOP2 catalytic inhibitor that targets both TOP2A and TOP2B, or topobexin, a new TOP2B selective catalytic inhibitor. Topobexin inhibition partially phenocopied a TOP2B deletion and thus providing an alternative to TOP2B gene knockout in many cell lines. In future, hiPSC derived CM with and without TOP2B and inhibition by topobexin ex vivo CM could be used to study anthracycline-induced cardiotoxicity and to screen for cardioprotectants. HighlightsO_LIUsed CRISPR-Cas9 to delete TOP2B from hiPSC C_LIO_LIProduced beating cardiomyocytes from both WT and TOP2B null hiPSC C_LIO_LITranscriptome analysis of WT and TOP2B null hiPSC and derived cardiomyocytes C_LIO_LIRNA seq showed he specific TOP2B inhibitor topobexin largely phenocopies TOP2B gene inactivation in iPSC derived cardiomyocytes. C_LIO_LITopobexin inhibition could be used as an alternative to a TOP2B gene knockout in many different cell types, speeding up the analysis of the function of TOP2B. C_LI

De novo vessel formation (vasculogenesis) in vitro is a key step in tissue engineering to preserve tissue viability for long-term assays and testing therapeutic agents. However, in vitro vasculogenesis is often unreliable due to differences in vascular-supporting cells, including endothelial cells and stromal cells such as smooth muscle cells (SMCs) and fibroblasts. Here, we developed a robust co-culture system of HUVECs and SMCs to generate stable vascular networks capable of maintaining tissue viability over extended periods. Given that SMC plasticity is a major limitation in supporting endothelial network formation, we systematically evaluated the effects of passage number, confluency, and freezing on primary SMC function. To overcome this limitation, we generated immortalized supportive SMCs, which preserved their vasculogenic gene program and functional capacity even at high passage. In addition, we identified and validated key genes associated with endothelial support, including CD248, C3, and FBLN1, all essential for vasculogenesis. Immortalized SMCs consistently maintained expression of these genes and supported robust vessel formation under variable culture conditions. Collectively, this study demonstrates that immortalized SMCs provide a stable, reproducible platform for endothelial-SMC co-cultures, enabling long-term vascularized tumor models suitable for functional studies and therapeutic screening.

Mesenchymal stem cells (MSCs) have garnered significant attention over the past three decades due to their robust regenerative potential, primarily mediated by their paracrine activity by releasing soluble bioactive factors and extracellular vesicles (EVs). The MSC secretome plays a pivotal role in wound healing by influencing cellular migration, inflammation, angiogenesis, extracellular matrix (ECM) remodeling, and re-epithelialization. SPARC (Secreted Protein Acidic and Rich in Cysteine), a multifunctional ECM glycoprotein involved in tissue repair and remodeling, regulates key processes such as cell migration, proliferation, angiogenesis, and survival. Despite its known role in ECM dynamics, the impact of SPARC expression on the regenerative properties of MSCs remains underexplored. In this study, we hypothesized that SPARC overexpression in MSCs enhances their secretomes regenerative capacity. Using lentiviral systems, we generated SPARC-overexpressing (+SPARC) and SPARC-knockdown (KD-SPARC) MSCs to investigate SPARCs role in wound healing. Conditioned media (CM) derived from these MSCs were analyzed in vitro for their effects on human skin keratinocytes and fibroblasts. Our results revealed that SPARC expression significantly influences cell-specific migration and cell cycle. Furthermore, in an in vivo wound healing model, CM from +SPARC MSCs accelerated regeneration, while SPARC absence in MSCs CM delayed the healing process. These findings underscore the critical role of SPARC in modulating MSC secretome composition and enhancing its regenerative efficacy. This study highlights SPARC as a promising therapeutic target for the development of advanced regenerative therapies aimed at improving cutaneous wound healing outcomes.

Optic fissure (OF) is a transient structure in the ventral optic cup, which acts as a conduit for periocular mesenchyme cells to enter the eye, forming hyaloid vasculature and retinal ganglion axons to exit. Optic fissure closes to form a continuous layer of retinal pigment epithelium and neural retina. Failure of OF closure results in coloboma, which is mostly genetic in nature. The severity of blindness depends on the tissue it effects and accounts for 10% of childhood blindness. In the current study, we describe coloboma pathogenesis caused by hippo effectors yap1 and wwtr1. Both the paralogs are expressed in the OF edges, possibly in the pioneer cells. wwtr1 homozygotes do not have coloboma, while yap1 homozygotes have coloboma and pigment defects which are exacerbated by absence of one copy of wwtr1 (yap1-/-; wwtr1+/-). The coloboma observed in these mutants is not due to defective optic cup morphogenesis nor an overgrown optic nerve. The pigment defects are more pronounced at the OF with complete absence of RPE specific transcription factors mitfA, tfec, and pigmentation gene dct. On the other hand, NR specific genes are upregulated and the unpigmented region at the OF have transdifferentiated retinal ganglion cells, amacrine, and photoreceptor cells. Our observations indicate that in the absence of yap1 and wwtr1, the cells at the OF cannot attain a conducive state to fuse nor they maintain the RPE specific fate and instead they transdifferentiate into unpigmented retina, causing a steric block for fusion, resulting in coloboma.

Optogenetics has emerged as a powerful technology for manipulating biological functions with high spatiotemporal resolution, yet the precise control of endogenous molecules remains a significant challenge. In this study, we developed Opto-MDMi, a dual-lock optogenetic platform designed to control the activity of endogenous p53, a master regulator of cell cycle and apoptosis. The p53 pathway is strictly governed by its negative regulators, MDM2 and MDMX, which inhibit p53 through direct binding and ubiquitination. Our system integrates two distinct light-responsive modules: Opto-MDMi (LOVTRAP), which regulates the nuclear translocation of p53-activating peptides, and Opto-MDMi (LOV2-PMI), which controls the binding activity of these peptides by photocaging them within the AsLOV2 domain. Through extensive in vitro screening and live-cell assays, we discovered that truncating the J helix of LOV2 effectively restricts the movement of fused inhibitory peptides, thereby masking their interaction with MDM2/MDMX under dark conditions. By combining these two regulatory layers into a dual-lock system, we achieved robust light-dependent activation of endogenous p53 while significantly suppressing basal activity in the dark. Our findings not only provide a potent tool for p53 research but also establish a general design principle for optogenetically regulating functional peptides with the LOV2 domain, offering a versatile framework for the future development of optogenetic actuators.

Down syndrome (DS) results from trisomy for human chromosome 21 and is the most frequent genetic cause of intellectual disability. No effective treatments currently exist that improve neurodevelopment and cognition. Atypical brain development in individuals with DS is apparent before birth, which suggests that the optimal time to begin administration of therapies is prenatally. Human neural progenitor cell (NPC) cultures provide a tractable in vitro model system to examine the effects of trisomy 21 (T21) on neurodevelopment and to measure the effects of pharmacological interventions. Here we report the results of preclinical studies evaluating 24 candidate therapies. RNA-Seq analyses found that euploid and T21 NPCs showed different transcriptomic responses to five candidate pharmacotherapies. The Rho-associated coiled-coil kinase (ROCK) inhibitor fasudil increased proliferation of T21 NPCs, reduced expression of inflammatory pathway genes in T21 NPCs, and reduced markers of inflammation in LPS-stimulated microglia model systems. These results demonstrate that fasudil can alter multiple T21-associated abnormalities in a beneficial manner, suggesting that fasudil warrants further study as a candidate prenatal pharmacotherapy for DS.

Vaccine adjuvants are critical for enhancing immune responses and sustaining antibody production. Although their safety profiles are well established, assessments have largely focused on metabolic and excretory organs such as the liver and kidneys, with limited attention to the heart. Here, we systematically evaluated the cardiac effects of five representative adjuvants in mice: alum, MF59, AS03, Sigma Adjuvant Systems, and lipid A. None of the adjuvants impaired baseline cardiac contractile function. Notably, lipid A uniquely enhanced mitochondrial respiratory capacity in rat and human induced pluripotent stem cell-derived cardiomyocytes and promoted mitochondrial membrane hyperpolarization. We next examined its therapeutic potential in a doxorubicin (Dox)-induced heart failure model characterized by mitochondrial dysfunction. Co-administration of lipid A with influenza hemagglutinin (HA) antigen significantly ameliorated cardiac dysfunction. In parallel, lipid A prevented the Dox-induced decline in anti-HA antibody titers, an effect associated with preservation of splenic B cell populations. Collectively, these findings reveal a previously unappreciated cytoprotective dimension of lipid A, demonstrating that it not only potentiates immune responses but also counteracts chemotherapy-induced functional decline by enhancing mitochondrial activity.

Bisphenol A (BPA) and Bisphenol S (BPS) exposure disrupt ovarian function and granulosa cell (GC) steroidogenesis. Extracellular vesicles (EVs) and their miRNA cargo, as mediators of cellular response to environmental stimuli, might be involved in fertility and folliculogenesis. This study explored modulation of microRNA expression after 48h BPA or BPS exposure (10 {micro}M) in ovine primary GC and EVs from corresponding conditioned medium (CM EVs). Small RNA sequencing of control (0h) and 48h treated GC, CM EVs as well as follicular fluid EVs allowed identification of 533 ovine miRNAs, including 129 new sequences. BPA did not alter miRNA expression in GC, while BPS decreased cellular oar-24b miR. In contrast, BPA modified expression of 4 miRNAs in CM-EVs, including 3 new sequences, and two miRNAs were modified by BPS. Both compounds reduced expression of sequence homologous to miR-1306. Further studies are required to decipher their roles in bisphenol toxicity in GC.

Pigs and chickens are not only the most important livestock species for global food production but also serve as key model organisms in various research disciplines. The pig is widely used in translational research due to its anatomical and physiological similarity to humans, providing valuable insights into immunology, metabolism, and disease mechanisms. In contrast, the chicken has become an essential model for studies related to poultry health, animal welfare, and developmental biology. Its externally developing embryo offers exceptional accessibility for experimental manipulation. Recent advances in genome editing technologies, particularly CRISPR/Cas9, have further expanded the potential of these species for functional genomic studies, although the efficient delivery of such tools remains a major challenge. By using virus-like particles (VLPs), we have been able to overcome this limitation. Here, we evaluated VLPs as delivery vehicles for genome engineering tools in pigs and chickens, two key livestock species at the human-animal interface. VLP-mediated delivery enabled efficient Cre recombination and high CRISPR/Cas9 editing rates in porcine cells, organoids, and oocytes, particularly when multiplexed. In chickens, VLPs supported robust Cre recombination and Cas9-mediated editing in cell culture, tracheal organ cultures, and in ovo. Reporter VLPs and dCas9 VLPs further demonstrated the versatility of this platform across porcine and avian systems. Together, these findings establish VLPs as an efficient and time-saving strategy for gene editing in livestock, with relevance for animal health, agricultural productivity, and translational One Health research.

Pericytes, specialized mural cells of capillaries, fulfill crucial physiological functions including promoting endothelial barrier function and regulating angiogenesis. Pericyte loss or dysfunction represents a central pathological feature in diabetic retinopathy (DR) and is increasingly recognized in neurodegenerative diseases as well as in poor stroke outcomes, underscoring an urgent need for therapies that restore pericyte function or promote their regeneration. Here, we utilized a Frizzled4 (FZD4) and Low-Density Lipoprotein Receptor-Related Protein 5 (LRP5) agonist antibody (F4L5.13) to investigate the functional consequences of mimicking {beta}-catenin-dependent signaling in CNS endothelial cells (ECs), which is physiologically induced by Norrin or WNT7A/B. In platelet-derived growth factor subunit B (Pdgfb) EC-specific knockout (ECKO) mice, a model of severe developmental pericyte deficiency with secondary blood-retina barrier (BRB) defects and hemorrhages, F4L5.13 significantly promoted retinal pericyte/mural cell proliferation and coverage, improved BRB function, reduced hemorrhages, and normalized vascular morphology. F4L5.13 restored Pdgfb mRNA expression levels from non-recombined cells in Pdgfb ECKO retinas. These findings highlight interactions of {beta}-catenin-dependent signaling and PDGFB production, identify a key pharmacodynamic action of F4L5.13 distinct from anti-VEGF therapies, and suggest that FZD4/LRP5 agonists may have uses as a regenerative pharmacology approach that promotes pericyte coverage in the neurovascular unit.

In vitro fertilization (IVF) is key for genetic improvement programs in bovine. However, embryos produced through IVF have lower developmental competence than those produced under in vivo conditions. Conventional sperm preparation for IVF typically relies on heparin for sperm capacitation but fails to replicate the finely tuned molecular environment of the oviduct, resulting in compromised embryonic competence. Here, we evaluated the effect of HyperBull, a novel capacitation technology, on bovine IVF outcomes using unsorted cryopreserved semen. In a split-sample design, 528 cumulus-oocyte complexes were co-incubated with either control or HyperBull capacitated spermatozoa from the same bull. While overall blastocyst rates were not significantly different between groups (34.21% HyperBull vs. 28.63% control, p=0.148), the proportion of hatched embryos was significantly higher in the HyperBull group (15.82% vs. 9.13%, p=0.016). These findings suggest that modulating capacitation signals prior to insemination enhances embryonic developmental competence, thereby improving readiness for implantation. HyperBull may thus represent a valuable tool to increase the efficiency of IVF programs.