Cell Proliferation

Epigenetics regulate cell-cycle kinetics, differentiation, apoptosis, and migration. Nuclear receptor-binding SET Domain (NSD) histone methyltransferases represent a family of oncoproteins with aberrant expression in cancer. Emerging reports suggest that NSD1 could be an attractive target as its expression is correlated with poor prognosis and tumorigenesis. Previously, we reported the target validation and structure-based virtual screening against NSD1, leading to the selection of several hit molecules with relatively high docking and MMGBSA delta G Bind scores. One of the best-fit molecules identified was 5-O-sulfamoyl adenosine (5-SA) and was compared with the S-Adenosyl-l-Cysteine (SAC), a structural analog of S-Adenosyl-l-Methionine (SAM) for its inhibitory activity against NSD1. IC50 values for 5-SA and SAC against NSD1 were 53.819 {micro}M and 115.003 {micro}M respectively. 5-SA significantly reduced the viability of DU145 and HepG2 cells with IC50 values calculated as 198{micro}M and 168.3 {micro}M respectively. It also reduced the RNA and protein expression levels of NSD1 and subsequently prevented dimethylation of lysine 36 on histone H3 (H3K36me2). Furthermore, 5-SA impeded proliferation, and migration, altered the cell cycle phase, and induced cell apoptosis. Interestingly, 5-SA potentiated the anticancer activity of 5-Fluorouracil (5-FU) against cancer cells. The xenograft model of prostate cancer also showed that 5-SA significantly reduced the tumor growth kinetics. However, the combination of 5-SA and 5-FU synergistically reduced tumor growth and improved survival of animals. To the best of our knowledge, we report for the first time that 5-SA mediated inhibition of NSD1 enhanced the tumor sensitivity to 5-FU and thereby, improved the tumor growth and progression. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/731397v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@e2f0bdorg.highwire.dtl.DTLVardef@12b2ca6org.highwire.dtl.DTLVardef@1807613org.highwire.dtl.DTLVardef@c7f8f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Astrocytes play a significant role in neuroprotection by internalizing neurodegenerative aggregates and facilitating their degradation. Recent studies indicate that -Synuclein (-SYN) protofibrils promote the transfer of pathogenic aggregates and dysfunctional mitochondria between astroglia via tunneling nanotubes (TNTs), which enhances cell survival and resistance to apoptosis. However, the underlying mechanism of TNT-driven apoptosis resistance remains unclear. We find that -SYN protofibrils induce aberrant mitochondria with decreased membrane potential ({Psi}m) and promote dynamic actin remodeling by relocating phosphorylated focal adhesion kinase (pFAK) to the nucleus, which triggers TNT formation in human astrocytoma cell lines and primary murine astrocytes. The important novel finding of this study is that pFAK in the nucleus co-localizes with Nanog, a crucial transcription factor for preserving stemness, and the interaction between pFAK and Nanog is critical for promoting p53 degradation via Mdm2-mediated ubiquitination and upregulating autophagy, thereby supporting the survival of astroglia exposed to toxic -SYN protofibrils. ROCK inhibitor y-27632 also drives TNT-formation via pFAK translocation to the nucleus, colocalizes with Nanog, and enhances stemness-related gene expression. Inhibiting TNT with the actin depolymerizing agent cytochalasin-D prevents pFAK co-localization with Nanog in the nucleus and fails to protect cells from -SYN-induced apoptosis. Nanog knockdown does not degrade p53 and hinders cell rescue from apoptosis. Furthermore, these transient TNTs transfer mitochondria to adjacent cells, potentially helping maintain metabolic stability. This study reveals that the TNT formation pathway promotes pFAK-Nanog interaction in the nucleus, leading to p53 degradation, which protects astroglia against -SYN proteotoxicity and prevents apoptosis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/727344v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@18d8675org.highwire.dtl.DTLVardef@76a383org.highwire.dtl.DTLVardef@e92202org.highwire.dtl.DTLVardef@1b7ee4b_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe CXCR2 receptor pathway plays a major role in inflammatory and invasive angiogenesis in human disease. ObjectiveWe evaluated AZD5069, a selective CXCR2 antagonist, as an angiogenesis inhibitor in human cell culture. MethodsHuman Umbilical Venous Endothelial Cells (HUVECs), Human Aortic Endothelial Cells (HAECs), and Human Pulmonary Artery Endothelial Cells (HPAECs) were cultured with standard in vitro techniques. AZD5069 (0, 8, 16, 32, 64, 128, 256 M) was evaluated as an angiogenesis inhibitor with fluorescent-labeled 5-Ethynyl-2-deoxyuridine (EdU) uptake to quantify endothelial cell proliferation, scratch assay to quantify endothelial cell migration, and Geltrex assay to quantify endothelial cell tubule and hub formation. AZD5069 cytotoxicity was evaluated with in situ terminal deoxynucleotidyl transferase 2-Deoxyuridine triphosphate- 5 (dUTP) nick-end labeling (TUNEL) to quantify apoptosis and membrane-impermeable cyanine dye uptake to quantify necrotic cell death. ResultsAZD5069 significantly reduced HUVEC, HAEC, and HPAEC proliferation, migration, tubule count, total tubule length, and node count with a dose-response. AZD5069 did not cause apoptosis nor necrotic cell death. ConclusionsAZD5069 inhibited angiogenesis without cytotoxicity in human endothelial cell culture. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility in cardiovascular, oncologic, and inflammatory disease. Condensed AbstractThe CXCR2 receptor pathway plays a major role regulating angiogenesis in inflammation and cancer. The CXCR2 receptor pathway has been evaluated in humans as a target for therapy in inflammatory disease and cancer but not as a therapeutic approach to block pathologic angiogenesis. AZD5069 is a clinical stage, direct CXCR2 antagonist. In human endothelial cell culture, AZD5069 inhibited angiogenesis without causing apoptosis or necrotic cell death. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility as a novel angiogenesis blocker in human disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/731993v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1ac8fb4org.highwire.dtl.DTLVardef@ea89forg.highwire.dtl.DTLVardef@607f94org.highwire.dtl.DTLVardef@157cec4_HPS_FORMAT_FIGEXP M_FIG Visual Abstract: AZD5069, a selective CXCR2 antagonist, significantly reduced endothelial cell proliferation, migration, and vascular tubule formation without causing necrotic or apoptotic cell death. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility in human cardiovascular, oncologic, and inflammatory disease with pathologic, dysregulated, or excessive angiogenesis. C_FIG

BackgroundAlcohol-induced osteonecrosis of the femoral head (AIONFH) is an orthopedic disorder from chronic alcohol abuse, characterized by disrupted femoral head blood supply, osteocyte death and structural collapse. Current hip-preserving therapy is unsatisfactory, and most patients eventually require total hip arthroplasty. Panax Notoginseng Saponins (PNS), the core active component of Panax notoginseng, exerts pro-angiogenic and anti-osteocyte apoptosis effects, but its specific therapeutic mechanism remains unclear. ObjectiveThis study used network pharmacology, molecular dynamics simulation and animal experiments to identify PNSs active components, core targets and key pathways for AIONFH, verify its in vivo efficacy, and provide a scientific basis for clinical application. MethodsPNS active components, their targets and AIONFH-related targets were screened from databases; intersection targets constructed an interaction network, core targets were screened by three machine learning algorithms, with concurrent GO and KEGG analysis. Molecular docking was performed between core targets and PNS components; Gromacs 2022 conducted 100 ns simulation to evaluate complex stability. AIONFH rat models were grouped with 4-week intragastric intervention; pathology, immunofluorescence and PCR were used for detection. Results and DiscussionNetwork pharmacology identified 127 PNS targets and 18 intersections with 672 AIONFH targets. Six core targets (including FGF2, HSD11B1) were screened; KEGG indicated VEGF pathway as key. Ginsenoside Re bound HSD11B1 with the lowest binding energy (-12.4 kcal/mol), and 100 ns simulation confirmed complex stability. Animal experiments showed PNS improved trabecular structure and regulated osteocyte activity. PNS treats AIONFH via multi-component, multi-target mode, core mechanism being osteocyte apoptosis inhibition. Results and DiscussionNetwork pharmacology screening identified 127 potential targets of PNS, and 18 potential intersection targets were obtained by overlapping with 672 AIONFH-related targets. Six core targets including FGF2 and HSD11B1 were screened out by machine learning, and KEGG analysis indicated that the VEGF pathway and other pathways were the key signaling pathways for PNS action. Molecular docking showed that Ginsenoside Re had the lowest binding energy with HSD11B1 (-12.4 kcal/mol), and 100 ns molecular dynamics simulation confirmed the stable conformation of this complex. Animal experiments demonstrated that PNS could improve trabecular bone structure and regulate osteocyte activity. In summary, PNS exerts a therapeutic effect on AIONFH through a multi-component, multi-target and multi-pathway mode, with the core mechanism of inhibiting osteocyte apoptosis.

There are currently few promising approaches for treatment of photoreceptor pathologies: for example, gene therapy to augment or replace mutated genes, has proven successful in preclinical studies, and some of these therapies are moving towards the clinic. Another approach aims to unlock the inherent stem-cell potential of non-neuronal retinal cells to regenerate neurons in situ. This line of research is based on the discovery that some vertebrates can restore even severely damaged retina from RPE with all the necessary cell types to regain full functionality. To determine whether this approach can be applied to humans, we established a robust in vitro culture system using fetal human RPE, and employed a barcode-multiplexed, single cell RNAseq based screen to find factors that would reprogram human RPE into photoreceptors. With this approach we were able to identify NEUROD1 as a complimentary factor to ASCL1. Their combined overexpression together with a treatment with bFGF and Activin A inhibitor produced RPE-derived neuronal cells with expression patterns characteristic of photoreceptors and other lineages.

Research on targeted genetic therapies for myocardial diseases, such as cardiomyopathies, currently focuses on (r)AAVs as the delivery method. Despite substantial efforts and advances in animal trials, predicting biodistribution and transduction efficacy in human tissue remains challenging due to interspecies differences in tissue tropism and the difficulty of accurately assessing alternative delivery routes and vector differences. The application in humans has also proven challenging, in part due to severe adverse events associated with systemic administration of (r)AAVs. This necessitates implementing alternative trial designs and stringent evaluation methods that minimize harm or risk to patients. Applying a predesigned vector carrying a gene-editing tool to a normothermic machine-perfused living beating heart in an ex vivo setting could overcome conventional obstacles and limitations. This can serve as a basis for safe and effective gene-therapy testing and assist in evaluating effects at the molecular level. Boxed-Breathing-Heart is a translational trial assessing the feasibility of ex vivo gene editing and gene translation in normothermic machine-perfused human hearts. Human hearts explanted from cardiomyopathy patients undergoing heart transplantation are donated for research and immediately placed in an Organ Care System, where they are surgically connected. The viability of the heart is maintained through normothermic perfusion of system solutions and donor blood. A predesigned AAV containing a CRISPR-Cas system is infused into the circulation and dispersed throughout the tissue via coronary perfusion. The changes at the cellular and molecular levels are assessed continuously via frequent sequential myocardial biopsies. Furthermore, after the pre-planned 72-hour perfusion, the heart is sectioned and analyzed using spatial and single-cell omics. The aim is to provide a proof-of-concept for genetic therapeutic options delivered to the human heart via AAV in an ex vivo perfusion setup. In summary, Boxed-Breathing-Heart provides an ex vivo translational platform for evaluating targeted cardiac gene therapies, enabling molecular analysis directly in human hearts and accelerating clinical translation without posing risks to patients.

Extracellular vesicles (EVs) are nanoscale, cell-secreted mediators of intercellular communication with growing promise as therapeutic agents. Manufacturing practices, including EV isolation and storage approaches, are critical determinants of product consistency, purity, and potency. In this study, neural stem cell (NSC)-derived EVs were isolated from conditioned NSC culture media via oscillator-based isolation (OSC), ultracentrifugation (1 or 2 hours), and ultrafiltration, and were stored lyophilized or cryopreserved. Nanoparticle yield, size distribution, and subpopulation composition were evaluated by nano-flow cytometry, quantifying total nanoparticles, membrane-bound EVs and CD63+ EVs. Purification was calculated via particle-to-protein ratios, morphology was evaluated by transmission electron microscopy, and potency was assessed using a microglia morphology assay. Particle yield was comparable across isolation methods, though protein clearance varied, with OSC demonstrating purification relative to conditioned media. Lyophilized samples retained structural integrity, size, and population profiles comparable to cryopreserved samples. Lyophilized and cryopreserved EVs exhibited dose-dependent immunomodulatory activity in our microglia morphology assay, with significant effects observed at 200,000 EVs per cell. These findings highlight the importance of isolation method in EV product quality and support lyophilization as a viable storage strategy which overcomes the logistical limitations of cryopreservation, thereby advancing the development of a robust pipeline for therapeutic EV manufacture.

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

A subset of extracellular vehicles (EVs) with particle diameters >200 nm, large vesicles (lEVs) contain mitochondria that increase recipient cell bioenergetics. To date, sequential centrifugation (SC) is the most reported protocol to separate lEVs from the smaller EVs (<200 nm)/exosomes. We have previously demonstrated that lEVs derived from brain endothelial cells (BECs) using the standard SC method transferred their innate mitochondria to recipient BECs, increased recipient BEC bioenergetics, reduced brain infarct volume, and improved behavioral outcomes in a mouse model of transient ischemic stroke. Despite their promising therapeutic activity, SC-isolated lEVs are likely a mixture of mitochondria-containing lEVs and non-mitochondria-containing lEVs. We hypothesized that subsequent purification of SC-isolated lEVs using density-gradient centrifugation (DGC) may yield a purer sample of mitochondria-containing lEVs. We established a DGC protocol to purify lEVs. In this pilot study, lEVs isolated using SC and DGC protocols were compared to determine their physicochemical characteristics and their effects on recipient BEC bioenergetics. SC-lEVs and DGC-lEVs both significantly restored ATP levels in OGD-injured BECs with no difference between groups. However, a Seahorse mitochondrial function assay revealed distinct functional effects: SC-lEVs did not significantly alter respiration, whereas DGC-lEVs induced a dose-dependent increase in oxygen consumption rate, indicating enhanced oxidative phosphorylation. These findings demonstrate that DGC purification yields a more mitochondria-enriched and functionally potent lEV preparation with an enhanced capacity to restore oxidative phosphorylation in ischemic BECs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/732469v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@12d5e90org.highwire.dtl.DTLVardef@19b44a5org.highwire.dtl.DTLVardef@b7ad75org.highwire.dtl.DTLVardef@dd1a3d_HPS_FORMAT_FIGEXP M_FIG C_FIG

New approach methodologies (NAMs), including induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) and retinal pigment epithelium (iRPE), are increasingly applied to study retinal disease mechanisms and therapeutic strategies. However, these models often remain relatively immature. Given the high lipid content and complex metabolism of the retina, it is unclear to what extent iPSC-derived systems recapitulate the human retinal lipidome. Here, we compared the lipidomic profiles of ROs and iRPE, collected at several differentiation stages, with those of post-mortem adult human macular, non-macular and RPE plus choroid (pmRPE). The lipidome of iRPE differed markedly from pmRPE, whereas prolonged differentiation of ROs resulted in a lipidomic profile increasingly resembling that of the post-mortem retina. Moreover, ROs showed similarities to both macular and non-macular lipidome. These findings show that iPSC-derived models can become valuable NAMs to study lipid-related retinal disorders and provide a framework to optimize differentiation protocols.

Photoreceptor transplantation is considered a disease-agnostic therapeutic strategy for retinal degenerative diseases with highly heterogenous genetic, molecular, and cellular pathologies. While integration of human photoreceptors enriched from stem cell-derived retinal organoids was noted in previous preclinical studies, the potential influence of retinal degeneration severity on transplantation efficiency has not been systematically assessed. Here, we employed mice presenting mild or severe retinal degeneration as recipients for human induced pluripotent stem cell-derived photoreceptors. Donor cells formed multi-cellular clusters that structurally integrated from 3 weeks post-transplantation (wpt) in mildly degenerated retinas, closely interacting with host Muller glia, resulting in proper maturation characterized by inner/outer segment and synapse formation by 26 wpt. In contrast, in severely degenerated hosts, donor photoreceptors remained mainly singularized and scattered in the subretinal space, showing limited structural integration or signs of maturation. Differential maturation of donor cells in mild vs. severe hosts was confirmed by single-cell RNA-sequencing analysis. However, transplantation at the beginning of the degeneration process of the severe model allowed structural integration and maturation of donor photoreceptors, despite complete loss of endogenous photoreceptors over time. The study thus shows that survival, integration, and maturation of donor photoreceptors depend on the degenerative retinal microenvironment shaping significantly transplantation efficiency.

ObjectiveTo investigate and clarify the role of Ginsenoside Ro (GRo) in diabetic cardiomyopathy (DiaCM) and to elucidate the molecular mechanism by which GRo ameliorates DiaCM. Methods[circled1] The construct of type 2 diabetic mouse model. The bought C57BL/6 male mice were housed in a specific pathogen-free (SPF) animal facility and randomly divided into control, STZ (model), STZ + GRo, and control+GRo groups. The STZ (model) and STZ + GRo groups were fed a high-fat and high-glucose diet combined with intraperitoneal injection of streptozotocin (STZ). The control and control + GRo groups were fed a normal diet, while the control + GRo and STZ + GRo groups were treated with GRo via oral gavage. Then, all groups were evaluated for cardiac function and structure by small animal echocardiography and histological staining including hematoxylin and eosin (HE) and Massons trichrome staining to screen and confirm diabetic cardiomyopathy in mice. Finally, immunofluorescence staining of cilia in mouse heart tissue was performed to determine whether GRo inhibits abnormal ciliary growth. [circled2] The construct of cell models. First, the CCK-8 (Cell Counting Kit-8) assay was used to separately evaluate the cytotoxicity of GRo and the combination of TGF-{beta}1 and PA in myocardial fibroblasts and cardiomyocytes. Subsequently, mouse myocardial fibroblast lines (MCFs) were treated with transforming growth factor-beta 1 (TGF-{beta}1), and H9c2 cardiomyocytes were treated with palmitic acid (PA). Both cell types then received the GRo treatment. [circled3] Molecular and cellular testing. Firstly, we measured serum levels of cardiac injury markers (CK-MB, MYO, and TNNI3), glutathione (GSH), and malondialdehyde (MDA). Secondly, we examined the expression of myocardial fibrosis-related genes (Col1a1, etc.), myocardial hypertrophy markers (Nppa, etc.), cilia-specific genes (Pkd1, etc.), and oxidative stress-related genes (Nrf2, etc.) in both animal and cell samples by Western blotting and RT-qPCR. Finally, we used immunofluorescence staining of myocardial fibroblasts to detect cilia length and phalloidin staining of cardiomyocytes to measure their cross-sectional area. [circled4] The correlation mechanism. Firstly, the cilia-specific inhibitory drug HIP-4 was used to disrupt cilia homeostasis by inhibiting cilia growth. Secondly, small activating RNA (saRNA) was used to upregulate the Pkd1 gene to verify whether GRo exerts its anti-fibrotic effects through the inhibition of PC1. Results[circled1] Animal level. A diabetic cardiomyopathy mouse model was successfully established by combining STZ injection with a high-fat and high-glucose diet, and treatment with GRo significantly ameliorated the associated symptoms. [circled2] Cellular level. We successfully established a myocardial fibrosis model by treating myocardial fibroblasts with TGF-{beta}1, and a myocardial hypertrophy model by treating cardiomyocytes with PA. Immunofluorescence staining demonstrated that GRo significantly decreased cilia length in the fibrosis model, while phalloidin staining showed that GRo significantly attenuated the increase in cardiomyocyte cross-sectional area. [circled3] Molecular level. Compared with the model group, GRo treatment significantly reduced serum levels of cardiac injury markers (CK-MB, MYO and TNNI3), glutathione (GSH) and malondialdehyde (MDA). Western blotting and RT-qPCR analyses of both animal and cell samples revealed that GRo markedly alleviated indicators of myocardial fibrosis and hypertrophy, while also suppressing cilia-specific genes and oxidative stress-related genes. Overall, GRo significantly ameliorated the markers associated with myocardial fibrosis and hypertrophy, and inhibited cilia-specific protein expression as well as oxidative stress parameters. [circled4] The correlation mechanism. The cilia-specific drug hedgehog pathway inhibitor 4 (HPI-4) was used to revealed that cilia homeostasis is closely linked to myocardial fibrosis and shortened cilia inhibit the fibrosis progression. Furthermore, upregulation of the Pkd1 gene by small activating RNA demonstrated that PC1 overexpression abrogates the therapeutic effect of GRo. Finally, GRo can alleviate DiaCM.

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.

Artery endothelial cells (ECs) arise through different pathways, including differentiation from mesodermal cells (vasculogenesis) or from already established vein or capillary plexus ECs (angiogenesis), the latter being most common during embryonic development and regeneration. Understanding the vein-to-artery (v2a) transition could improve revascularization therapies, but progress is limited by a lack of human models. Here, we develop a human pluripotent stem cell (hPSC) differentiation protocol that models the v2a EC conversion. Comparing v2a and mesoderm-to-artery (m2a) transcriptomes with publicly available single cell RNA sequencing (scRNA-seq) data from human embryos showed they reflected angiogenesis- and vasculogenesis-derived artery ECs, respectively. This reductionist system revealed that VEGF activation alongside PI3K inhibition was sufficient for vein ECs to acquire arterial identity within 48 hours. We model a critical step in vascular development and define the minimal signals required for artery differentiation from veins, providing a framework to promote this conversion in revascularization or therapeutic contexts.

Zebrafish (Danio rerio) possess remarkable regenerative capacity, making them an ideal model for studying the molecular mechanisms underlying tissue regeneration. In this article we report the identification of blastema linked exosome like extracellular vesicles (EVs) in zebrafish, that to the vesicles were plausibly being translocated in the proximo-distal axis through filipodia. We further thoroughly examined the exosome like EVs isolated from regenerating tissues of zebrafish caudal fins to characterize their nucleic acid cargo and evaluate their potential regulatory functions in regeneration. Caudal fins were amputated and allowed to regenerate and exosome like EVs isolated from blastema tissues displayed increased abundance compared to non-amputated controls. RNA sequencing identified a dynamic cluster of EV linked microRNAs (miRs). These differentially expressed miRs, including dre-miR-21, dre-miR-200b, dre-miR-218a and dre-let-7e were upregulated and associated with promoting proliferation, migration, differentiation, and tumour suppression pathways. Moreover, dre-miR-100, dre-miR-146a and dre-miR-200c regulated osteogenic differentiation, inflammatory signalling, epithelial-mesenchymal transition, and cell adhesion. Regeneration is generally believed to be coordinated only by local morphogen diffusion. Through this study it is indicative that filipodia bound EVs might have a pivotal role in long-range communication between blastema and the proximal tissues during the regeneration process. A detailed analyses of the miR targets and their validation could potentially lead to novel advancement and solutions in the field of regeneration and regenerative medicine in the near future.

Cell polarity complexes are essential for embryogenesis, but their regulatory mechanisms during early developmental transitions remain incompletely understood. Here, we individually deleted the Crumbs, Par, and Scrib polarity complexes in mouse embryonic stem cells (mESCs). While loss of any single complex did not affect pluripotency or proliferation, deletion of Par complex disrupted the naive-to-primed transition and impaired subsequent differentiation, particularly lumen formation in neural tube organoids. Mechanistically, Par complex deficiency led to hyperphosphorylation of focal adhesion kinase (FAK) at the primed stage, driving a morphological shift from flat monolayer clusters to dome-shaped colonies. FAK inhibition rescued the aberrant morphology. Upstream, Par complex loss increased AKT phosphorylation, which remodeled extracellular matrix (ECM) and regulated integrin signaling via FURIN-LEFTY, ultimately modulating FAK activity. In addition, conditioned medium from wild-type cells partially rescued differentiation defects in Par knockout cells in a LEFTY-dependent manner. These phenotypes were consistently observed in naive-to-primed transition, neural stem cell differentiation, embryoid body formation, teratoma assays, and neural tube organoid differentiation. Together, these findings establish a Par complex-AKT-FURIN-LEFTY-ECM-integrin-FAK signaling cascade that links apical-basal polarity to early lineage specification and morphogenesis, providing a mechanistic framework for how polarity cues are translated into developmental outcomes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722465v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1c62c46org.highwire.dtl.DTLVardef@184d5b5org.highwire.dtl.DTLVardef@1ea9017org.highwire.dtl.DTLVardef@9a0318_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementThis study elucidates the molecular mechanism by which the Par complex regulates the establishment of cell polarity. The authors demonstrate that the Par complex promotes the expression of the protein convertase FURIN via AKT signaling, thereby enhancing the maturation and secretion of LEFTY protein. This process remodels the ECM and modulates integrin signaling, ultimately regulating FAK activity and controlling the establishment of cell polarity. These findings reveal how polarity cues govern early lineage specification and morphogenesis, with implications across multiple developmental contexts.

BackgroundMetazoan adenosine-to-inosine (A-to-I) mRNA editing temporospatially diversifies the neuronal transcriptome and proteome. The limited read length from next-generation sequencing (NGS) constrains the quantification of the potentially differential editing levels across different splicing isoforms, restricting our understanding of the extent to which RNA editing contributes to molecular diversity and its interplay with splicing. MethodsWe employed reverse transcription nested PCR (RT-nPCR) and developed a novel interfering-Primer PCR (iPrimer PCR) technique to distinguish different transcripts of any gene. We selected multiple essential genes exhibiting RNA editing in coding sequences (CDSs) or untranslated regions (UTRs) for isoform-specific amplification and Sanger sequencing. ResultsNine different Adar isoforms together with pre-mRNA had distinct editing levels at the S>G auto-recoding site, which was predicted to have isoform-specific effects on catalytic activities. Although pre-mRNA editing might exert isoform-dependent promotion/suppression of splicing, closely located editing sites, such as those in neuronal genes qvr and stj, still exhibited high correlation in editing levels due to co-editing. iPrimer strategy further discovered differential recoding levels between the long/short 3UTR isoforms of gene jef. ConclusionsWe provide the first comprehensive solution for isoform-specific PCR amplification of any gene, enabling quantification of RNA editing level of different isoforms. Our results offer insights into how RNA editing interplays with splicing, and highlight its complicated role in expanding molecular diversity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/725286v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ebc82org.highwire.dtl.DTLVardef@1ea365dorg.highwire.dtl.DTLVardef@1971aceorg.highwire.dtl.DTLVardef@160d053_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed isoform-specific PCR followed by Sanger sequencing, and achieved the quantification of differential RNA editing levels in different transcripts of a gene.

Background: Chronic and non-healing wounds remain a major clinical challenge with limited therapeutic options. Angiogenesis and inflammation are central to tissue repair, and mesenchymal stem cells (MSC) contribute to these processes through their trophic and immunomodulatory secretome. Cannabidiol (CBD) exhibits antioxidant and immunomodulatory properties. However, whether CBD-rich Cannabis sativa extract stimulate MSC toward a pro-angiogenic secretome remains unclear. Purpose: This study aims to determine whether purified CBD or a phytochemically CBD-rich full spectrum extract stimulate umbilical cord-derived human MSC (UC-hMSC) to secrete pro-angiogenic factors and enhance endothelial responses relevant to wound healing. Methods: UC-hMSC were preconditioned with either purified CBD or a CBD-rich full-spectrum extract. Transcriptional changes were assessed by qPCR. The functional impact of the resulting secretome was evaluated in vitro using HUVEC-based proliferation and tube formation assays, and in vivo through the chick chorioallantoic membrane assay. To explore underlying mechanisms, we examined HIF-1 stabilization and VEGFA release in UC-hMSC, and VEGFR-2/ERK signaling in HUVEC. Results: Purified CBD and full-spectrum CBD extract preconditioned UC-hMSC secretomes, increased HUVEC proliferation, tube formation, and enhanced vascular branching in the CAM assay. Mechanistic analyses indicated activation of the HIF-1/VEGF axis in UC-hMSC, and ERK1/2 activation in HUVEC that was sensitive to VEGFR-2 blockade. Conclusion: Purified CBD and CBD-rich full-spectrum extract prime UC-hMSC toward a pro-angiogenic secretome that promotes endothelial activation and neovascularization. These findings suggest that cannabinoid-based preconditioning of UC-hMSC involves the HIF-1/VEGF axis and VEGFR-2/ERK signaling pathways in endothelial cells, supporting further investigation of this approach in wound healing and regenerative therapies.

Collateral arteries are natural bypasses that can reroute blood flow around arterial blockages, limiting tissue injury during stroke and coronary artery disease. Despite their clinical effectiveness, therapeutic strategies to stimulate collateral artery growth remain unavailable due to our limited understanding of their developmental mechanisms. Remarkably, guinea pigs display exceptionally dense collateral artery networks across various organs, resulting in complete resistance to ischemic damage in the brain and heart. In this study, we compared single-cell RNA sequencing (scRNA-seq) from guinea pig and mouse tissues to identify endothelial cell (EC) gene expression patterns associated with extensive collateral artery development. We then developed an in vivo Perturb-seq platform in mice to test whether genes differentially expressed in guinea pigs influence artery EC specification. This pipeline identified artery repressors that were downregulated in guinea pigs and increased pial collateral abundance when inhibited in mice. Downstream analysis suggests that artery repressors, including WNT and hypoxia response genes, function in two capillary EC subsets--Esm1+ pre-artery and Apln+ angiogenic tip cells. Reduced activity of these repressors allows more ECs to acquire arterial identity, potentiating collateral artery formation. Collectively, our study establishes a strategy for discovering the genes underlying species-specific traits, suggests that guinea pigs have collaterals due to decreased activity of artery inhibitor pathways and hypoxia responses, and identifies novel targets for stimulating collateral artery formation (Graphical abstract). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/721711v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1d6f264org.highwire.dtl.DTLVardef@c3ad35org.highwire.dtl.DTLVardef@a0af7dorg.highwire.dtl.DTLVardef@1614c61_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and PurposeCardiotoxicity is a major cause for drug failure throughout the drug development process, with particular concern for action potential prolongation and arrhythmia. Hence, such liabilities are heavily considered during the early phases of drug design to pre vent dangerous compounds from progressing. New approach methodologies (NAMs) that efficiently examine this risk early in the discovery pipeline should help streamline drug development programs. We developed a cardiac NAM, a 384-well open bath platform consisting of cardiac tissue derived from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, enabling high-throughput drug screening while maintaining the structural and functional complexity of 3D cardiac micromuscles. MethodsWe dramatically increased throughput without compromising physiological relevance provided by the 3D micromuscle structure. Our 384-well open bath high-throughput platform allowed evaluation of multiple compounds at a time, enabling us to study the CiPA (comprehensive in vitro proarrhythmia assay) drug panel for proarrhythmia screening. We obtained phenotypic fingerprints of all 28 compounds (9 low, 11 intermediate, and 8 high arrhythmia risk; https://cipaproject.org) in dose-escalation studies around their respective clinical concentrations. The analysis was augmented with an in silico pipeline that used phenotypic biomarkers to invert data into a mathematical model of cellular currents to infer which ion channels were affected upon drug exposure, and then trained a ML model to predict channel block. Results and ConclusionsWe found accurate detection of arrhythmic potential for most of the compounds, and the in silico model inversions were consistent with published values of compound channel block. All the high risk compounds showed action potential duration (APD) prolongation coupled with either action potential abnormalities, early afterdepolarizations (EADs), or beat cessation. For the intermediate risk group, 9 out of 11 compounds caused APD prolongation alone or in combination with EADs while 2 others showed either beat cessation or beat rate change. Augmentation of APD analysis with detailed biophysical modeling and ML tools provided meaningful insight into the mechanisms involved in APD changes. Overall, our cardiac NAM allowed for fast and relevant screening for mechanistic understanding of APD prolongation and proarrhythmic activity, at massively increased throughput compared to other 3D micromuscle models. SummaryCardiotoxicity testing is critical in drug development to prevent arrhythmogenic side effects. Current stringent regulations have greatly reduced market withdrawals; however, these strict evaluations often lead to costly late-stage failures and loss of promising candidates as false positives. We developed a cardiac new approach methodology (NAM), a 384-well open bath cardiac micromuscle platform created from hiPSC-derived cardiomyocytes, enabling high-throughput drug screening while maintaining the structural and functional complexity of 3D cardiac micromuscles. Using the comprehensive in vitro proarrhythmia assay (CiPA) drug panel, we validated the system to accurately detect proarrhythmic potential. Our assay provided phenotypic fingerprints based on mechanical and electrophysiological biomarkers. Integration with computational modeling offered insights into multi-ion channel effects (MICE). Particularly, we identified sodium channel block contributions as a significant factor for poor risk prediction based on traditional parameters. The combined experimental and computational platform can enhance early drug screening, thereby reducing late-stage failures and promoting the progression of low-risk compounds with complex electrophysiological profiles.