Main Cell Types Research Articles

Enhanced US/CT/MR imaging of integrin αvβ3 for liver fibrosis staging in rat.

Liver fibrosis is a global health challenge with high morbidity and mortality rates, and diagnostic sensitivity of liver fibrosis tests can be increased using multimodal molecular agents. We designed cyclic arginine-glycine-aspartic acid (cRGD)-modified nanoparticles (NPs) using ultrasound (US)/computed tomography (CT)/magnetic resonance (MR) triple-modality imaging to evaluate liver fibrosis stages. In vitro and in vivo studies were conducted using primary hepatic stellate cells (HSCs) and a rat model of liver fibrosis induced by carbon tetrachloride (CCl4). Our results showed cRGD-poly(lactic-co-glycolic acid)-Fe3O4-perfluorocarbon bromide (cRGD-PLGA-Fe3O4-PFOB) NPs were preferentially internalised by activated HSCs (aHSCs). The main cell types expressing integrin αvβ3 during liver fibrogenesis were the aHSCs. The protein levels of αv and β3 expressed on aHSCs increased with the progression of liver fibrosis. After intravenous injection of cRGD-PLGA-Fe3O4-PFOB NPs, the echo intensity (EI) values, CT values, and T2 values of liver parenchyma correlated well with liver fibrosis severity. cRGD-PLGA-Fe3O4-PFOB NPs as multifunction contrast agents showed great potential to reflect the degree of HSC activation and distinguish among different liver fibrotic stages. The ligand-directed and integrin αvβ3-mediated accumulation provides active and passive targeting capabilities, permitting the targeted multimodal imaging of cRGD-PLGA-Fe3O4-PFOB NPs, which delivers accurate non-invasive diagnosis and real-time monitoring of liver fibrosis development.

96P Goblet cell differentiation in colorectal cancer

In the large intestine, the multipotent stem cells are located at the base of the crypt and differentiate into three main cell types: enterocytes, goblet cells, and enteroendocrine cells. Goblet cells’ main function is the synthesis and secretion of mucins. Genetic and epigenetic changes that provide survival advantages for stem or progenitor cells resulting in the deregulation of cellular differentiation are major causes of all carcinomas.

In vitro propagation of XXY human Klinefelter spermatogonial stem cells: A step towards new fertility opportunities.

Klinefelter Syndrome (KS) is characterized by a masculine phenotype, supernumerary sex chromosomes (47, XXY), and impaired fertility due to loss of spermatogonial stem cells (SSCs). Early testicular cryopreservation could be an option for future fertility treatments in these patients, including SSCs transplantation or in vitro spermatogenesis. It is critically essential to adapt current in vitro SSCs propagation systems as a fertility option for KS patients. KS human testicular samples (13,15- and 17-year-old non-mosaic KS boys) were donated by patients enrolled in an experimental testicular tissue banking program. Testicular cells were isolated from cryopreserved tissue and propagated in long-term culture for 110 days. Cell-specific gene expression confirmed the presence of all four main cell types found in testes: Spermatogonia, Sertoli, Leydig, and Peritubular cells. A population of ZBTB16+ undifferentiated spermatogonia was identified throughout the culture using digital PCR. Flow cytometric analysis also detected an HLA-/CD9+/CD49f+ population, indicating maintenance of a stem cell subpopulation among the spermatogonial cells. FISH staining for chromosomes X and Y showed most cells containing an XXY karyotype with a smaller number containing either XY or XX. Both XY and XX populations were able to be enriched by magnetic sorting for CD9 as a spermatogonia marker. Molecular karyotyping demonstrated genomic stability of the cultured cells, over time. Finally, single-cell RNAseq analysis confirmed transcription of ID4, TCN2, and NANOS 3 within a population of putative SSCs population. This is the first study showing successful isolation and long-term in vitro propagation of human KS testicular cells. These findings could inform the development of therapeutic fertility options for KS patients, either through in vitro spermatogenesis or transplantation of SSC, in vivo.

SFRP4+ stromal cell subpopulation with IGF1 signaling in human endometrial regeneration.

Our understanding of full-thickness endometrial regeneration after injury is limited by an incomplete molecular characterization of the cell populations responsible for the organ functions. To help fill this knowledge gap, we characterized 10,551 cells of full-thickness normal human uterine from two menstrual phases (proliferative and secretory phase) using unbiased single cell RNA-sequencing. We dissected cell heterogeneity of main cell types (epithelial, stromal, endothelial, and immune cells) of the full thickness uterine tissues, cell population architectures of human uterus cells across the menstrual cycle. We identified an SFRP4+ stromal cell subpopulation that was highly enriched in the regenerative stage of the human endometria during the menstrual cycle, and the SFRP4+ stromal cells could significantly enhance the proliferation of human endometrial epithelial organoid in vitro, and promote the regeneration of endometrial epithelial glands and full-thickness endometrial injury through IGF1 signaling pathway in vivo. Our cell atlas of full-thickness uterine tissues revealed the cellular heterogeneities, cell population architectures, and their cell–cell communications during the monthly regeneration of the human endometria, which provide insight into the biology of human endometrial regeneration and the development of regenerative medicine treatments against endometrial damage and intrauterine adhesion.

Define the Two Molecular Subtypes of Epithelioid Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a fatal disease of respiratory system. Despite the availability of invasive biomarkers with promising results, there are still significant diagnostic and therapeutic challenges in the treatment of MPM. One of three main mesothelioma cell types, epithelioid mesothelioma makes up approximately 70% of all mesothelioma cases. Different observational findings are under process, but the molecular heterogeneity and pathogenesis of epithelioid malignant pleural mesothelioma (eMPM) are still not well understood. Through molecular analysis, expression profiling data were used to determine the possibility and optimal number of eMPM molecular subtypes. Next, clinicopathological characteristics and different molecular pathways of each subtype were analyzed to prospect the clinical applications and advanced mechanisms of eMPM. In this study, we identified two distinct epithelioid malignant pleural mesothelioma subtypes with distinct gene expression patterns. Subtype I eMPMs were involved in steroid hormone biosynthesis, porphyrin and chlorophyll metabolism, and drug metabolism, while subtype II eMPMs were involved in rational metabolism, tyrosine metabolism, and chemical carcinogenesis pathways. Additionally, we identified potential subtype-specific therapeutic targets, including CCNE1, EPHA3, RNF43, ROS1, and RSPO2 for subtype I and CDKN2A and RET for subtype II. Considering the need for potent diagnostic and therapeutic biomarkers for eMPM, we are anticipating that our findings will help both in exploring underlying mechanisms in the development of eMPM and in designing targeted therapy for eMPM.

Single-nucleus RNA Sequencing reveals the mechanism of cigarette smoke exposure on diminished ovarian reserve in mice

The systematic toxicological mechanism of cigarette smoke (CS) on ovarian reserve has not been extensively investigated. Female 8-week-old C57BL/6 mice at peak fertility were exposed to CS or indoor air only for 30 days (100 mice per group) and the effects of CS on ovarian reserve were assessed using Single-Nucleus RNA Sequencing (snRNA-seq). In addition, further biochemical experiments, including immunohistochemical staining, ELISA, immunofluorescence staining, transmission electron microscopy, cell counting kit-8 assay, flow cytometry analysis, senescence-associated β-galactosidase staining, and western blotting, were accomplished to confirm the snRNA-seq results. We identified nine main cell types in adult ovaries and the cell-type-specific differentially expressed genes (DEGs) induced by CS exposure. Western blot results verified that down-regulation of antioxidant genes (Gpx1 and Wnt10b) and the steroid biosynthesis gene (Fdx1) occurred in both ovarian tissue and human granulosa cell-like tumor cell line (KGN cells) after CS exposure. Five percent cigarette smoke extract (CSE) effectively stimulated the production of reactive oxygen species (ROS), DNA damage, cellular senescence and markedly inhibited KGN cell proliferation by inducing G1-phase cell cycle arrest. Moreover, down-regulation of Gja1, Lama1 and the Ferroptosis indicator (Gpx4) in granulosa cells plays a significant role in ultrastructural changes in the ovary induced by CS exposure. These observations suggest that CS exposure impaired ovarian follicle reserve might be caused by REDOX imbalance in granulosa cells. The current study systematically determined the damage caused by CS in mouse ovaries and provides a theoretical basis for early clinical prediction, diagnosis and intervention of CS exposure-associated primary ovarian insufficiency (POI), and is of great significance in improving female reproductive health.

Integrated multi-omics reveals the activated retinal microglia with intracellular metabolic reprogramming contributes to inflammation in STZ-induced early diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of visual impairment and blindness among working-age people. Inflammation is recognized as a critical driver of the DR process. However, the main retina-specific cell type producing pro-inflammatory cytokines and its mechanism underlying DR are still unclear. Here, we used single-cell sequencing to identify microglia with metabolic pathway alterations that were the main source of IL-1β in STZ-induced DR mice. To profile the full extent of local metabolic shifts in activated microglia and to reveal the metabolic microenvironment contributing to immune mechanisms, we performed integrated metabolomics, lipidomics, and RNA profiling analyses in microglia cell line samples representative of the DR microenvironment. The results showed that activated microglia with IL-1β increase exhibited a metabolic bias favoring glycolysis, purine metabolism, and triacylglycerol synthesis, but less Tricarboxylic acid (TCA). In addition, some of these especially glycolysis was necessary to facilitate their pro-inflammation. These findings suggest that activated microglia with intracellular metabolic reprogramming in retina may contribute to pro-inflammation in the early DR.

1694P Single cell characterization of longitudinal biopsies from breast cancer patients treated with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib

The recent introduction of CDK4/6 inhibitors has been one of the most pivotal breakthroughs in breast cancer therapy in the last decades. A growing body of evidence proposes that CDK4/6 inhibitors potentially influence the recruitment of immune cells in the tumor microenvironment, but also induce a senescent-like phenotype in the tumor cells. The NEOLETRIB-study is a multicenter, single-arm, open-label, neoadjuvant, phase II trial. Locally advanced breast cancer patients belonging to the luminal-A and luminal-B subtypes, received neoadjuvant therapy for 6 months with ribociclib (600 mg daily, 21 days on / 7 days off) and letrozole (2.5 mg daily). Pre-treatment, on-treatment (end of the first 21 days of therapy) and end-of-treatment biopsies were subjected to single-cell transcriptome using the Chromium Single-Cell v2 5′ Chemistry. Raw gene expression matrices were analyzed with the Seurat package (v4.0.2). First, empty droplets and droplets containing two cells were filtered out using the EmptyDrops and scDblFinder algorithms respectively. In addition, cell barcodes with < 500 UMIs, < 300 expressed genes, > 5000 expressed genes or > 15% of reads mapping to mitochondrial RNA were filtered out. The gene expression of the remaining good quality cells was normalized. Variably expressed genes were used to construct principal components (PCs). Significant PCs were further used to cluster cells. We identified 8 main cell types: T cells, B cells, epithelial cells, fibroblasts, endothelial cells, mast cells and dendritic cells. To discriminate between cancer and normal epithelial cells, we estimated for each epithelial cell the copy number variations using the InferCNV algorithm. Furthermore, to identify specific and specialized cell subtype, we clustered each of the eight cell type independently and annotated the clusters obtained using validated marker genes. We also compare our annotations with publish methods identifying cell states in single cell data (Luca et al., Cell 2021). We highlight how scRNA-seq and our analytic pipelines are allowing us to study the tumor microenvironment of breast cancer biopsies at a very high resolution.

Autophagy Plays Multiple Roles in the Soft-Tissue Healing and Osseointegration in Dental Implant Surgery-A Narrative Review.

Dental endo-osseous implants have become a widely used treatment for replacing missing teeth. Dental implants are placed into a surgically created osteotomy in alveolar bone, the healing of the soft tissue lesion and the osseointegration of the implant being key elements to long-term success. Autophagy is considered the major intracellular degradation system, playing important roles in various cellular processes involved in dental implant integration. The aim of this review is an exploration of autophagy roles in the main cell types involved in the healing and remodeling of soft tissue lesions and implant osseointegration, post-implant surgery. We have focused on the autophagy pathway in macrophages, endothelial cells; osteoclasts, osteoblasts; fibroblasts, myofibroblasts and keratinocytes. In macrophages, autophagy modulates innate and adaptive immune responses playing a key role in osteo-immunity. Autophagy induction in endothelial cells promotes apoptosis resistance, cell survival, and protection against oxidative stress damage. The autophagic machinery is also involved in transporting stromal vesicles containing mineralization-related factors to the extracellular matrix and regulating osteoblasts’ functions. Alveolar bone remodeling is achieved by immune cells differentiation into osteoclasts; autophagy plays an important and active role in this process. Autophagy downregulation in fibroblasts induces apoptosis, leading to better wound healing by improving excessive deposition of extracellular matrix and inhibiting fibrosis progression. Autophagy seems to be a dual actor on the scene of dental implant surgery, imposing further research in order to completely reveal its positive features which may be essential for clinical efficacy.

Neutrophils in Atlantic salmon (Salmo salar L.) are MHC class II+ and secret IL-12p40 upon bacterial exposure

Antigen-presentation via major histocompatibility complex (MHC) to T cells is the key event to initiate adaptive immune responses. In teleosts, as in mammals, the main types of professional antigen-presenting cells (APCs) are dendritic cells (DCs), monocytes/macrophages, and B cells. In the current study, flow cytometry, immunostaining and qPCR have been used to show that neutrophils in the teleost fish Atlantic salmon (Salmo salar L.) have antigen-presenting properties. The neutrophils were positive for MHC class II, CD83 and CD80/86, and upon in vitro bacterial exposure, gene expression analysis of purified neutrophils showed that IL-12p40, which is essential for proliferation of naïve T cells, was highly upregulated at both 6 and 24 h post bacterial exposure. Based on presence of MHC class II and upregulation of molecules involved in antigen presentation and T cell activation, we suggest that neutrophils in Atlantic salmon have potential to function as professional APCs. This work makes an important basis for further exploring the potential of using neutrophils to develop new, targeted immunoprophylactic measures.

Accelerated aging-related transcriptome alterations in neurovascular unit cells in the brain of Alzheimer’s disease

Alzheimer’s disease (AD) is the most common cause of dementia with no effective therapies. Aging is a dominant risk factor for AD. The neurovascular unit (NVU) plays an important role in maintaining homeostasis of the brain microenvironment. The accelerated aging of NVU cells may directly impair NVU function and contribute to AD pathogenesis. However, the expression patterns of aging-related genes (AGs) in NVU cells of AD remain unclear. In this study, we performed single-nucleus transcriptome analysis of 61,768 nuclei from prefrontal cortical samples of patients with AD and normal control (NC) subjects. Eight main cell types were identified, including astrocytes, microglia, excitatory neurons, inhibitory neurons, oligodendrocytes, oligodendrocyte precursor cells, pericytes, and endothelial cells. Transcriptomic analysis identified the expression patterns of AGs in NVU cells of AD. Gene set enrichment analysis confirmed the key aging-associated cellular pathways enriched in microglia and oligodendrocytes. These aging-related transcriptomic changes in NVU were cross-validated using bulk transcriptome data. The least absolute shrinkage and selection operator regression method was used to select the crucial AGs most associated with AD: IGF1R, MXI1, RB1, PPARA, NFE2L2, STAT5B, FOS, PRKCD, YWHAZ, HTT, MAPK9, HSPA9, SDHC, PRKDC, and PDPK1. This 15-gene model performed well in discriminating AD from NC samples. Among them, IGF1R, MXI1, PPARA, YWHAZ, and MAPK9 strongly correlated with pathologic progression in AD, were identified as critical regulators of AD. Although most AGs showed similar trends of expression changes in different types of NVU cells in AD, certain AGs were expressed in a cell-specific manner. Our comprehensive analysis of brain NVU from patients with AD reveals previously unknown molecular changes associated with aging that may underlie the functional dysregulation of NVU, providing important insights for exploring potential cell-specific therapeutic targets to restore brain homeostasis in AD.

Single-cell sequencing reveals heterogeneity between pancreatic adenosquamous carcinoma and pancreatic ductal adenocarcinoma with prognostic value.

Pancreatic adenosquamous carcinoma (ASPC) is a rare subtype of pancreatic cancer with lethal malignancy, and few studies have focused on the heterogeneity of ASPC. Here, we performed a single-cell sequencing procedure on pancreatic tumor tissue from an ASPC patient and a patient with high-grade intraductal papillary mucinous neoplasm (IPMN). Through the combined analysis of single-cell sequencing data from five pancreatic ductal adenocarcinoma (PDAC) patients, one IPMN patient, and one ASPC patient in a public database, we identified 11 main types of cells, including macrophages, B cells, cancer stem cells, ductal cells, fibroblasts, endo/stellate cells, neutrophils, acinar cells, T cells, natural killer (NK) cells, dendritic cells, and mast cells. Then, the different characteristics and differentiation paths of the immune microenvironment among IPMN, ASPC, and PDAC in macrophages, T cells, and cancer-associated fibroblasts (CAFs) were identified through multiple bioinformatics analyses. Two novel special cancer-associated fibroblasts were identified as nCAFs and imCAFs. Then, cancer cells in duct cells were identified using the infercnv software. Two ASPC-specific subgroups of cancer cells with squamous cell features were identified. Finally, the identified specific CAFs and cancer cells were mapped to TCGA-PAAD cohort through the cibersoftx software. All of these identified subgroups were calculated to have a significant prognostic value in pancreatic cancer patients. These findings will promote the clinical application of single-cell sequencing data of pancreatic cancer and deepen our understanding of ASPC.

Human Cytomegalovirus and Human Herpesvirus 6 Coinfection of Dermal Fibroblasts Enhances the Pro-Inflammatory Pathway Predisposing to Fibrosis: The Possible Impact on Systemic Sclerosis.

Systemic sclerosis (SSc) is a severe autoimmune disease likely triggered by genetic and environmental factors, including viral infections. Human cytomegalovirus (HCMV) and human herpesvirus 6A species (HHV-6A) have been associated with SSc, based on in vivo and in vitro evidence, but the data are still inconclusive. Furthermore, despite both viruses being highly prevalent in humans and able to exacerbate each other’s effects, no data are available on their joint effects. Hence, we aimed to study their simultaneous impact on the expression of cell factors correlated with fibrosis and apoptosis in in vitro coinfected fibroblasts, representing the main target cell type in SSc. The results, obtained by a microarray detecting 84 fibrosis/apoptosis-associated factors, indicated that coinfected cells underwent higher and more sustained expression of fibrosis-associated parameters compared with single-infected cells. Thus, the data, for the first time, suggest that HCMV and HHV-6A may cooperate in inducing alterations potentially leading to cell fibrosis, thus further supporting their joint role in SSc. However, further work is required to definitively answer whether β-herpesviruses are causally linked to the disease and to enable the possible use of targeted antiviral treatments to improve clinical outcomes.

Identification of Human Retinal Organoid Cell Differentiation-Related Genes via Single-Cell Sequencing Data Analysis.

Objective To study the development process of the human retina, we analyzed the development track of main cell types and transitional cell populations, identifying the retinal organoid cell differentiation-related genes (RDRGs). Methods Single-cell RNA sequencing data (scRNA-Seq) of human retinal organoids were downloaded from Gene Expression Omnibus (GEO) database in this study. Data were processed with quality analysis and analysis of variance. Principal component analysis and t-distributed stochastic neighbor embedding were used to conduct dimension reduction analysis and type annotation for the screened data. Marker genes and RDRGs were identified by differential analysis. Cell differentiation characteristics were determined by trajectory analysis. Enrichment pathways were analyzed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG), and functional modules were obtained by protein-protein interaction (PPI) network analysis. Results iPSCs were mainly located at the root of differentiation trajectory, while neurons and astrocytes were distributed in different branches, respectively. Meanwhile, 220 RDRGs were obtained. They were involved in the biological functions related to vision and visual development, as well as significantly enriched in signaling pathways associated with retinal vascular development and retinal neuroregulation. Protein-protein interaction network construction and functional subnetwork analysis were conducted on RDRGs, and two functional submodules were obtained. The enrichment analysis presented that the two submodules played a vital role in retinal development, visual perception, and cell respiration. Conclusions This study identified RDRGs and revealed the biological functions involved in these genes, which are expected to provide evidence for researching retinal development and diseases.

Abstract P2064: High Resolution Single Cell Analysis Of The Developing Human Heart

The human heart is composed of various cell types with distinct embryonic origins and functions. Having an understanding of the components of the developing heart at a molecular level should provide better insights into its function in health and disease. Current single cell studies of human fetal hearts are limited by low cell numbers and poor cell type resolution. Here, we aimed to increase the number of cells studied while adding a spatial resolution. We analysed human hearts aged between post conception weeks of 8 and 11, where apex and base in each were processed, providing spatial resolution on cells constituting the myocardium and the great vessels. Our analysis suggests 6 main cell types in the developing hearts with a total of 28 subtypes and provides a differential expression signature for similar cell types with different origins and anatomical locations, including the smooth muscle cells and endothelial cells of the myocardium and the great vessels. Through immunohistochemistry, we validated our findings on these subtypes, as well as on the cells that were less clearly described before, such sinoatrial node cells or resident macrophages. Cell to cell interaction analysis inferred distinct molecular crosstalk across subtypes, including the cells of the great vessels or the vessels of the myocardium. Our findings suggest a list of transcription factors that might be involved in governing the identity of these cells. Collectively, our analysis provides a better understanding of human heart development, and will inform studies into disease modelling and disease progression.

New transcriptome and clinical findings of platelet-activating factor in chronic spontaneous urticaria: Pathogenic and treatment relevance.

The objective of the study was to assess the pathogenic and treatment relevance of Platelet Activating factor (PAF) in chronic spontaneous urticaria (CSU). The expression and cellular location of PAF receptor (PAFR) and serum levels of PAF and PAF acetylhydrolase (PAF-AH) in patients with moderate/severe CSU (n=45) and healthy controls (HCs, n=17) were studied. Skin samples from the active wheal (LS-CSU, 13 samples for qPCR and 33 for immunohistochemistry) and non-lesional skin (NLS-CSU, 13 samples) of CSU patients and HCs (13 samples and 5 for immunohistochemistry) were analyzed. Serum PAF and PAF-AH levels were measured by ELISA and compared between HC (10 samples) and CSU patients (25 samples) and, among them, between those refractory and non-refractory to second-generation H1 -antihistamines (sgAH). PAFR mRNA expression was significantly higher in LS-CSU versus HCs (p=0.014). PAFR positive staining in immunohistochemistry was mainly found in the epidermal basal layer in HCs, whereas it was broadly present along the epidermis in LS-CSU samples. Endothelial cells showed PAFR expression exclusively in LS-CSU and NLS-CSU samples. PAFR expression was observed in the nerves of HC, LS-CSU, and NLS-CSU samples. Double PAFR/CD43 expression showed that T-lymphocytes were the main cell type from the wheal inflammatory infiltrate expressing PAFR. A significantly lower PAF-AH/PAF ratio was observed in sgAH non-responders versus responders (6.1 vs. 12.6; p=0.049). Our study confirms that PAF is a mediator of wheal pathogenesis in CSU. The significantly lower PAF-AH/PAF ratio in sgAH non-responders vs responders suggests that PAF could be a potential biomarker of sgAH refractoriness.

The cytotoxic effect of graphene oxide nanoparticles on bacterial cells

The results of research on the introduction of nanotechnology in various fields of scientific activities are presented. In medical and veterinary practice, nanoparticles of various forms of carbon are considered promising, because they have a wide arsenal of biomodulatory effects on the body, exhibiting little ecotoxic and organismotoxic effects. Their antibacterial effect is of great importance for the use in medicine. Microorganisms are one of the objects of study of the cytotoxic properties of new medicinal products. The cytotoxic effect of oxidized graphene nanoparticles on the main types of bacterial cells was evaluated by light and atomic force microscopy. Light microscopy allowed to establish that the effect of subinhibitory concentrations of nanoparticles of oxidized graphene, achieved by studying the colonies of microorganisms on the border of their growth inhibition, can lead to tinctorial transversion in Gram-positive microorganisms, in particular Staphylococcus aureus, while in Gram-negative E. coli such a phenomenon is not observed. Using the method of atomic force microscopy, it was found that toxic concentrations of oxidized graphene nanoparticles lead to morphological degradation, the degree of which depends on the exposure time of nanoparticles. Morphological degradation of cells in the main types of bacteria (cocci, bacilli), accompanied by a decrease in cell contours, was observed at 30 min exposure. When the exposure was increased from 30 to 90 min, complete morphological destruction of the bacterial cells and decay of the bacterial population composition were observed. The cytotoxic concentration of oxidized graphene nanoparticles is more than 75 µg·ml-1, as determined by the results of microscopy of test culture samples (Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538).

Identification and implication of tissue-enriched ligands in epithelial–endothelial crosstalk during pancreas development

Development of the pancreas is driven by an intrinsic program coordinated with signals from other cell types in the epithelial environment. These intercellular communications have been so far challenging to study because of the low concentration, localized production and diversity of the signals released. Here, we combined scRNAseq data with a computational interactomic approach to identify signals involved in the reciprocal interactions between the various cell types of the developing pancreas. This in silico approach yielded 40,607 potential ligand-target interactions between the different main pancreatic cell types. Among this vast network of interactions, we focused on three ligands potentially involved in communications between epithelial and endothelial cells. BMP7 and WNT7B, expressed by pancreatic epithelial cells and predicted to target endothelial cells, and SEMA6D, involved in the reverse interaction. In situ hybridization confirmed the localized expression of Bmp7 in the pancreatic epithelial tip cells and of Wnt7b in the trunk cells. On the contrary, Sema6d was enriched in endothelial cells. Functional experiments on ex vivo cultured pancreatic explants indicated that tip cell-produced BMP7 limited development of endothelial cells. This work identified ligands with a restricted tissular and cellular distribution and highlighted the role of BMP7 in the intercellular communications contributing to vessel development and organization during pancreas organogenesis.

768 Dermal fibroblast expression of lef1 is critical to normal skin and hair development and regenerative wound healing in mice

Fibroblasts are the main cell type in the dermis and are major contributors to the wound healing process. During skin development, papillary fibroblasts from the upper dermis uniquely give rise to specialized hair follicle-supporting fibroblasts like the dermal papillae, which controls hair development and cycling, as well as the arrector pili muscles that grant hair its contractile functions. Neonatal papillary fibroblasts are also the only fibroblasts which are supportive of hair follicle regeneration, which we have previously shown to occur naturally in neonatal wound healing and in chamber grafting experiments in adult wounds.

Tri‐culture of spatially organizing human skeletal muscle cells, endothelial cells, and fibroblasts enhances contractile force and vascular perfusion of skeletal muscle tissues

Constructing engineered human skeletal muscle tissues that resemble the function and microstructure of human skeletal muscles is key to utilizing them in a variety of applications such as drug development, disease modeling, regenerative medicine, and engineering biological machines. However, current in vitro skeletal muscle tissues are far inferior to native muscles in terms of contractile function and lack essential cues for muscle functions, particularly heterotypic cell-cell interactions between myoblasts, endothelial cells, and fibroblasts. Here, we develop an engineered muscle tissue with a coaxial three-layered tubular structure composed of an inner endothelial cell layer, an endomysium-like layer with fibroblasts in the middle, and an outer skeletal muscle cell layer, similar to the architecture of native skeletal muscles. Engineered skeletal muscle tissues with three spatially organized cell types produced thicker myotubes and lowered Young's modulus through extracellular matrix remodeling, resulting in 43% stronger contractile force. Furthermore, we demonstrated that fibroblasts localized in the endomysium layer induced angiogenic sprouting of endothelial cells into the muscle layer more effectively than fibroblasts homogeneously distributed in the muscle layer. This layered tri-culture system enables a structured spatial configuration of the three main cell types of skeletal muscle and promotes desired paracrine signaling, resulting in improved angiogenesis and increased contractile force. This research offers new insights to efficiently obtain new human skeletal muscle models, transplantable tissues, and actuators for biological machines.