Bone and entheseal targets for growth factors in diffuse idiopathic skeletal hyperostosis.

INTRODUCTION: Diffuse idiopathic skeletal hyperostosis (DISH) is a common condition of the adult skeleton where new bone growth occurs in entheseal and bony regions. The cause for the new bone growth is unclear but many lines of evidence point to a role for growth factors linked to abnormal metabolism in these patients. The bone targets for these presumed growth factors are poorly defined. This review summarises the clinical evidence relevant to the sites of origin of new bone formation in DISH to better define potential cellular targets for bone growth in DISH. METHODS: This is a narrative review of relevant papers identified from searches of PubMed and online journals. RESULTS: Sites of new bone growth in the enthesis were identified in patients with DISH, with likely cellular targets for growth factors being mesenchymal stem cells in the outer part of the enthesis. Similar undifferentiated skeletal stem cells are present in the outer annulus fibrosis and in the bony eminences of vertebral bodies and other bones, with the potential for response to growth factors. CONCLUSION: Mesenchymal stem cells are present in specific entheseal and bony locations that are likely responsive to putative growth factors leading to new bone formation characteristic of DISH. Further study of these regions in the context of metabolic abnormalities in DISH will allow for better understanding of the pathophysiology of this common condition.

Bone regeneration driven by a nano-hydroxyapatite/chitosan composite bioaerogel for periodontal regeneration.

The most recent progress in reconstructive therapy for the management of periodontitis and peri-implantitis bone defects has relied on the development of highly porous biodegradable bioaerogels for guided bone regeneration. The objective of this work was to evaluate in vitro the osteoinduction of periodontal-originating cells (human dental follicle mesenchymal cells, DFMSCs) promoted by a nano-hydroxyapatite/chitosan (nHAp/CS) bioaerogel, which was purified and sterilized by a sustainable technique (supercritical CO2). Moreover, the in vivo bone regeneration capacity of the nHAp/CS bioaerogel was preliminarily assessed as a proof-of-concept on a rat calvaria bone defect model. The quantification of DNA content of DFMSCs seeded upon nHAp/CS and CS scaffolds (control material) showed a significant increase from the 14th to the 21st day of culture. These results were corroborated through confocal laser scanning microscopy analysis (CLSM). Furthermore, the alkaline phosphatase (ALP) activity increased significantly on the 21st day, similarly for both materials. Moreover, the presence of nHAp promoted a significantly higher expression of osteogenic genes after 21 days when compared to CS scaffolds and control. CLSM images of 21 days of culture also showed an increased deposition of OPN over the nHAp/CS surface. The in vivo bone formation was assessed by microCT and histological analysis. The in vivo evaluation showed a significant increase in bone volume in the nHAp/CS test group when compared to CS and the empty control, as well as higher new bone formation and calcium deposition within the nHAp/CS structure. Overall, the present study showed that the nHAp/CS bioaerogel could offer a potential solution for periodontal and peri-implant bone regeneration treatments since the in vitro results demonstrated that it provided favorable conditions for DFMSC proliferation and osteogenic differentiation, while the in vivo outcomes confirmed that it promoted higher bone ingrowth.

Stromal KITL/SCF promotes pancreas tissue homeostasis and restrains tumor progression.

Components of normal tissue architecture serve as barriers to tumor progression. Inflammatory and wound-healing programs are requisite features of solid tumorigenesis, wherein alterations to immune and non-immune stromal elements enable loss of homeostasis during tumor evolution. The precise mechanisms by which normal stromal cell states limit tissue plasticity and tumorigenesis, and which are lost during tumor progression, remain largely unknown. Here we show that healthy pancreatic mesenchyme expresses the paracrine signaling molecule KITL, also known as stem cell factor, and identify loss of stromal KITL during tumorigenesis as tumor-promoting. Genetic inhibition of mesenchymal KITL in the contexts of homeostasis, injury, and cancer together indicate a role for KITL signaling in maintenance of pancreas tissue architecture, such that loss of the stromal KITL pool increased tumor growth and reduced survival of tumor-bearing mice. Together, these findings implicate loss of mesenchymal KITL as a mechanism for establishing a tumor-permissive microenvironment.

Comprehensive identification, isolation, and culture of human breast cell types.

Tissues are formed and shaped by cells of many different types and are orchestrated through countless interactions. Deciphering a tissue's biological complexity thus requires studying it at cell-level resolution, where molecular and biochemical features of different cell types can be explored and thoroughly dissected. Unfortunately, the lack of comprehensive methods to identify, isolate, and culture each cell type from many tissues has impeded progress. Here, we present a method for the breadth of cell types composing the human breast. Our goal has long been to understand the essence of each of these different breast cell types, to reveal the underlying biology explaining their intrinsic features, the consequences of interactions, and their contributions to the tissue. This biological exploration has required cell purification, deep-RNA sequencing-and a thorough dissection of the genes and pathways defining each cell type. Whereas the molecular analysis is presented in an adjoining article, we present here an exhaustive cellular dissection of the human breast and explore its cellular composition and histological organization. Moreover, we introduce a novel FACS antibody panel and rigorous gating strategy capable of isolating each of the twelve major breast cell types to purity. Finally, we describe the creation of primary cell models from nearly every breast cell type-some the first of their kind- and submit these as critical tools for studying the dynamic cellular interactions within breast tissues and tumors. Together, this body of work delivers a unique perspective of the breast, revealing insights into its cellular, molecular, and biochemical composition.

Distal Femur Cemented Modular Prosthesis Failure Causing a Medical Device-Related Adverse Event: A Report of a Rare Case.

A distal femoral cemented modular prosthesis is a viable option for post-bone tumor and limb salvage procedures. The major reasons for implant failures are the poor quality of implants, mechanical stress, biochemical reactions, and extended period of the implant in vivo use. Rare incidences have been reported of distal femur prosthesis implant malfunctioning in a subject having osteosarcoma. Common adverse events associated with implant failure include surgical site infections, swelling, pain, revision of the surgical procedure, cyst formation, and build-up of metal debris on soft tissues. Our case report summarizes gross malfunctioning of a distal femur cemented modular prosthesis experienced by a 24-year-old post-operated osteosarcoma patient who developed excruciating sudden pain and the inability to bear weight on the right leg, with the sudden onset of these symptoms developing while turning in bed.

A novel perspective of calvarial development: the cranial morphogenesis and differentiation regulated by dura mater.

There are lasting concerns on calvarial development because cranium not only accommodates the growing brain, but also safeguards it from exogenous strikes. In the past decades, most studies attributed the dynamic expansion and remodeling of cranium to the proliferation of osteoprecursors in cranial primordium, and the proliferation of osteoprogenitors at the osteogenic front of cranial suture mesenchyme. Further investigations identified series genes expressed in suture mesenchymal cells as the markers of the progenitors, precursors and postnatal stem cells in cranium. However, similar to many other organs, it is suggested that the reciprocal interactions among different tissues also play essential roles in calvarial development. Actually, there are increasing evidence indicating that dura mater (DM) is indispensable for the calvarial morphogenesis and osteogenesis by secreting multiple growth factors, cytokines and extracellular matrix (ECM). Thus, in this review, we first briefly introduce the development of cranium, suture and DM, and then, comprehensively summarize the latest studies exploring the involvement of ECM in DM and cranium development. Eventually, we discussed the reciprocal interactions between calvarium and DM in calvarial development. Actually, our review provides a novel perspective for cranium development by integrating previous classical researches with a spotlight on the mutual interplay between the developing DM and cranium.

Requirement of Pdgfrα+ cells for calvarial bone repair.

Platelet-derived growth factor receptor α (PDGFRα) is often considered as a general marker of mesenchymal cells and fibroblasts, but also shows expression in a portion of osteoprogenitor cells. Within the skeleton, Pdgfrα+ mesenchymal cells have been identified in bone marrow and periosteum of long bones, where they play a crucial role in participating in fracture repair. A similar examination of Pdgfrα+ cells in calvarial bone healing has not been examined. Here, we utilize Pdgfrα-CreERTM;mT/mG reporter animals to examine the contribution of Pdgfrα+ mesenchymal cells to calvarial bone repair through histology and single-cell RNA sequencing (scRNA-Seq). Results showed that Pdgfrα+ mesenchymal cells are present in several cell clusters by scRNA-Seq, and by histology a dramatic increase in Pdgfrα+ cells populated the defect site at early timepoints to give rise to healed bone tissue overtime. Notably, diphtheria toxin-mediated ablation of Pdgfrα reporter+ cells resulted in significantly impaired calvarial bone healing. Our findings suggest that Pdgfrα-expressing cells within the calvarial niche play a critical role in the process of calvarial bone repair.

Exploring the role of DNMT1 in dental papilla cell fate specification during mouse tooth germ development through integrated single-cell transcriptomics and bulk RNA sequencing.

OBJECTIVES: This study aimed to investigate the regulatory mechanisms governing dental mesenchymal cell commitment during tooth development, focusing on odontoblast differentiation and the role of epigenetic regulation in this process. METHODS: We performed single-cell RNA sequencing (scRNA-seq) of dental cells from embryonic day 14.5 (E14.5) mice to understand the heterogeneity of developing tooth germ cells. Computational analyses including gene regulatory network (GRN) assessment were conducted. We validated our findings using immunohistochemistry (IHC) and in vitro loss-of-function analyses using the DNA methyltransferase 1 (DNMT1) inhibitor Gsk-3484862 in primary dental mesenchymal cells (DMCs) isolated from E14.5 mouse tooth germs. Bulk RNA-seq of Gsk-3484862-treated DMCs was performed to identify potential downstream targets of DNMT1. RESULTS: scRNA-seq analysis revealed diverse cell populations within the tooth germs, including epithelial, mesenchymal, immune, and muscle cells. Using single-cell regulatory network inference and clustering (SCENIC), we identified Dnmt1 as a key regulator of early odontoblast development. IHC analysis showed the ubiquitous expression of DNMT1 in the dental papilla and epithelium. Bulk RNA-seq of cultured DMCs showed that Gsk-3484862 treatment upregulated odontoblast-related genes, whereas genes associated with cell division and the cell cycle were downregulated. Integrated analysis of bulk RNA-seq data with scRNA-seq SCENIC profiles was used to identify the potential Dnmt1 target genes. CONCLUSIONS: Dnmt1 may negatively affect odontoblast commitment and differentiation during tooth development. These findings contribute to a better understanding of the molecular mechanisms underlying tooth development and future development of hard-tissue regenerative therapies.

Emilin2 marks the target region for mesenchymal cell accumulation in bone regeneration.

BACKGROUND: Regeneration of injured tissue is dependent on stem/progenitor cells, which can undergo proliferation and maturation processes to replace the lost cells and extracellular matrix (ECM). Bone has a higher regenerative capacity than other tissues, with abundant mesenchymal progenitor cells in the bone marrow, periosteum, and surrounding muscle. However, the treatment of bone fractures is not always successful; a marked number of clinical case reports have described nonunion or delayed healing for various reasons. Supplementation of exogenous stem cells by stem cell therapy is anticipated to improve treatment outcomes; however, there are several drawbacks including the need for special devices for the expansion of stem cells outside the body, low rate of cell viability in the body after transplantation, and oncological complications. The use of endogenous stem/progenitor cells, instead of exogenous cells, would be a possible solution, but it is unclear how these cells migrate towards the injury site. METHODS: The chemoattractant capacity of the elastin microfibril interface located protein 2 (Emilin2), generated by macrophages, was identified by the migration assay and LC-MS/MS. The functions of Emilin2 in bone regeneration were further studied using Emilin2-/- mice. RESULTS: The results show that in response to bone injury, there was an increase in Emilin2, an ECM protein. Produced by macrophages, Emilin2 exhibited chemoattractant properties towards mesenchymal cells. Emilin2-/- mice underwent delayed bone regeneration, with a decrease in mesenchymal cells after injury. Local administration of recombinant Emilin2 protein enhanced bone regeneration. CONCLUSION: Emilin2 plays a crucial role in bone regeneration by increasing mesenchymal cells. Therefore, Emilin2 can be used for the treatment of bone fracture by recruiting endogenous progenitor cells.

p38 Signaling Mediates Naringin-Induced Osteogenic Differentiation of Porcine Metanephric Mesenchymal Cells.

OBJECTIVE: To explore the potential of metanephric mesenchymal cells (MMCs) for osteogenesis and naringin's ability to enhance this process and its molecular mechanism. METHODS: Porcine MMCs at 70 days of gestation were used as tool cells, cultured in osteogenic induction medium, identified by immunocytochemistry staining. Osteogenic potential of porcine MMCs and naringin's ability to enhance this process was tested by detecting changes in cell viability, alkaline phosphatase (ALP) activity, the expression of runt-related transcription factor 2 (Runx2), osteopontin (OPN) and osteocalcin (OCN), and the formation of mineralized nodules, and the application of the p38 signaling pathway inhibitor SB203580 vitiated the osteogenesis-promoting effect of naringin. RESULTS: Immunocytochemical staining showed that the cells were Vimentin and Six2(+), E-cadherin and CK-18(-). Naringin can activate the p38 signaling pathway to enhance the osteogenesis of porcine MMCs by increasing cell viability, ALP activity, the expressions of Runx2, OPN and OCN, and the formation of mineralized nodules (P<0.05). The application of p38 signaling pathway inhibitor SB203580 vitiated the osteogenesis-promoting effect of naringin, manifested by decreased ALP activity, the expressions of Runx2, OPN and OCN, and the formation of mineralized nodules (P<0.05). CONCLUSION: Naringin, the active ingredient of Chinese herbal medicine Rhizoma Drynariae for nourishing Shen (Kidney) and strengthening bone, enhances the osteogenic differentiation of renal MMCs through the p38 signaling pathway.

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts.

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study using genetic mouse models, we dissected the roles of bone morphogenetic protein (BMP) receptor 1a (Bmpr1a)-mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

Congenital disorders are medical conditions that are present from birth. Although many congenital disorders are rare, they can have a severe impact on the quality of life of those affected. For example, congenital pulmonary airway malformation (CPAM) is a rare congenital disorder that occurs in around 1 out of every 25,000 pregnancies. In CPAM, abnormal, fluid-filled sac-like pockets of tissue, known as cysts, form within the lungs of unborn babies. After birth, these cysts become air-filled and do not behave like normal lung tissue and stop a baby's lungs from working properly. In severe cases, babies with CPAM need surgery immediately after birth. We still do not understand exactly what the underlying causes of CPAM might be. CPAM is not considered to be hereditary ­ that is, it does not appear to be passed down in families ­ nor is it obviously linked to any environmental factors. CPAM is also very difficult to study, because researchers cannot access tissue samples during the critical early stages of the disease. To overcome these difficulties, Luo et al. wanted to find a way to study CPAM in the laboratory. First, they developed a non-human animal 'model' that naturally forms CPAM-like lung cysts, using genetically modified mice where the gene for the signaling molecule Bmpr1a had been deleted in lung cells. Normally, Bmpr1a is part of a set of the molecular instructions, collectively termed BMP signaling, which guide healthy lung development early in life. However, mouse embryos lacking Bmpr1a developed abnormal lung cysts that were similar to those found in CPAM patients, suggesting that problems with BMP signalling might also trigger CPAM in humans. Luo et al. also identified several other genes in the Bmpr1a-deficient mouse lungs that had abnormal patterns of activity. All these genes were known to be controlled by BMP signaling, and to play a role in the development and organisation of lung tissue. This suggests that when these genes are not controlled properly, they could drive formation of CPAM cysts when BMP signaling is compromised. This work is a significant advance in the tools available to study CPAM. Luo et al.'s results also shed new light on the molecular mechanisms underpinning this rare disorder. In the future, Luo et al. hope this knowledge will help us develop better treatments for CPAM, or even help to prevent it altogether.

Molecular Pathways and Animal Models of Tricuspid Atresia and Univentricular Heart.

The process of valve formation is a complex process that involves intricate interplay between various pathways at precise times. Although we have not completely elucidated the molecular pathways that lead to normal valve formation, we have identified a few major players in this process. We are now able to implicate TGF-ß, BMP, and NOTCH as suspects in tricuspid atresia (TA), as well as their downstream targets: NKX2-5, TBX5, NFATC1, GATA4, and SOX9. We know that the TGF-ß and the BMP pathways converge on the SMAD4 molecule, and we believe that this molecule plays a very important role to tie both pathways to TA. Similarly, we look at the NOTCH pathway and identify the HEY2 as a potential link between this pathway and TA. Another transcription factor that has been implicated in TA is NFATC1. While several mouse models exist that include part of the TA abnormality as their phenotype, no true mouse model can be said to represent TA. Bridging this gap will surely shed light on this complex molecular pathway and allow for better understanding of the disease process.

An Optimized Protocol for the Generation of Alveolospheres from Wild-Type Mice.

Organoid models have become an integral part of the research methodology in the lung field. These systems allow for the study of progenitor and stem cell self-renewal, self-organization, and differentiation. Distinct models of lung organoids mimicking various anatomical regions of mature lungs have emerged in parallel to the increased gain of knowledge regarding epithelial stem and progenitor cell populations and the corresponding mesenchymal cells that populate the in vivo niche. In the distal lung, type 2 alveolar epithelial cells (AEC2s) represent a stem cell population that is engaged in regenerative mechanisms in response to various insults. These cells self-renew and give rise to AEC1s that carry out gas exchange. Multiple experimental protocols allowing the generation of alveolar organoids, or alveolospheres, from murine lungs have been described. Among the drawbacks have been the requirement of transgenic mice allowing the isolation of AEC2s with high viability and purity, and the occasional emergence of bronchiolar and bronchioalveolar organoids. Here, we provide a refined gating strategy and an optimized protocol for the generation of alveolospheres from wild-type mice. Our approach not only overcomes the need for transgenic mice to generate such organoids, but also yields a pure culture of alveolospheres that is devoid of bronchiolar and bronchioalveolar organoids. Our protocol contributes to the standardization of this important research tool.

Adipose-derived stromal vascular fraction cells to treat long-term pulmonary sequelae of coronavirus disease 2019: 12-month follow-up.

BACKGROUND AIMS: Long coronavirus disease (COVID) is estimated to occur in up to 20% of patients with coronavirus disease 2019 (COVID-19) infections, with many having persistent pulmonary symptoms. Mesenchymal stromal cells (MSCs) have been shown to have powerful immunomodulatory and anti-fibrotic properties. Autologous adipose-derived (AD) stromal vascular fraction (SVF) contains MSC and other healing cell components and can be obtained by small-volume lipoaspiration and administered on the same day. This study was designed to study the safety of AD SVF infused intravenously to treat the pulmonary symptoms of long COVID. METHODS: Five subjects with persistent cough and dyspnea after hospitalization and subsequent discharge for COVID-19 pneumonia were treated with 40 million intravenous autologous AD SVF cells and followed for 12 months, to include with pulmonary function tests and computed tomography scans of the lung. RESULTS: SVF infusion was safe, with no significant adverse events related to the infusion out to 12 months. Four subjects had improvements in pulmonary symptoms, pulmonary function tests, and computed tomography scans, with some improvement noted as soon as 1 month after SVF treatment. CONCLUSIONS: It is not possible to distinguish between naturally occurring improvement or improvement caused by SVF treatment in this small, uncontrolled study. However, the results support further study of autologous AD SVF as a treatment for long COVID.

Osteogenic differentiation of human mesenchymal stem cells on electroactive substrates.

This study investigates the effect of electroactivity and electrical charge distribution on the biological response of human bone marrow stem cells (hBMSCs) cultured in monolayer on flat poly(vinylidene fluoride), PVDF, substrates. Differences in cell behaviour, including proliferation, expression of multipotency markers CD90, CD105 and CD73, and expression of genes characteristic of different mesenchymal lineages, were observed both during expansion in basal medium before reaching confluence and in confluent cultures in osteogenic induction medium. The crystallisation of PVDF in the electrically neutral α-phase or in the electroactive phase ß, both unpoled and poled, has been found to have an important influence on the biological response. In addition, the presence of a permanent positive or negative surface electrical charge distribution in phase ß substrates has also shown a significant effect on cell behaviour.

Single-cell analysis of mesenchymal cells in permeable neural vasculature reveals novel diverse subpopulations of fibroblasts.

BACKGROUND: In the choroid plexus and pituitary gland, vasculature is known to have a permeable, fenestrated phenotype which allows for the free passage of molecules in contrast to the blood brain barrier observed in the rest of the CNS. The endothelium of these compartments, along with secretory, neural-lineage cells (choroid epithelium and pituitary endocrine cells) have been studied in detail, but less attention has been given to the perivascular mesenchymal cells of these compartments. METHODS: The Hic1CreERT2 Rosa26LSL-TdTomato mouse model was used in conjunction with a PdgfraH2B-EGFP mouse model to examine mesenchymal cells, which can be subdivided into Pdgfra+ fibroblasts and Pdgfra- pericytes within the choroid plexus (CP) and pituitary gland (PG), by histological, immunofluorescence staining and single-cell RNA-sequencing analyses. RESULTS: We found that both CP and PG possess substantial populations of distinct Hic1+ mesenchymal cells, including an abundance of Pdgfra+ fibroblasts. Within the pituitary, we identified distinct subpopulations of Hic1+ fibroblasts in the glandular anterior pituitary and the neurosecretory posterior pituitary. We also identified multiple distinct markers of CP, PG, and the meningeal mesenchymal compartment, including alkaline phosphatase, indole-n-methyltransferase and CD34. CONCLUSIONS: Novel, distinct subpopulations of mesenchymal cells can be found in permeable vascular interfaces, including the CP, PG, and meninges, and make distinct contributions to both organs through the production of structural proteins, enzymes, transporters, and trophic molecules.

High quality repair of osteochondral defects in rats using the extracellular matrix of antler stem cells.

BACKGROUND: Cartilage defects are some of the most common causes of arthritis. Cartilage lesions caused by inflammation, trauma or degenerative disease normally result in osteochondral defects. Previous studies have shown that decellularized extracellular matrix (ECM) derived from autologous, allogenic, or xenogeneic mesenchymal stromal cells (MSCs) can effectively restore osteochondral integrity. AIM: To determine whether the decellularized ECM of antler reserve mesenchymal cells (RMCs), a xenogeneic material from antler stem cells, is superior to the currently available treatments for osteochondral defects. METHODS: We isolated the RMCs from a 60-d-old sika deer antler and cultured them in vitro to 70% confluence; 50 mg/mL L-ascorbic acid was then added to the medium to stimulate ECM deposition. Decellularized sheets of adipocyte-derived MSCs (aMSCs) and antlerogenic periosteal cells (another type of antler stem cells) were used as the controls. Three weeks after ascorbic acid stimulation, the ECM sheets were harvested and applied to the osteochondral defects in rat knee joints. RESULTS: The defects were successfully repaired by applying the ECM-sheets. The highest quality of repair was achieved in the RMC-ECM group both in vitro (including cell attachment and proliferation), and in vivo (including the simultaneous regeneration of well-vascularized subchondral bone and avascular articular hyaline cartilage integrated with surrounding native tissues). Notably, the antler-stem-cell-derived ECM (xenogeneic) performed better than the aMSC-ECM (allogenic), while the ECM of the active antler stem cells was superior to that of the quiescent antler stem cells. CONCLUSION: Decellularized xenogeneic ECM derived from the antler stem cell, particularly the active form (RMC-ECM), can achieve high quality repair/reconstruction of osteochondral defects, suggesting that selection of decellularized ECM for such repair should be focused more on bioactivity rather than kinship.

Mesenchymal cells support the early retention of primary alveolar type 2 cells on acellular mouse lung scaffolds.

Objectives: Tissue engineering approaches via repopulation of acellular biological grafts provide an exciting opportunity to generate lung grafts for transplantation. Alveolar type 2 (AT2) cells are a promising cell source for re-epithelialization. There are however inherent limitations with respect to their survival and growth, thus impeding their usability for tissue engineering applications. This study investigates the use of mesenchymal stromal cells to support primary AT2 cells for recellularization of mouse lung scaffolds. Methods: AT2 cells and bone marrow-derived mesenchymal cells (BMC) were co-delivered to decellularized mouse lung scaffolds. Recellularized lungs were evaluated for cell surface coverage, viability, and differentiation at 1 and 4 days after cell seeding. Recellularization was evaluated via histological analysis and immunofluorescence. Results: Simultaneous delivery of AT2 and BMC into acellular lung scaffolds resulted in enhanced cell surface coverage and reduced AT2 cell apoptosis in the recellularized scaffolds at Day 1 but not Day 4. AT2 cell number decreased after 4 days in both of AT2 only and codelivery groups suggesting limited expansion potential in the scaffold. After retention in the scaffold, AT2 cells differentiated into Aqp5-expressing cells. Conclusions: Our results indicate that BMC support AT2 cell survival during the initial attachment and engraftment phase of recellularization. While our findings suggest only a short-term beneficial effect of BMC, our study demonstrates that AT2 cells can be delivered and retained in acellular lung scaffolds; thus with preconditioning and supporting cells, may be used for re-epithelialization. Selection and characterization of appropriate cell sources for use in recellularization, will be critical for ultimate clinical application.

Mesothelial cells with mesenchymal features enhance peritoneal dissemination by forming a protumorigenic microenvironment.

Malignant ascites accompanied by peritoneal dissemination contain various factors and cell populations as well as cancer cells; however, how the tumor microenvironment is shaped in ascites remains unclear. Single-cell proteomic profiling and a comprehensive proteomic analysis are conducted to comprehensively characterize malignant ascites. Here, we find defects in immune effectors along with immunosuppressive cell accumulation in ascites of patients with gastric cancer (GC) and identify five distinct subpopulations of CD45(-)/EpCAM(-) cells. Mesothelial cells with mesenchymal features in CD45(-)/EpCAM(-) cells are the predominant source of chemokines involved in immunosuppressive myeloid cell (IMC) recruitment. Moreover, mesothelial-mesenchymal transition (MMT)-induced mesothelial cells strongly express extracellular matrix (ECM)-related genes, including tenascin-C (TNC), enhancing metastatic colonization. These findings highlight the definite roles of the mesenchymal cell population in the development of a protumorigenic microenvironment to promote peritoneal dissemination.

The Tumor Immune Microenvironment plays a Key Role in Driving the Progression of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an epithelial cancer distinguished by bile duct cell differentiation and is also a fibroproliferative tumor. It is characterized by a dense mesenchyme and a complex tumor immune microenvironment (TME). The TME comprises both cellular and non-cellular components. The celluar component includes CCA cells, immune cells and mesenchymal cells represented by the cancer-associated fibroblasts (CAFs), while the non-cellular component is represented by mesenchymal elements such as the extracellular matrix (ECM). Recent studies have demonstrated the important role of the TME in the development, progression, and treatment resistance of CCA. These cell-associated prognostic markers as well as intercellular connections, may serve as potential therapeutic targets and could inspire new treatment approaches for CCA in the future. This paper aims to summarize the current understanding of CCA's immune microenvironment, focusing on immune cells, mesenchymal cells, ECM, intercellular interactions, and metabolism within the microenvironment.