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To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90) + HLA-DRA hi sublining fibroblasts, IL1B + pro-inflammatory monocytes, ITGAX + TBX21 + autoimmune-associated B cells and PDCD1 + peripheral helper T (T PH ) cells and follicular helper T (T FH ) cells. We defined distinct subsets of CD8 + T cells characterized by GZMK + , GZMB + , and GNLY + phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1 + HLA-DRA hi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis. Defining cell types and their activation status in rheumatoid arthritis (RA) is critical to understanding this disease. Raychaudhuri and colleagues leverage several single-cell -omics approaches to define the cellular processes and pathways in the human RA joint.

Group 2 innate lymphoid cells (ILC2s) regulate inflammation and immunity in mammalian tissues 1 , 2 . Although ILC2s are found in cancers of these tissues 3 , their roles in cancer immunity and immunotherapy are unclear. Here we show that ILC2s infiltrate pancreatic ductal adenocarcinomas (PDACs) to activate tissue-specific tumour immunity. Interleukin-33 (IL33) activates tumour ILC2s (TILC2s) and CD8 + T cells in orthotopic pancreatic tumours but not heterotopic skin tumours in mice to restrict pancreas-specific tumour growth. Resting and activated TILC2s express the inhibitory checkpoint receptor PD-1. Antibody-mediated PD-1 blockade relieves ILC2 cell-intrinsic PD-1 inhibition to expand TILC2s, augment anti-tumour immunity, and enhance tumour control, identifying activated TILC2s as targets of anti-PD-1 immunotherapy. Finally, both PD-1 + TILC2s and PD-1 + T cells are present in most human PDACs. Our results identify ILC2s as anti-cancer immune cells for PDAC immunotherapy. More broadly, ILC2s emerge as tissue-specific enhancers of cancer immunity that amplify the efficacy of anti-PD-1 immunotherapy. As ILC2s and T cells co-exist in human cancers and share stimulatory and inhibitory pathways, immunotherapeutic strategies to collectively target anti-cancer ILC2s and T cells may be broadly applicable. Tumour-infiltrating group 2 innate lymphoid cells prime CD8 + T cells and amplify the anti-tumour effects of PD-1 blockade in pancreatic ductal adenocarcinoma.

The synovium is a mesenchymal tissue composed mainly of fibroblasts, with a lining and sublining that surround the joints. In rheumatoid arthritis the synovial tissue undergoes marked hyperplasia, becomes inflamed and invasive, and destroys the joint 1 , 2 . It has recently been shown that a subset of fibroblasts in the sublining undergoes a major expansion in rheumatoid arthritis that is linked to disease activity 3 – 5 ; however, the molecular mechanism by which these fibroblasts differentiate and expand is unknown. Here we identify a critical role for NOTCH3 signalling in the differentiation of perivascular and sublining fibroblasts that express CD90 (encoded by THY1 ). Using single-cell RNA sequencing and synovial tissue organoids, we found that NOTCH3 signalling drives both transcriptional and spatial gradients—emanating from vascular endothelial cells outwards—in fibroblasts. In active rheumatoid arthritis, NOTCH3 and Notch target genes are markedly upregulated in synovial fibroblasts. In mice, the genetic deletion of Notch3 or the blockade of NOTCH3 signalling attenuates inflammation and prevents joint damage in inflammatory arthritis. Our results indicate that synovial fibroblasts exhibit a positional identity that is regulated by endothelium-derived Notch signalling, and that this stromal crosstalk pathway underlies inflammation and pathology in inflammatory arthritis. NOTCH3 signalling is shown to be the underlying driver of the differentiation and expansion of a subset of synovial fibroblasts implicated in the pathogenesis of rheumatoid arthritis.

Cell membrane-coated nanoparticles are emerging as a new type of promising nanomaterials for immune evasion and targeted delivery. An underlying premise is that the unique biological functions of natural cell membranes can be conferred on the inherent physiochemical properties of nanoparticles by coating them with a cell membrane. However, the extent to which the membrane protein properties are preserved on these nanoparticles and the consequent bio—nano interactions are largely unexplored. Here, we synthesized two mesenchymal stem cell (MSC) membrane-coated silica nanoparticles (MCSNs), which have similar sizes but distinctly different stiffness values (MPa and GPa). Unexpectedly, a much lower macrophage uptake, but much higher cancer cell uptake, was found with the soft MCSNs compared with the stiff MCSNs. Intriguingly, we discovered that the soft MCSNs enabled the forming of a more protein-rich membrane coating and that coating had a high content of the MSC chemokine CXCR4 and MSC surface marker CD90. This led to the soft MCSNs enhancing cancer cell uptake mediated by the CD90/integrin receptor-mediated pathway and CXCR4/SDF-1 pathways. These findings provide a major step forward in our fundamental understanding of how the combination of nanoparticle elasticity and membrane coating may be used to facilitate bio—nano interactions and pave the way forward in the development of more effective cancer nanomedicines.

Multipotent mesenchymal stromal cells (MSCs) can be considered an accessible therapeutic tool for regenerative medicine. Here, we compared the growth kinetics, immunophenotypic and immunomodulatory properties, gene expression and secretome profile of MSCs derived from human adult bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and Wharton’s jelly (WJ-MSCs) cultured in clinically-relevant conditions, with the focus on the neuroregenerative potential. All the cell types were positive for CD10/CD29/CD44/CD73/CD90/CD105/HLA-ABC and negative for CD14/CD45/CD235a/CD271/HLA-DR/VEGFR2 markers, but they differed in the expression of CD34/CD133/CD146/SSEA-4/MSCA-1/CD271/HLA-DR markers. BM-MSCs displayed the highest immunomodulatory activity compared to AT- and WJ-MSCs. On the other hand, BM-MSCs secreted the lower content and had the lower gene expression of neurotrophic growth factors compared to other cell lines, which may be caused by the higher sensitivity of BM-MSCs to nutrient limitations. Despite the differences in growth factor secretion, the MSC secretome derived from all cell sources had a pronounced neurotrophic potential to stimulate the neurite outgrowth of DRG-neurons and reduce the cell death of neural stem/progenitor cells after H 2 O 2 treatment. Overall, our study provides important information for the transfer of basic MSC research towards clinical-grade manufacturing and therapeutic applications.

The antileukemic effect of inhibiting bromodomain and extra-terminal domain-containing (BET-containing) proteins (BETPs) such as BRD4 has largely been largely attributed to transcriptional downregulation of cellular anabolic and antiapoptotic processes, but its effect on the bone marrow microenvironment, a sanctuary favoring the persistence of leukemic stem/progenitor cells, is unexplored. Sustained degradation of BETP with the small-molecule BET proteolysistargeting chimera (PROTAC) ARV-825 resulted in a marked downregulation of surface CXCR4 and CD44, key proteins in leukemia-microenvironment interactions, in acute myeloid leukemia (AML) cells. Abrogation of surface CXCR4 expression impaired SDF-1[alpha]-directed migration and was mediated through transcriptional downregulation of PIM1 kinase, which in turn phosphorylates CXCR4 and facilitates its surface localization. Downregulation of CD44, including isoforms CD44v8- 10 impaired cystine uptake, lowered intracellular reduced glutathione, and increased oxidative stress. More important, BETP degradation markedly decreased the [CD34.sup.+][CD38.sup.-][CD90.sup.-][CD45RA.sup.+] leukemic stem cell population and, alone or in combination with cytarabine, prolonged survival in a mouse model of human leukemia that included AML patient-derived xenografts (AML-PDX). Gene expression profiling and single-cell proteomics confirmed a downregulation of the gene signatures associated with \"sternness\" in AML and Wnt/[beta]-catenin and Myc pathways. Hence, BETP degradation by ARV-825 simultaneously targets cell-intrinsic signaling, stromal interactions, and metabolism in AML.

BackgroundWe have previously reported that CD14+ dendritic-shaped cells show a dendritic morphology under the electron microscopy and engage in a pseudoemperipolesis phenomenon with lymphocytes. CD14+ dendritic-shaped cells express CD90 in the perivascular areas of RA synovial tissues.ObjectivesTo examine the differentiation potential of CD14+CD90+ cells into dendritic cells and their relationship to chronic inflammation in RA.MethodsRA synovial tissues harvested at the time of joint surgeries were used after obtaining informed consent. The harvested tissues were cultured in vitro, and CD14+CD90+ cells were sorted using flow cytometry. Subsequently, the cells were divided into CD14+CD90+ and non-CD14+CD90+ cell groups and cultured in dendritic cell differentiation medium. The populations of cells expressing CD83 and HLA-DR were examined by flow cytometry to determine their differentiation potential into dendritic cells.ResultsAt day 7 after dendritic cell differentiation culture, the group of CD14+CD90+ cells had a higher percentage of cells expressing CD83 and HLA-DR than the group of non-CD14+CD90+ cells.ConclusionCD14+ dendritic-shaped cells detected in RA synovial tissues are considered to be derived from CD14+CD90+ cells in the perivascular areas, which may be involved in RA inflammation as dendritic cells.References[1]Ochi T, Sawai T, Murakami K, Kamataki A, Uzuki M, Tomita T, et al. Nurse-like cells in rheumatoid arthritis: Formation of survival niches cooperating between the cell types. Mod Rheum 2018; 29: 1-5.[2]Kurose R, Satoh T, Kurose A, Satoh Y, Ishibashi Y, Wakai Y, et al. Association of CD90 expression by CD14+ dendritic-shaped cells in rheumatoid arthritis synovial tissue with chronic inflammation. ACR Open Rheumatology 2022; 0: 1-10.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva‐derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK‐8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT‐qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT‐qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90‐positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.

Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that, in human, the first lineage restriction events occur within the CD19 − CD34 + CD38 − CD45RA − CD49f + CD90 + (49f + ) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution, we observe a continuous but polarised organisation of the 49f + compartment, where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9A hi CD34 lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics, whereas CLEC9A lo CD34 hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state, distinct from lymphoid-primed multipotent progenitors, representing the earliest entry point into lymphoid commitment. Human blood cells all develop from haematopoietic stem cells (HSCs), classically thought to be multipotent. Here the authors show, using single-cell RNA-seq and functional assays, that loss of erythroid potential and commitment to the myelo-lymphoid lineage occurs within the purest HSC compartment to date.

Easy isolation, lack of ethical issues, high proliferation, multi-lineage differentiation potential and immunomodulatory properties of umbilical cord (UC)-derived mesenchymal stem cells (MSCs) make them a valuable tool in stem cell research. Recently, Wharton’s jelly (WJ) was proven as the best MSC source among various compartments of UC. However, it is still unclear whether or not Wharton’s jelly-derived MSCs (WJMSCs) from different parts of the whole cord exhibit the same characteristics. There may be varied MSCs present in different parts of WJ throughout the length of the UC. For this purpose, using an explant attachment method, WJMSCs were isolated from three different parts of the UC, mainly present towards the placenta (mother part), the center of the whole cord (central part) and the part attached to the fetus (baby part). WJMSCs from all three parts were maintained in normal growth conditions (10% ADMEM) and analyzed for mesenchymal markers, pluripotent genes, proliferation rate and tri-lineage differentiation potential. All WJMSCs were highly proliferative, positively expressed CD90, CD105, CD73 and vimentin, while not expressing CD34, CD45, CD14, CD19 or HLA-DR, differentiated into adipocytes, osteocytes and chondrocytes and expressed pluripotency markers OCT-4, SOX-2 and NANOG at gene and protein levels. Furthermore, MSCs derived from all the parts were shown to have potency towards hepatocyte-like cell differentiation. Human bone marrow-derived MSCs were used as a positive control. Finally, we conclude that WJMSCs derived from all the parts are valuable sources and can be efficiently used in various fields of regenerative medicine.