Migratory chondroprogenitors retain superior intrinsic chondrogenic potential for regenerative cartilage repair as compared to human fibronectin derived chondroprogenitors

Cell-based therapy for articular hyaline cartilage regeneration predominantly involves the use of mesenchymal stem cells and chondrocytes. However, the regenerated repair tissue is suboptimal due to the formation of mixed hyaline and fibrocartilage, resulting in inferior long-term functional outcomes. Current preclinical research points towards the potential use of cartilage-derived chondroprogenitors as a viable option for cartilage healing. Fibronectin adhesion assay-derived chondroprogenitors (FAA-CP) and migratory chondroprogenitors (MCP) exhibit features suitable for neocartilage formation but are isolated using distinct protocols. In order to assess superiority between the two cell groups, this study was the first attempt to compare human FAA-CPs with MCPs in normoxic and hypoxic culture conditions, investigating their growth characteristics, surface marker profile and trilineage potency. Their chondrogenic potential was assessed using mRNA expression for markers of chondrogenesis and hypertrophy, glycosaminoglycan content (GAG), and histological staining. MCPs displayed lower levels of hypertrophy markers (RUNX2 and COL1A1), with normoxia-MCP exhibiting significantly higher levels of chondrogenic markers (Aggrecan and COL2A1/COL1A1 ratio), thus showing superior potential towards cartilage repair. Upon chondrogenic induction, normoxia-MCPs also showed significantly higher levels of GAG/DNA with stronger staining. Focused research using MCPs is required as they can be suitable contenders for the generation of hyaline-like repair tissue.

Comparative lipid profiling of murine and human atherosclerotic plaques using high-resolution MALDI MSI

The distribution of atherosclerotic lesions in the aorta and its branches of ApoE knockout (ApoE−/−) mice is like that of patients with atherosclerosis. By using high-resolution MALDI mass spectrometry imaging (MSI), we aimed at characterizing universally applicable physiological biomarkers by comparing the murine lipid marker profile with that of human atherosclerotic arteries. Therefore, the aorta or carotid artery of male ApoE−/− mice at different ages, human arteries with documented atherosclerotic changes originated from amputated limbs, and corresponding controls were analysed. Obtained data were subjected to multivariate statistical analysis to identify potential biomarkers. Thirty-one m/z values corresponding to individual lipid species of cholesterol esters, lysophosphatidylcholines, lysophosphatidylethanolamines, and cholesterol derivatives were found to be specific in aortic atherosclerotic plaques of old ApoE−/− mice. The lipid composition at related vessel positions of young ApoE−/− mice was more comparable with wild-type mice. Twenty-six m/z values of the murine lipid markers were found in human atherosclerotic peripheral arteries but also control vessels and showed a more patient-dependent diverse distribution. Extensive data analysis without marker preselection based on mouse data revealed lysophosphatidylcholine and glucosylated cholesterol species, the latter not being detected in the murine atherosclerotic tissue, as specific potential novel human atherosclerotic vessel markers. Despite the heterogeneous lipid profile of atherosclerotic peripheral arteries derived from human patients, we identified lipids specifically colocalized to atherosclerotic human tissue and plaques in ApoE−/− mice. These data highlight species-dependent differences in lipid profiles between peripheral artery disease and aortic atherosclerosis.

Defining Cardiac Cell Populations and Relative Cellular Composition of the Early Fetal Human Heart

The human heart is primarily composed of cardiomyocytes, fibroblasts, endothelial and smooth muscle cells. Reliable identification of fetal cardiac cell types using protein markers is important for understanding cardiac development and delineating the cellular composition of the human heart during early development, which remains largely unknown. The aim of this study was to use immunohistochemistry (IHC), flow cytometry and RT-PCR analyses to investigate the expression and specificity of commonly used cardiac cell markers in the early human fetal heart (8-12 post-conception weeks). The expression of previously reported protein markers for the detection of cardiomyocytes (Myosin Heavy Chain (MHC) and Troponin I (cTnI)), fibroblasts (DDR2, Thy1, Vimentin), endothelial cells (CD31) and smooth muscle cells (α-SMA) were assessed. Flow cytometry revealed two distinct populations of cTnI expressing cells based on fluorescence intensity: cTnI High and cTnI Low . MHC positive cardiomyocytes were cTnI High , whereas MHC negative non-myocyte cells were cTnI Low . cTnI expression in non-myocytes was further confirmed by IHC and RT-PCR analyses, suggesting troponins are not cardiomyocyte-specific and may play distinct roles in non-muscle cells during early development. Vimentin was confirmed to be enriched in cultured fibroblast populations and flow cytometry revealed Vim High and Vim Low cell populations in the fetal heart. MHC positive cardiomyocytes were Vim Low whilst CD31 positive endothelial cells were Vim High . Based on the markers investigated, we estimate fetal human cardiomyocyte populations comprise 75-80% of total cardiac cells and exhibit the following marker profile: α-MHC + /cTnI High /Vim Low . For the non-cardiomyocyte population, we estimate they comprise 20-25% of total cardiac cells and exhibit the following marker profile: α-MHC - /cTnI Low /Vim High . Our study suggests the marker profiles and proportions of fetal cardiac populations are distinct from that of the adult heart.

AB0022 AUTOREACTIVE B CELLS IN RHEUMATOID ARTHRITIS DISPLAY AN ACTIVATED PHENOTYPE OF RECENT ANTIGEN EXPOSURE

Background:Rheumatoid arthritis, in particular ACPA+ RA, is characterized by frequent disease flares and poor chances to achieve DMARD-free sustained remission. Recently, we have shown that ACPA-expressing memory B cells (MBC) remain in a persistently activated state throughout disease, even in patients in DMARD-induced clinical remission.(1) The reasons why the ACPA B cell response is continuously activated are unknown, as well as why the response does not revert to a resting, ‘quiescent’ state. We hypothesized that continuous antigen exposure in germinal centres drives ACPA B cell activation, leading to a ‘recent germinal centre emigrant’ phenotype of these cells in the circulation.Objectives:To understand whether the activated phenotype of ACPA-expressing B cells could be induced by recent antigen exposure, to thereby discern the processes of immune activation that remain active in patients even in clinical remission and to argue whether these processes could be targets for therapeutic intervention.Methods:ACPA-expressing B cells were identified in peripheral blood of RA patients by flow cytometry during different stages of disease and characterized by a panel of activation- and germinal centre related markers (CD80, CD86, CD32, CD95, Ki-67). In addition, three healthy donors received a TT booster vaccination. TT-specific MBC were identified in blood at different timepoints (before vaccination and up to 22 weeks after vaccination) and analysed phenotypically over time.Results:The majority of ACPA-expressing B cells strongly expressed CD95 and the co-stimulatory marker CD80. A part was also positive for the proliferation marker Ki-67 (on average 30%), and most cells downregulated the inhibitory marker CD32. TT-specific MBC adopted a comparable phenotype after booster vaccination, but most markers returned to the pre-vaccination expression level gradually over time. These effects were antigen-dependent because the phenotype of TT-negative B cells remained unchanged. The phenotypic composition of the proliferating ACPA-positive B cell pool most closely corresponded to a stimulation history of 1-2 weeks after antigen exposure. Notably, none of the Ki-67 negative ACPA-specific MBC showed phenotypic quiescence, indicating either a short life-time (in circulation) after antigen encounter or persistent additional factors of activation.Figure 1.Ki-67 expression on ACPA-specific MBC in RA (A) and on TT-specific MBC in 3 healthy donors before and after booster vaccination (B).Conclusion:ACPA-expressing MBC phenotypically resemble TT-specific MBC after recent (1-2 weeks) booster vaccination, reflecting the phenotype of recent germinal centre emigrants, and remain activated, whereas TT-specific MBC lose this marker profile over time. These observations suggest that ACPA-expressing MBC either home to tissue or survive shortly in the circulation, or that additional factors drive or program these cells to persistent activation. Transcriptomic profiling and analysis of the homing marker profile may help to answer these questions. Furthermore, it will be important to understand the association of persistent activation of ACPA-expressing B cells in clinical remission and the risk for disease flares upon treatment discontinuation.References:[1]Kristyanto H, Blomberg NJ, Slot LM, van der Voort EIH, Kerkman PF, Bakker A, et al. Persistently activated, proliferative memory autoreactive B cells promote inflammation in rheumatoid arthritis. Sci Transl Med. 2020;12(570).Disclosure of Interests:Nienke Blomberg: None declared, Hendy Kristyanto: None declared, Thomas Huizinga Grant/research support from: Gilead, Rene Toes: None declared, Hans Ulrich Scherer Grant/research support from: Pfizer, Lilly, Sanofi, BMS

Monocyte Subset Recruitment Marker Profile Is Inversely Associated With Blood ApoA1 Levels

Dyslipidemia promotes development of the atherosclerotic plaques that characterise cardiovascular disease. Plaque progression requires the influx of monocytes into the vessel wall, but whether dyslipidemia is associated with an increased potential of monocytes to extravasate is largely unknown. Here (using flow cytometry) we examined recruitment marker expression on monocytes from generally healthy individuals who differed in lipid profile. Comparisons were made between monocyte subsets, participants and relative to participants’ lipid levels. Monocyte subsets differed significantly in their expression of recruitment markers, with highest expression being on either the classical or non-classical subsets. However, these inter-subset differences were largely overshadowed by considerable inter-participant differences with some participants having higher levels of recruitment markers on all three monocyte subsets. Furthermore, when the expression of one recruitment marker was high, so too was that of most of the other markers, with substantial correlations evident between the markers. The inter-participant differences were explained by lipid levels. Most notably, there was a significant inverse correlation for most markers with ApoA1 levels. Our results indicate that dyslipidemia, in particular low levels of ApoA1, is associated with an increased potential of all monocyte subsets to extravasate, and to do so using a wider repertoire of recruitment markers than currently appreciated.

Monolayer platform to generate and purify human primordial germ cells in vitro provides new insights into germline specification

Generating primordial germ cells (PGCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but currently entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days. We used our simplified differentiation platform and single-cell RNA-sequencing to uncover new insights into PGC specification. Transient WNT activation for 12 hours followed by WNT inhibition specified PGCs; by contrast, sustained WNT instead induced primitive streak. Thus, somatic (primitive streak) and PGCs are related—yet distinct—lineages segregated by temporally-dynamic signaling. Pluripotency factors including NANOG are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCs can be easily purified by virtue of their CXCR4+PDGFRA−GARP− surface-marker profile and single-cell RNA-sequencing reveals that they harbor strong transcriptional similarities with fetal PGCs.

Lysophospholipids as Predictive Markers of ST-Elevation Myocardial Infarction (STEMI) and Non-ST-Elevation Myocardial Infarction (NSTEMI)

The present study explored patterns of circulating metabolites and proteins that can predict future risk for ST-elevation myocardial infarction (STEMI) and non-ST-elevation myocardial infarction (NSTEMI). We conducted a prospective nested case-control study in northern Sweden in individuals who developed STEMI (N = 50) and NSTEMI (N = 50) within 5 years and individually matched controls (N = 100). Fasted plasma samples were subjected to multiplatform mass spectrometry-based metabolomics and multiplex protein analyses. Multivariate analyses were used to elucidate infarction-specific metabolite and protein risk profiles associated with future incident STEMI and NSTEMI. We found that altered lysophosphatidylcholine (LPC) to lysophosphatidylethanolamine (LPE) ratio predicted STEMI and NSTEMI events in different ways. In STEMI, lysophospholipids (mainly LPEs) were lower, whereas in NSTEMI, lysophospholipids (mainly LPEs) were higher. We found a similar response for all detected lysophospholipids but significant alterations only for those containing linoleic acid (C18:2, p < 0.05). Patients with STEMI had higher secretoglobin family 3A member 2 and tartrate-resistant acid phosphate type 5 and lower platelet-derived growth factor subunit A, which are proteins associated with atherosclerosis severity and plaque development mediated via altered phospholipid metabolism. In contrast, patients with NSTEMI had higher levels of proteins associated with inflammation and macrophage activation, including interleukin 6, C-reactive protein, chemerin, and cathepsin X and D. The STEMI risk marker profile includes factors closely related to the development of unstable plaque, including a higher LPC:LPE ratio, whereas NSTEMI is characterized by a lower LPC:LPE ratio and increased inflammation.