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57. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Prof. Dr. med.

Vorsitz

Prof. Dr.

PD Dr. Dr.

PD Dr.

The epigenetic landscape of activating histone modifications during erythropoiesis is dependent on the ABO blood group and accounts for differences in the erythroid potential in carriers of blood group B versus A and O

Immunogenetics and Basic Immunology

Die epigenetische Lokalisation der aktivierenden Histonmodifikationen während der Erythropoese ist abhängig von der ABO-Blutgruppe und erklärt die Unterschiede im erythroiden Potenzial von Trägern der Blutgruppe B im Vergleich zu A und O

The ABO blood group (BG) system is the most important factor in clinical transfusion and transplantation medicine and its antigens are involved in the development of various human diseases. Previously, we demonstrated that the ABO BG directly affects red blood cell (RBC) homeostasis through BG-dependent modulation of erythropoiesis at the microRNA level and corresponding transcription factors (TFs). Furthermore, it was suggested that ABO transcription may be regulated epigenetically. Since, histone modifications play a crucial role in the regulation of erythropoiesis we investigated the regulatory role of histone modifications for the ABO BG phenotype.Hematopoietic stem cells carrying different ABO BG phenotype were differentiated into erythroid precursor cells for 5 days. Chromatin immunoprecipitation coupled with high-throughput sequencing analyses using antibodies against activating histone modifications H3K9ac, H3K27ac, and H3K4me3 were performed in triplicates.On day five of erythroid differentiation, the number of blood group specific H3K9ac modifications in cells carrying BG B were around 10x and 5x higher compared to BG A and O, respectively. In contrast, the number of H3K4me3 modifications was increased in cells carrying BG A and O compared to BG B. Furthermore, H3K27ac modifications dominated in cells of BG O. Whereas, in cells carrying BG B an increased number of H3K9ac and H3K4me3 peaks were located in the gene body (particular in introns), in cells carrying BG A these modifications were predominantly located in the promotor-TSS (transcription start site) region (&lt;1kb). H3K27ac modifications were less present independent of the BG phenotype, whereas in cells carrying BG B and O a higher number of peaks were found in intergenic regions. In cells of BG A H3K27ac modifications were more present in promotor-TSS regions.Our data suggest for the first time that histone modifications are associated with the ABO BG resulting in differences in the regions that are accessible for the respective transcription factors. This might be an explanation for the blood group-dependent differences in the regulation of erythropoiesis. Further exploration of the epigenetic regulation of the ABO BG dependent modulation of erythropoiesis holds great potential for shedding light on various diseases, including myocardial infarction, thrombosis, and infection, where associations between incidence and the ABO blood group have been reported.We have no conflict of interest.

Abstract lecture

Prof.

Conditioned medium from Adipose Stromal Cells reduces cisplatin-induced proximal tubular epithelial cell apoptosis by downregulation of miR-181a-5p

Regenerative Cell Therapy

Konditioniertes Medium von Fettgewebsstromazellen reduziert die Cisplatin-induzierte Apoptose proximaler tubulärer Epithelzellen durch Herunterregulierung von miR-181a-5p

Cisplatin is a widely used effective cancer chemotherapeutic drug. However, nephrotoxicity is one of its limiting side effects. Cisplatin nephrotoxicity, especially apoptosis of proximal epithelial cells, appears associated with aberrant expression of microRNAs (miRNAs). By trophic factors, adipose stromal cells (ASC) exert a protective effect inhibiting apoptosis and promoting tissue regeneration. We hypothesize that this protective effect acts by modulating the miRNA expression in injured renal cells by miRNA-mediated post-transcriptional gene regulation.To mimic in vitro injury and ASC-mediated protection, proximal tubule epithelial cells (PTECs) were treated with cisplatin for 1h and then treated for a further 23h with cisplatin plus ASC-derived conditioned medium (CM-ASC). Viability, migratory capacity in a scratch wound healing assay, apoptosis and p53 expression were assessed. Further, mRNAs prepared from PTECs treated with/without cisplatin and with/without ASC-CM were assessed by small-RNASeq. Candidate miRNAs were functionally validated in apoptosis assays.Cisplatin-treated PTECs reduced viability, migratory capacity and induced apoptosis in line with increased p53 expression. ASC-CM ameliorated this cisplatin toxicity; especially migratory activity and apoptosis with p53 levels back to physiological levels. Small-RNAseq analysis identified differentially expressed miRNAs in all groups, particularly miR-181a-5p, known to play a role in apoptosis. Functional testing revealed that inhibition of miR-181a-5p led to a reduction in apoptosis accentuated by ASC-CM. In contrast, a miR-181a mimic did not affect cellular apoptosis.Our results demonstrate that ASC-CM per se exerts a protective effect reducing cisplatin cytotoxicity in renal proximal tubule cells. ASC-CM counteracted the change in expression of microRNA induced by cisplatin and identified miR-181a as a possible therapeutic target to prevent cisplatin-induced cytotoxicity.This project has received funding from the European Union"s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813839.

Priming of MSC with inflammatory and damage related factors: a model to study changes of gene and protein patterns

Priming von MSC mit inflammatorischen und schädigungsbezogenen Faktoren: ein Modell zur Untersuchung von Veränderungen der Gen- und Proteinmuster

Mesenchymal stromal cells (MSCs) are a major cell source for regenerative medicine in various therapeutic approaches. Recent studies show that priming of MSCs can improve their regenerative potential. We primed MSCs with a mix of inflammatory-associated molecules (IC) and a mix of damage-associated proteins (DC) to investigate how each priming affects MSC"s transcriptome and protein expression. Moreover, we tried to identify which single factor of the IC is the main mediator of the IC effects.MSCs were incubated with IC, consisting of IL-1β, TNF-α, IL-6, CXCL8, C3a, C5a and DC, consisting of HMGB1 and histones for 6 hours and 24 hours. Differently expressed gene analysis of these datasets in comparison with an unstimulated control for each incubation period were performed. Proteins of 20 selected immunomodulatory genes upregulated (fold change ≥ 3, false discovery rate ≤ 0.01) by IC were analysed by measurement of supernatant concentrations with magnetic-bead-based multiplex assays. Pathway analysis of differently expressed genes was conducted. Main mediators of the IC were identified in silico and via incubating MSCs with the individual IC components and measurement of certain supernatant protein concentrations.IC and DC priming resulted in significant transcriptomic changes: After 6 and 24 hours 447 genes were upregulated by IC, 77 by DC and 44 by IC and DC. DC and IC induced different patterns with stronger effects by IC in terms of number of affected genes and the magnitude of fold-change. In contrast to DC, IC increased concentration of 13 of 20 selected proteins (CCL7, CCL20, CXCL1, CXCL5, CXCL6, CXCL8, MMP3, IL-6, IL-27, CSF2, CSF3, LIF). Upstream regulator analysis revealed IL-1β and TNF-α as main mediators of IC effects. The canonical pathways affected by IC with highest Z-scores (QIAGEN IPA core analysis) were IL-17A signaling in fibroblasts, IL-17 signaling and IL-6 signaling. DC only affected TREM1-Signaling after both 6 and 24 hours.Priming of MSCs with inflammatory related molecules affects the transcriptome and protein expression. All upregulated proteins analysed in the supernatant of IC primed MSCs are relevant for immune response and have autocrine and paracrine effects. Therefore, we conclude that priming of MSCs with IC might improve their regenerative potential in inflammatory diseases. Future research should focus on reduction of IC components to ideally a single priming agent to optimize MSCs for therapy.The authors declare no commercial, proprietary or financial interest.SE, MW, MK, MR and HS work for the University Hospital Ulm. AG works for the Ulm University. This work is supported by Deutsche Forschungsgemeinschaft grant CRC1149 &quot;Danger Response, Disturbance Factors and Regenerative Potential after Acute Trauma&quot;, project number 251293561. The materials presented and views expressed here are the responsibility of the authors only.

Prof. Dr. rer. nat.

Clonal Immortalized Mesenchymal Stromal Cells: An ideal cell source for a reproducible and standardized production of therapeutic extracellular vesicles

Clonal Immortalized Mesenchymal Stromal Cells: an ideal cell source for a reproducible and standardized production of therapeutic extracellular vesicles

Within the last decade, we and others have successfully demonstrated the therapeutic efficacy of extracellular vesicles (EVs) prepared from the conditioned media of primary mesenchymal stromal cells (MSCs). Among others, our MSC-EV preparations suppressed disease symptoms of a treatment-resistant graft-versus-host disease (GvHD) patient and improved the outcomes in ischemic stroke and hypoxia-induced neonatal encephalopathy (HIE) animals notably.Nevertheless, the considerable variability among donors and the limited lifespan of primary MSCs in vitro present substantial obstacles in translating primary MSC products successfully into the market.To tackle this challenge, we have devised strategies addressing standardization, reproducibility, and scalability in EV production. Specifically, we have developed methods to immortalize primary MSCs and to successfully establish monoclonal MSC lines. Cells of resulting clonal immortalized MSC lines (ciMSCs) maintain their authentic and healthy bona fide MSC characteristics and still allow manufacturing of therapeutically active EV products.Up to now, we have confirmed in various in vivo and in vitro assays that ciMSC-EVs conserved their function. Among others like their counterparts obtained from primary MSCs, they can suppress T cell activation in a multi-donor mixed lymphocyte reaction assay in a reproducible and standardized manner. Additionally, these ciMSC-EVs exhibit the ability to alleviate symptoms in ischemic stroke and HIE models, mirroring the efficacy of primary MSC-EVs.Based on these findings, we conclude that ciMSC-EVs harbor therapeutic potentials, and ciMSCs represent an ideal cell source for the standardized, reproducible, and scalable manufacturing of EV-based therapeutics.None

Effect of MSCA1 expression on characteristics and functionality of mesenchymal stromal cells

Einfluss der MSCA1-Expression auf Eigenschaften und Funktionalität mesenchymaler Stromazellen

The therapeutic potential of mesenchymal stromal cells (MSC) has been shown in multiple diseases. However, the heterogeneity of MSC and thus the existence of MSC subpopulations may influence their potency. Among other markers, mesenchymal stem cell antigen-1 (MSCA1) can be used to distinguish subpopulations. Since this marker is associated with the regulation of different cellular programs, the effect of MSCA1 expression levels on MSC characteristics and functionality was investigated.MSC subpopulations were sorted by FACS according to their MSCA1 expression. Applied sort criteria were 5-10% of MSC with highest or lowest expression (MSCA1 high or low). Control groups included the overall MSC population, either exposed to the FACS procedure (Sort Ctrl) or untreated (Ctrl). The MSC were expanded for two passages. Basic expansion parameters, cell viability, and the differentiation potential were analyzed. The expression of identity (CD73, CD90, CD105), purity (CD14, CD34, CD45, MHC II), and additional markers (e.g., MSCA1, CD146) was determined. Functional analyses included secretion of bioactive molecules (e.g., ACTH, Osteopontin (OPN), Tumor necrosis factor-inducible gene 6 (TSG-6)) and inhibition of T cell proliferation.No differences were obtained between the groups regarding basic MSC characteristics (viability, potential for ex vivo expansion, differentiation potential, expression of identity and purity markers). A stable expression of MSCA1 could be shown which remained significantly higher for MSCA1 high compared to MSCA1 low for two passages. Moreover, the expression of CD146 was significantly increased for MSCA1 high, while the expression of other markers was unaffected. An altered secretome of MSC subpopulations was indicated by a significantly higher secretion of OPN and ACTH by MSCA1 low and TSG-6 by MSCA1 high, respectively (Figure 1). No difference in the inhibitory potential of T cells was observed between MSCA1 high and low subpopulations.MSC subpopulations could be distinguished by MSCA1 expression with consistent basic MSC characteristics. Altered properties were observed regarding the secretion of factors relevant for e.g., bone formation or wound healing. Gene expression analyses are ongoing to further elucidate potential differences in the potency of MSC subpopulations. This may help in improving the efficiency of MSC therapy by selecting for subpopulations with distinct features, thereby reducing the heterogeneity of MSC.The authors declare no commercial, proprietary or financial interest. VJ, KG, SE, MR and HS received funding from the Bundesministerium für Verteidigung, the European Union"s Seventh Framework Programme and the European Union"s Network HORIZON2020. The materials presented and views expressed here are the responsibility of the authors only. The European Union Commission takes no responsibility for any use made of the information set out.

Ectopic expression of CD39, a key regulator of the purinergic signaling pathway, increased the immunomodulatory properties of MSC-derived extracellular vesicles

Die ektopische Expression von CD39, einem wichtigen Regulator des purinergen Signalwegs, verstärkte die immunmodulatorischen Eigenschaften von extrazellulären Vesikeln, die von MSC stammen

Mesenchymal stromal cells (MSCs) exert significant therapeutic effects through extracellular vesicles (EVs), which possess immunomodulatory functions. A key mechanism involves the MSC marker CD73. CD73, in collaboration with CD39, which MSCs do not naturally express, regulates the purinergic pathway by converting proinflammatory extracellular ATP (eATP) into anti-inflammatory adenosine. This project investigates whether ectopic expression of CD39 in immortalized MSCs can enhance the therapeutic potency of MSC-EV preparations.We genetically modified immortalized MSCs to express CD39 using a lentiviral transduction strategy. Post-transduction, we confirmed that the transgene expression did not affect the MSCs' cellular properties and harvested their EVs for comprehensive characterization. A biochemical calorimetric assay was employed to evaluate the function of CD39 in the MSC-EV preparations by monitoring the hydrolysis of eATP into adenosine. To assess immunomodulatory properties, especially in suppressing activated T cells, we used a multi-donor mixed lymphocyte reaction assay (mdMLR). This assay combined mononuclear cells from 12 healthy donors to ensure robust allogenic immune responses.MSC-EV preparations from CD39-expressing MSCs efficiently hydrolyzed ATP into adenosine, unlike those from original MSCs. In the mdMLR assay, without eATP, both MSC-EV types exhibited similar immunomodulatory activities, suppressing activated CD4 and CD8 T cells equally. However, with eATP, the immunomodulatory capability of CD39+ MSC-EV preparations significantly increased, surpassing that of original MSC-EV preparations.Incorporating CD39 into immortalized MSCs markedly enhances the immunomodulatory capabilities of MSC-EV preparations, particularly in the presence of eATP. We are currently evaluating the therapeutic potential of CD39+ MSC-EVs in murine ischemic stroke and acute Graft-versus-Host Disease (GvHD) models.The authors declare no conflicts of interest related to this study. The research was conducted independently, and no funding bodies had any influence on the design, collection, analysis, or interpretation of data, nor on the decision to publish the results. All experiments involving lentiviral transduction and animal models were performed in compliance with ethical standards and approved by the relevant institutional review boards.

Abstract lecture (Abstract winner)

Raum 27

Regenerative cell therapy

Abstractsitzungen

Regenerative cell therapy

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Vorsitz

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