Differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on T, B and NK cell functions

Mesenchymal stromal cells (MSCs) are multipotent cells, immunomodulatory stem cells that are currently used for regenerative medicine and treatment of a number of inflammatory diseases, thanks to their ability to significantly influence tissue microenvironments through the secretion of large variety of soluble factors. Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease. Here, we used a standardized methodological approach to dissect the immunomodulatory effects exerted by MSC-derived EVs on unfractionated peripheral blood mononuclear cells and purified T, B and NK cells. We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division. Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases

Extracellular Vesicles Mediate Mesenchymal Stromal Cell-Dependent Regulation of B Cell PI3K-AKT Signaling Pathway and Actin Cytoskeleton

Mesenchymal stromal cells (MSCs) are adult, multipotent cells of mesodermal origin representing the progenitors of all stromal tissues. MSCs possess significant and broad immunomodulatory functions affecting both adaptive and innate immune responses once MSCs are primed by the inflammatory microenvironment. Recently, the role of extracellular vesicles (EVs) in mediating the therapeutic effects of MSCs has been recognized. Nevertheless, the molecular mechanisms responsible for the immunomodulatory properties of MSC-derived EVs (MSC-EVs) are still poorly characterized. Therefore, we carried out a molecular characterization of MSC-EV content by high-throughput approaches. We analyzed miRNA and protein expression profile in cellular and vesicular compartments both in normal and inflammatory conditions. We found several proteins and miRNAs involved in immunological processes, such as MOES, LG3BP, PTX3, and S10A6 proteins, miR-155-5p, and miR-497-5p. Different in silico approaches were also performed to correlate miRNA and protein expression profile and then to evaluate the putative molecules or pathways involved in immunoregulatory properties mediated by MSC-EVs. PI3K-AKT signaling pathway and the regulation of actin cytoskeleton were identified and functionally validated in vitro as key mediators of MSC/B cell communication mediated by MSC-EVs. In conclusion, we identified different molecules and pathways responsible for immunoregulatory properties mediated by MSC-EVs, thus identifying novel therapeutic targets as safer and more useful alternatives to cell or EV-based therapeutic approaches

Notch signalling drives bone marrow stromal cell-mediated chemoresistance in acute myeloid leukemia

Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB.These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML

Quality controls in Advanced Therapy Medicinal Products:safety assessment of Mesenchymal Stromal Cells to treat human bone defects

Negli ultimi 25 anni, c\u2019\ue8 stato un crescente interesse nei confronti della medicina rigenerativa e questo ha portato allo sviluppo di una nuova categoria di prodotti medicinali basati sull\u2019utilizzo di cellule, che vengono definiti come \u201cTerapie Avanzate\u201d. Il Regolamento N.1394/2007 del Parlamento Europeo, ha decretato l\u2019esistenza di tre tipi di Prodotti Medicinali di Terapia Avanzata: \u201cProdotti di terapia genica\u201d, \u201cProdotti di terapia cellulare somatica\u201d e \u201cProdotti di ingegneria tissutale\u201d. Lo scopo delle terapie cellulari \ue8 quello di prevenire o curare patologie attraverso l\u2019effetto farmacologico, immunologico o metabolico esercitato dalle cellule. Le Cellule Stromali Mesenchimali (MSC) rappresentano i candidati ideali per questo tipo di terapie, per le seguenti motivazioni: sono cellule multipotenti facilmente estraibili da diversi tessuti; si espandono rapidamente in coltura e producono fattori trofici che modulano l\u2019infiammazione, il rimodellamento tissutale e l\u2019apoptosi; hanno propriet\ue0 immunomodulatorie. L\u2019impiego di queste cellule in trial clinici richiede la dimostrazione della loro efficacia in esperimenti in vivo, e la loro produzione, come per ogni tipo di farmaco iniettabile, deve avvenire nel rispetto delle norme GMP (Norme di Buona Fabbricazione), le quali regolano i processi di produzione, la caratterizzazione dei prodotti finiti e i controlli di qualit\ue0. Tutte le fasi del processo di produzione delle MSC devono essere standardizzate per garantire una qualit\ue0 e sicurezza cellulare riproducibili. Questo progetto \ue8 mirato a dimostrare la capacit\ue0 di rigenerazione ossea delle cellule stromali mesenchimali ed ha come scopo primario la standardizzazione dei controlli di qualit\ue0 necessari per la loro applicazione clinica. Partendo dai dati preclinici ottenuti in collaborazione con il gruppo di Burastero G., che aveva dimostrato in vivo la capacit\ue0 delle MSC di favorire la rigenerazione ossea, abbiamo sviluppato un protocollo clinico di fase I che prevede il trattamento di difetti dell\u2019osso scafoide con le MSC autologhe espanse ex-vivo in associazione ad Orthoss\uae (biomateriale inorganico di origine bovina) e BMP-7 (proteina osteoinduttiva ricombinante umana). La produzione delle MSC per uso clinico verr\ue0 effettuata nella Cell Factory di Verona, afferente al Dipartimento Interaziendale di Medicina Trasfusionale presso il Policlinico G.B. Rossi. Durante questi anni, \ue8 stato messo a punto un pannello di saggi su campioni di midollo osseo da 5 donatori sani espansi in Cell Factory; i risultati ottenuti sono i seguenti: 1) le analisi della vitalit\ue0 e del fenotipo hanno dimostrato il mantenimento dell\u2019identit\ue0 delle MSC al termine del processo di espansione; 2) la ricerca di virus, micoplasma, funghi o specie microbiche ha rivelato l\u2019assenza di contaminazione e quindi la generazione di un prodotto sterile; 3) in seguito all\u2019aggiunta di stimoli specifici, le MSC prodotte sono in grado di differenziare in adipociti ed osteoblasti; 4) la capacit\ue0 clonogenica viene mantenuta anche dopo espansione; 5) le propriet\ue0 immunomodulatorie delle MSC nei confronti dei principali effettori immuni sono state confermate anche dopo espansione; 6) l\u2019analisi dell\u2019espressione genica e il test di mutagenesi hanno dimostrato l\u2019assenza di trasformazione neoplastica al termine del processo produttivo; 7) la valutazione del cariotipo non ha evidenziato aberrazioni cromosomiche nel prodotto cellulare. I suddetti test sono stati convalidati e dichiarati come controlli di qualit\ue0 definitivi del protocollo clinico \u201cRicostruzione di deficit dell\u2019osso scafoide mediante l\u2019uso di cellule staminali mesenchimali autologhe espanse ex-vivo\u201d(Eudract N\ub02012-002424-33), che \ue8 stato sottoposto all\u2019approvazione dell\u2019ISS (Istituto Superiore di Sanit\ue0) e dell\u2019AIFA (Agenzia Italiana del Farmaco), che sono gli organi preposti al controllo delle sperimentazioni cliniche.Over the last 25 years, there has been a growing interest towards regenerative medicine and this has led to the development of a new category of medicinal products based on the use of cells, grouped under the name of \u201cAdvanced Therapies\u201d. The European Regulation N\ub01394/2007 classified three kinds of Advanced Therapy Medicinal Products: \u201cgene therapy medicinal products\u201d, \u201csomatic cell therapy medicinal products\u201d and \u201ctissue engineered products\u201d. Aim of cellular therapies in regenerative medicine is to treat or prevent human diseases through the pharmacological, immunological or metabolic action of the cells. Mesenchymal Stromal Cells (MSC) are key candidates for cellular therapies for different reasons: they are multipotent cells easy to obtain from different tissues; they expand rapidly in culture and produce trophic factors which modulate inflammation, tissue remodeling and apoptosis; they possess immune regulatory properties. The employment of these cells in clinical trials requires in vivo studies to test their efficacy, and their manufacturing, like any kind of injectable drug, must follow GMP (Good Manufacturing Practice) rules, which regulate precisely processes, final product characterization and quality controls. All steps in MSC manufacturing need to be standardized to ensure a reproducible cellular quality and potency assessment. Aim of this work is the demonstration of the safety of MSCs for bone regeneration and is focused on the standardization of quality controls necessary for their use in patients. Starting from pre-clinical data obtained in collaboration with the group of Burastero G., who performed in vivo experiments in animals demonstrating the ability of MSCs to induce bone regeneration, we developed a phase I clinical protocol for the treatment of scaphoid bone defects with autologous MSCs ex-vivo expanded, associated with Orthoss\uae ( an inorganic bone matrix of bovine origin) and BMP-7 (Osigraft, osteoinductive human recombinant protein). The production of clinical-grade MSCs will be carried out in the Cell Factory of the AOUI of Verona, located at \u201cU.O.C. di Medicina Trasfusionale\u201d of the Policlinico G.B. Rossi, Verona. We set up a panel of assays using five different bone marrow samples from healthy donors expanded in Cell Factory and we obtained the following results: 1) the analyses of viability and phenotype showed the preservation of the identity of MSC after the expansion; 2) the analysis on the presence of endotoxin, viruses, fungi or microbial species demonstrated no contamination and a sterile cellular product; 3) after the addiction of specific stimuli, the MSCs were able to differentiate into osteoblasts and adipocytes; 4) the clonogenicity of MSCs after expansion was maintained; 5) the immunomodulatory properties towards the immune effector cells were confirmed; 6) the gene expression analysis and the mutagenesis test demonstrated the absence of neoplastic transformation after the expansion process; 7) the karyotype analysis showed no chromosomal aberrations in the cellular product. All these validated tests have become the definitive quality controls of the clinical protocol \u201cRicostruzione di deficit dell\u2019osso scafoide mediante l\u2019uso di cellule staminali mesenchimali autologhe espanse ex-vivo\u201d(Eudract N\ub02012-002424-33), which has been submitted to the approval of the responsible organs, i.e. ISS (Istituto Superiore di Sanit\ue0) and AIFA (Agenzia Italiana del Farmaco)

Inhibition of GSK-3 Signalling Enhances Sensitivity of Non-Promyelocitic Acute Myeloid Leukemia (AML) Cell to Chemotherapy

Background: GSK-3 is a serine-threonine kinase involved in metabolic regulation as well as in the control of many pathways associated to cancer development, including Notch Wnt/\u3b2-catenin, Hedgehog, and AKT. It has been demonstrated that association of GSK-3 inhibitors with All-trans-retinoic acid (ATRA) significantly improves ATRA-mediated differentiation and cell death of acute promyelocytic (APL) leukaemia cells. However, little is currently known about the contribution of GSK-3 role to non-promyelocytic AML cell response to treatment with chemotherapeutic agents. Aims: In this study, we aim to validate GSK-3 signalling as potent successful therapeutic target in non-promyelocytic AML. For this purpose we tested different GSK-3 for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatments. Methods: GSK-3 expression was analyzed by Western blot or flow cytometry inAML cell lines (HL-60, THP1, U937) or primary non-promyelocyticAML blast cells (30 samples). AML cellscultured alone or in presence ofhuman bone marrow mesenchymal stromal cells (hBM-MSCs) were treated with GSK-3 inhibitors, including LiCL, AR-A014418, SB 216763, in association or not with Cytarabine (Ara-C) or Idarubicin. Cell proliferation and cell death were measured by CFSE dilution and TOPRO-3/Annexin-V staining, respectively. Results: Flow cytometry and Western blot analysis in AML samples revealed high expression levels of all GSK-3forms, including total GSK-3\u3b1, (Ser21) GSK-3\u3b1, total GSK-3\u3b2, and (Ser 21) GSK-3\u3b2; theseforms were all down-modulated when AML cells were cultured in presence of hBM-MSCs, thus suggesting that GSK-3 plays an important role in transducting micro-environmental signals in AML cells interacting with bone marrow stroma. The treatment of AML cells with increasing concentrations of each GSK-3 inhibitors decreased AML cell viability in a dose-dependent manner; interestingly, hBM-MSCs or peripheral blood mononuclear cells were less sensitive to GSK-3inhibitors. The addition of each inhibitor increased dramatically the AML cell apoptotic rate induced by the addition of Ara-C or Idarubicin in vitro. Notably, LiCl and AR-A014418 were capable of abrogating hBM-MSC-mediated AML cell resistance to apoptosis induced by Ara-C or Idarubicin. Conclusion: Overall our data clearly demonstrated that inhibition of GSK-3 reduced proliferation and chemoresistance of non promyelocytic AML cells. Thus GSK-3 inhibition represents a therapeutic strategy not only for APL but also for other AML subtypes

Notch singalling inhibition as a multi-target therapy to overcome bone marrow microenvironment-mediated drug resistance in AML

Background: Several pro-survival proteins promoting resistance to chemother- apy, such as BCl-2, STAT3,NF\u3baB and AKT,are over-expressed in AML cells, thus representing the basis for targeted therapies. However, only multi-target drug strategy may lead to the modulation of the pro-survival protein network, due to the simultaneous activation of alternative pathways. Notch signalling is a master developmental pathway that controls tumour cell survival by interacting with pro-survival proteins, such as \u3b2-catenin, BCL-2, STAT3, NF\u3baB, and AKT, thus representing an ideal target to interfere with all these pathways in different cancer systems. We recently showed that Notch inhibition was capable of abrogating microen- vironment-mediated AML cell chemo-resistance (P550, EHA20); however, little is known about the mechanism involved. Aims: We studied the mechanisms underlying microenvironmental, Notch- mediated AML chemo-resistance by investigating the contribution of BCl-2, STAT3, NF\u3baB and AKT. Using in silico and in vitro approaches we analyzed the expression changes of these proteins in ex-vivo AML cell samples in condition of pharmacological or genetical Notch down-regulation, as well as in AML cells either cultured alone or co-cultured with human bone marrow mesenchymal stromal cells (hBM-MSCs) in presence of chemoterapeutic agents, such as cytarabine (Ara-C) and Idarabucin. Methods: Cells were obtained from bone marrow (33) and peripheral blood (22) samples of AML patients. hBM-MSCs were expanded from bone marrow of 20 healthy donors (BM-MSCs) and 20 AML patients (BM-MSCs*). Gene set enrich- ment analysis (GSEA) were perform using GEOR tools on AML expression array of 304 patients previously deposited in Gene Expression Omnibus (GSE10358). Genetic inhibition of Notch signalling was achieved in AML cell lines (HL-60 and THP1) by infecting cells with lentiviral particles carrying shRNA for either RBP- jK or MALM1, two mediators of Notch signaling. Pharmacological Inhibition of Notch in AML was achieved by using Gamma secretase Inhibitors (GSIs), Notch transcription factors Inhibitor SAHM1, and combination of Notch blocking anti- bodies.Ara-C, and idarubicin were added to culture supernatants at different concentrations. Cell viability was evaluated by Annexin-V/Propidium Iodide (PI). Protein levels were analyzed by intracellular staining with corresponding fluo- rophore conjugated antibodies, followed by flow cytometry analysis. Results: In silico Gene set enrichment analysis and flow cytometry analysis showed that AML samples highly expressed Notch1, Jagged1, STAT3, NF\u3ba-B and AKT genes and proteins. Notably, higher levels of Notch1 were found in patients with poor cytogenetic prognosis, while STAT3, NF-\u3baB and AKT were uniformly expressed by AML patients. Protein analysis revealed low levels of pro-survival proteins AKT, STAT3 and NF-\u3baB in RBP-jk and MALM1 knock- down cells, as compared to control cells infected with non specific shRNA. We then verified that genetic (shRNA) and pharmacological inhibition of Notch, by using either GSIs or Notch receptor blocking antibodies, was capable of sen- sitizing AML cells, either cultured alone or in presence of hBM-MSCs, to ARA- C or idarubucine. Additionally, we found that hBM-MSC-dependent induction of AML chemoresistance was associated to increase of AKT, NF-\u3baB and STAT3 protein levels in AML cells. Similarly, Notch inhibition with GSIs prevented hBM- MSC-mediated increase of AKT, NF-\u3baB and STAT3, thus restoring sensibility of AML cells to Idarubucin treatment. Summary/Conclusions: These results suggest that inhibition of Notch sig- nalling is sufficient to reduce protein levels of AKT, STAT3 and NF-\u3baB proteins involved in AML chemoresistance, thus making the pro-survival core network controlled by Notch a potential target for specific Notch targeted therapies

Role of Wnt/\u3b2-Catenin Signalling in Acute Myeloid Leukemia (AML) Cell Response to Chemotherapy

Background: Growing evidences from both preclinical and clinical investigations reveal the critical role of Wnt signalling for the development of many cancers and for their response to chemotherapy. Although recent studies suggest that aberrant Wnt signalling can be involved in the neoplastic myeloid cell growth, the contribution of the Wnt/\u3b2-catenin pathway to AML survival and chemoresistance is still unclear. Aims: In this study, we investigated the contribution of WNT/\u3b2-CATENIN signalling to AML survival and chemoresistance. For this purpose we tested different modulators of Wnt/\u3b2-Catenin pathway for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatment. Methods: AML primary blast cells(30 samples) or AML cell lines cultured alone or in presence of human bone marrow mesenchymal stromal cells (hBM-MSCs), were treated with with Cytarabine (Ara-C) or Idarubicin, in presence or absence of Wnt modulators, including ligands (Wnt3a, Wnt5a/5b), Porcupine inhibitors (IWP-2), LRP6 inhibitors (Niclosamide), or antagonists of TCF/\u3b2-catenin (PKF118-310, PNU-74654). Results: In silico analysis showed the enrichment of Wnt signalling components in AML samples. Western Blot and flow cytometry showed the presence of total \u3b2-catenin only in about 2/3 of primary samples analyzed, while . \u3b2-catenin positive samples had different degree of activation of the pathway, as revealed by the expression of active forms of \u3b2-catenin, including (Ser675)\u3b2-catenin and non-phospho-(Ser33/37/Thr41) \u3b2-catenin. Notably, we found that active forms of \u3b2-catenin increased in AML samples in co-culture with hBM-MSCs, thus suggesting that Wnt signalling could be involved in the crosstalk between bone marrow stroma and AML cells. The addition of Wnt or pharmacological inhibitors, such as IWP-2, PNU-74654 and Niclosamide, to the culture medium of \u3b2-catenin-positive AML samples, either cultured alone or in co-culture with hBM-MSCs, reduced AML cell proliferation with slight effect on cell death. When associated to Idarubicin, all Wnt inhibitors except IWP-2 synergycally induced a dramatic cell death in AML cells in both culture conditions. However, when Idarubicin was replaced by Ara-C the synergism was observed only with Niclosamide and PKF. Cell death was mainly due to apoptosis, as shown by Annexin-V staining. Conclusion: Overall our data show that Wnt inhibitors reduce proliferation and chemoresistance of AML cells in culture or co-culture with bone marrow stroma cells. Wnt/\u3b2-catenin signalling may represent a potential therapeutic strategy to improve AML treatment, overcoming bone marrow stromal-mediated anti-apoptotic and chemoresistance effects

Remdesivir plus dexamethasone versus dexamethasone alone for the treatment of COVID-19 patients requiring supplemental O2 therapy: a prospective controlled non-randomized study

Background: Remdesivir is an antiviral used to treat COVID-19 which improves some clinical outcomes. Dexamethasone has been shown to be effective in reducing mortality. It has been hypothesized that combination of these two drugs can improve mortality. We evaluated the effect of combination on mortality of COVID-19 patients requiring O2 therapy. Methods: A prospective quasi-experimental study, including two independent, sequential controlled cohorts, one received remdesivir-dexamethasone and the other dexamethasone alone, was designed. All COVID-19 patients requiring supplemental O2 therapy were enrolled consecutively. The sample size to power mortality was a priori calculated. The primary endpoints were 30-day mortality and viral clearance differences. Secondary endpoints were differences in hospitalization times, improvement in respiratory failure (PO2/FiO2) and inflammatory indices (fibrinogen, CRP, neutrophil/lymphocyte ratio, D-Dimer). Kaplan-Meier curves and the log-rank test were used to evaluate significant differences in mortality between groups. Results: 151 COVID-19 patients were enrolled (remdesivir/dexamethasone group, 76 and dexamethasone alone,75). No differences in demographic, clinical and laboratory characteristics were observed between the two groups at baseline. Faster viral clearance occurred in the remdesivir/dexamethasone group compared to dexamethasone alone (median 6 vs 16 days; p<0.001). 30-days mortality in the remdesivir/dexamethasone group was 1.3%, while in dexamethasone alone was 16% (p<0.005). In the remdesivir/dexamethasone group compared to dexamethasone alone there was a reduction in hospitalization days (p<0.0001) and a faster improvement in both respiratory function and inflammatory markers. Conclusions: Remdesivir/dexamethasone treatment is associated with significant reduction in mortality, length of hospitalization, and faster SARS-CoV-2 clearance, compared to dexamethasone alone

EFFECTS OF A NOVEL CERAMIC BIOMATERIAL ON IMMUNE MODULATORY PROPERTIES AND DIFFERENTIATION POTENTIAL OF MESENCHYMAL STROMAL CELLS

International audienceThe aim of this study was to assess the immune modulatory properties of human mesenchymal stromal cells obtained from bone marrow (BM-MSCs), fat (ASCs) and cord blood (CB-MSCs) in the presence of a novel hydroxyapatite and tricalcium-phosphate (HA/TCP) biomaterial as scaffold for MSC delivery. In resting conditions, a short-term culture with HA/TCP did not modulate the anti-apoptotic and suppressive features of the various MSC types towards T, B and NK cells; in addition, when primed with inflammatory cytokines, MSC maintained or not on HA/TCP similarlyincreased their suppressive capacities. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype with up-regulation of OSTERIX and OSTEOCALCIN, similarly to what obtained with dexamethasone and, to a higher extent, BMP-4 treatment. MSC-derived osteoblasts did not trigger immune cell activation, but were less efficient than undifferentiated MSCs in inhibiting stimulated T and NK cells. Interestingly, their suppressive machinery included not only the activation of IDO, which plays a central role in T-cell inhibition, but also COX-2 that was not significantly involved in immune modulatory effect of human undifferentiated MSCs. COX-2 is significantly involved in bone healing, suggesting that its induction by HA/TCP could also contribute to the therapeutic activity of MSC for bone tissue engineering