Mesenchymal stromal cells induce regulatory T cells via epigenetic conversion of human conventional CD4 T cells in vitro

© 2020 The Authors. S TEM CELLS published by Wiley Periodicals LLC on behalf of AlphaMed Press. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.Regulatory T cells (Treg) play a critical role in immune tolerance. The scarcity of Treg therapy clinical trials in humans has been largely due to the difficulty in obtaining sufficient Treg numbers. We performed a preclinical investigation on the potential of mesenchymal stromal cells (MSCs) to expand Treg in vitro to support future clinical trials. Human peripheral blood mononuclear cells from healthy donors were cocultured with allogeneic bone marrow-derived MSCs expanded under xenogeneic-free conditions. Our data show an increase in the counts and frequency of CD4+ CD25high Foxp3+ CD127low Treg cells (4- and 6-fold, respectively) after a 14-day coculture. However, natural Treg do not proliferate in coculture with MSCs. When purified conventional CD4 T cells (Tcon) are cocultured with MSCs, only cells that acquire a Treg-like phenotype proliferate. These MSC-induced Treg-like cells also resemble Treg functionally, since they suppress autologous Tcon proliferation. Importantly, the DNA methylation profile of MSC-induced Treg-like cells more closely resembles that of natural Treg than of Tcon, indicating that this population is stable. The expression of PD-1 is higher in Treg-like cells than in Tcon, whereas the frequency of PDL-1 increases in MSCs after coculture. TGF-β levels are also significantly increased MSC cocultures. Overall, our data suggest that Treg enrichment by MSCs results from Tcon conversion into Treg-like cells, rather than to expansion of natural Treg, possibly through mechanisms involving TGF-β and/or PD-1/PDL-1 expression. This MSC-induced Treg population closely resembles natural Treg in terms of phenotype, suppressive ability, and methylation profile.This project received funding from: Fundação para a Ciência e Tecnologia, Portugal, under the Harvard Medical School-Portugal Program project Induction of Immune Tolerance in Human Allogeneic Hematopoietic Stem Cell Transplantation (HMSP-ICT/0001/2011) and UID/BIM/50005/2019, project funded by Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado. We also acknowledge the funding received from POR Lisboa 2020 through the project PRECISE – Accelerating progress toward the new era of precision medicine (project no. 16394).info:eu-repo/semantics/publishedVersio

MAGIC: A European program to push the insertion of maskless lithography

n/

Antibody response elicited by the SARS-CoV-2 vaccine booster in patients with multiple sclerosis: Who gains from it?

Background and purpose: Although two doses of COVID-19 vaccine elicited a protective humoral response in most persons with multiple sclerosis (pwMS), a significant group of them treated with immunosuppressive disease-modifying therapies (DMTs) showed less efficient responses. Methods: This prospective multicenter observational study evaluates differences in immune response after a third vaccine dose in pwMS. Results: Four hundred seventy-three pwMS were analyzed. Compared to untreated patients, there was a 50-fold decrease (95% confidence interval [CI] = 14.3–100.0, p < 0.001) in serum SARS-CoV-2 antibody levels in those on rituximab, a 20-fold decrease (95% CI = 8.3–50.0, p < 0.001) in those on ocrelizumab, and a 2.3-fold decrease (95% CI = 1.2–4.6, p = 0.015) in those on fingolimod. As compared to the antibody levels after the second vaccine dose, patients on the anti-CD20 drugs rituximab and ocrelizumab showed a 2.3-fold lower gain (95% CI = 1.4–3.8, p = 0.001), whereas those on fingolimod showed a 1.7-fold higher gain (95% CI = 1.1–2.7, p = 0.012), compared to patients treated with other DMTs. Conclusions: All pwMS increased their serum SARS-CoV-2 antibody levels after the third vaccine dose. The mean antibody values of patients treated with ocrelizumab/rituximab remained well below the empirical "protective threshold" for risk of infection identified in the CovaXiMS study (>659 binding antibody units/mL), whereas for patients treated with fingolimod this value was significantly closer to the cutoff

From Promise to Reality: Bioengineering Strategies to Enhance the Therapeutic Potential of Extracellular Vesicles

Extracellular vesicles (EVs) have been the focus of great attention over the last decade, considering their promising application as next-generation therapeutics. EVs have emerged as relevant mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. Given their natural ability to shuttle messages between cells, EVs have been explored both as inherent therapeutics in regenerative medicine and as drug delivery vehicles targeting multiple diseases. However, bioengineering strategies are required to harness the full potential of EVs for therapeutic use. For that purpose, a good understanding of EV biology, from their biogenesis to the way they are able to shuttle messages and establish interactions with recipient cells, is needed. Here, we review the current state-of-the-art on EV biology, complemented by representative examples of EVs roles in several pathophysiological processes, as well as the intrinsic therapeutic properties of EVs and paradigmatic strategies to produce and develop engineered EVs as next-generation drug delivery systems

SPA therapy of upper respiratory tract inflammations

The upper airway respiratory diseases (i.e. common cold, allergic rhinitis, nonallergic/vasomotor rhinitis, acute and chronic rhinosinusitis and nasal polyposis) in which nasal congestion is a common symptom are often undertreated due to the frequent inadequate efficacy and safety concern with current therapies. In scientific literature, few studies seem to support the hypothesis that nasal inhalatory treatment with thermal water promotes the improvement of nasal symptoms, even if the mechanisms by which the improvement from SPA therapy can be expected remain debated. A prospective comparative study with a pre-post design has been performed consecutively enrolling 33 (males 70\ua0%) patients of both genders older than 12\ua0years of age, affected by chronic sinonasal inflammation. All patients underwent a 14-days course of radioactive water warm vapour inhalations followed by nasal aerosol of the same thermal water 10\ua0min each once/day at Merano Therme. At the beginning and end of the study, in all the subjects, nasal function evaluation by active anterior rhinomanometry, mucociliary transport time (MCTt) determination and nasal cytology were performed. After the inhalatory treatment, the mucociliary function was improved and the pathologic mucociliary transport times recorded at the beginning of the study being significantly reduced to physiologic ones. Besides, before treatment, the cytologic picture showed an inflammatory cell infiltration (eosinophils, neutrophils with/without bacteria, mast cells) in 37\ua0% of patients; after therapy in 66\ua0% of these patients, the rhinocytogram was normal. Our results suggest, according to the literature data, that SPA therapy with radioactive water could represent an alternative choice in chronic inflammatory diseases of the upper airways, nonresponsive to pharmacological therapy

Hypothermic Preservation of Adipose-Derived Mesenchymal Stromal Cells as a Viable Solution for the Storage and Distribution of Cell Therapy Products

Cell and gene therapies (CGT) have reached new therapeutic targets but have noticeably high prices. Solutions to reduce production costs might be found in CGT storage and transportation since they typically involve cryopreservation, which is a heavily burdened process. Encapsulation at hypothermic temperatures (e.g., 2–8 °C) could be a feasible alternative. Adipose tissue-derived mesenchymal stromal cells (MSC(AT)) expanded using fetal bovine serum (FBS)- (MSC-FBS) or human platelet lysate (HPL)-supplemented mediums (MSC-HPL) were encapsulated in alginate beads for 30 min, 5 days, and 12 days. After bead release, cell recovery and viability were determined to assess encapsulation performance. MSC identity was verified by flow cytometry, and a set of assays was performed to evaluate functionality. MSC(AT) were able to survive encapsulated for a standard transportation period of 5 days, with recovery values of 56 ± 5% for MSC-FBS and 77 ± 6% for MSC-HPL (which is a negligible drop compared to earlier timepoints). Importantly, MSC function did not suffer from encapsulation, with recovered cells showing robust differentiation potential, expression of immunomodulatory molecules, and hematopoietic support capacity. MSC(AT) encapsulation was proven possible for a remarkable 12 day period. There is currently no solution to completely replace cryopreservation in CGT logistics and supply chain, although encapsulation has shown potential to act as a serious competitor

Scalable manufacturing of human mesenchymal stem/stromal cells and derived exosomes in the single-use, vertical-wheel bioreactor system using a human platelet lysate culture supplement

Mesenchymal stem/stromal cells (MSC) hold great promise for tissue engineering and regenerative medicine settings due to their multilineage differentiation potential and their intrinsic immunomodulatory and trophic activities. Large cell doses (\u3e1x106 cells/kg) are however required for clinical implementation and the success in obtaining those cell numbers is dependent on efficient ex vivo expansion protocols able to comply with Good Manufacturing microcarrier-based cultures in scalable bioreactors using serum-/xenogeneic-free (S/XF) culture components. In this context, a S/XF microcarrier-based culture system was successfully established for the expansion of human UCM and AT MSC using an innovative disposable bioreactor system utilizing the Vertical-Wheel™ technology (PBS-0.1 MAG with maximum working volume of 100 mL, PBS Biotech) combined with a commercially available fibrinogen-depleted human platelet lysate-based culture supplement (UltraGRO™-PURE, AventaCell BioMedical). By optimizing the agitation and feeding regimes, UCM and AT MSC were successfully expanded to maximum cell densities of 5.3 ± 0.4 x 105 cell/mL (n=3) and 3.6 ± 0.7 x 105 cell/mL (n=3), respectively, after 7 days of culture (cell viability ≥ 94%), while maintaining their identity. Recently, increasing evidence has proposed extracellular vesicles (EVs), as exosomes, as mediators of many of the MSC-associated therapeutic features. Exosomes are small EVs (30-150nm) of endocytic origin, involved in intercellular communication, through transfer of a cargo of proteins and RNAs. In this context, the platform established for the expansion of MSC is under optimization for exosome production. Dynamic culture systems, as the one presented herein, are expected to allow a higher exosome titer, as well as a better control when fine-tuning the exosomes\u27 properties, by changing culture conditions (e.g. shear, oxygen). Preliminary results have shown that human MSC expanded in the Vertical-Wheel™ bioreactor system allowed to obtain a population of EVs with a more homogeneous size distribution profile, when compared to cells cultured in traditional static systems. Overall, we demonstrate that this culture system is able to robustly manufacture human MSC and MSC-based exosomes towards the development of novel therapeutic products