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

Monitoring the ex-vivo expansion of human mesenchymal stem/stromal cells in xeno-free microcarrier-based reactor systems by MIR spectroscopy

Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell-based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex-vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi-analytical methods, some of them time-consuming. The present work evaluates the use of mid-infrared (MIR) spectroscopy, through rapid and economic high-throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno-free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R2 ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R2 of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes

Scalable production of human mesenchymal stromal cell-derived extracellular vesicles under serum-/xeno-free conditions in a microcarrier-based bioreactor culture system

Copyright © 2020 de Almeida Fuzeta, Bernardes, Oliveira, Costa, Fernandes-Platzgummer, Farinha, Rodrigues, Jung, Tseng, Milligan, Lee, Castanho, Gaspar, Cabral and da Silva. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Mesenchymal stromal cells (MSC) hold great promise for tissue engineering and cell-based therapies due to their multilineage differentiation potential and intrinsic immunomodulatory and trophic activities. Over the past years, increasing evidence has proposed extracellular vesicles (EVs) as mediators of many of the MSC-associated therapeutic features. EVs have emerged as mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. EVs are derived from cell membranes, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. In this work, EVs were produced by MSC isolated from different human tissue sources [bone marrow (BM), adipose tissue (AT), and umbilical cord matrix (UCM)]. A serum-/xeno-free microcarrier-based culture system was implemented in a Vertical-WheelTM bioreactor (VWBR), employing a human platelet lysate culture supplement (UltraGROTM-PURE), toward the scalable production of MSC-derived EVs (MSC-EVs). The morphology and structure of the manufactured EVs were assessed by atomic force microscopy, while EV protein markers were successfully identified in EVs by Western blot, and EV surface charge was maintained relatively constant (between −15.5 ± 1.6 mV and −19.4 ± 1.4 mV), as determined by zeta potential measurements. When compared to traditional culture systems under static conditions (T-flasks), the VWBR system allowed the production of EVs at higher concentration (i.e., EV concentration in the conditioned medium) (5.7-fold increase overall) and productivity (i.e., amount of EVs generated per cell) (3-fold increase overall). BM, AT and UCM MSC cultured in the VWBR system yielded an average of 2.8 ± 0.1 × 1011, 3.1 ± 1.3 × 1011, and 4.1 ± 1.7 × 1011 EV particles (n = 3), respectively, in a 60 mL final volume. This bioreactor system also allowed to obtain a more robust MSC-EV production, regarding their purity, compared to static culture. Overall, we demonstrate that this scalable culture system can robustly manufacture EVs from MSC derived from different tissue sources, toward the development of novel therapeutic products.unding received by iBB-Institute for Bioengineering and Biosciences from the Portuguese Foundation for Science and Technology (FCT) (UID/BIO/04565/2020) and through the projects PTDC/EQU-EQU/31651/2017, PTDC/BBB-BQB/1693/2014, and PTDC/BTM-SAL/31057/2017 is acknowledged. Funding received from POR de Lisboa 2020 through the project PRECISE – Accelerating progress toward the new era of precision medicine (Project N. 16394) is also acknowledged. MAF (PD/BD/128328/2017) and FO (PD/BD/135046/2017) acknowledge FCT for the Ph.D. fellowships and DG (SFRH/BPD/109010/2015) for the Post-Doctoral fellowship.info:eu-repo/semantics/publishedVersio

Neurodifferentiation and neuroprotection potential of mesenchymal stromal cell-derived secretome produced in different dynamic systems

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterized by the degeneration of the dopamine (DA) neurons in the substantia nigra pars compacta, leading to a loss of DA in the basal ganglia. The presence of aggregates of alpha-synuclein (α-synuclein) is seen as the main contributor to the pathogenesis and progression of PD. Evidence suggests that the secretome of mesenchymal stromal cells (MSC) could be a potential cell-free therapy for PD. However, to accelerate the integration of this therapy in the clinical setting, there is still the need to develop a protocol for the large-scale production of secretome under good manufacturing practices (GMP) guidelines. Bioreactors have the capacity to produce large quantities of secretomes in a scalable manner, surpassing the limitations of planar static culture systems. However, few studies focused on the influence of the culture system used to expand MSC, on the secretome composition. In this work, we studied the capacity of the secretome produced by bone marrow-derived mesenchymal stromal cells (BMSC) expanded in a spinner flask (SP) and in a Vertical-Wheel™ bioreactor (VWBR) system, to induce neurodifferentiation of human neural progenitor cells (hNPCs) and to prevent dopaminergic neuron degeneration caused by the overexpression of α-synuclein in one Caenorhabditis elegans model of PD. Results showed that secretomes from both systems were able to induce neurodifferentiation, though the secretome produced in the SP system had a greater effect. Additionally, in the conditions of our study, only the secretome produced in SP had a neuroprotective potential. Lastly, the secretomes had different profiles regarding the presence and/or specific intensity of different molecules, namely, interleukin (IL)-6, IL-4, matrix metalloproteinase-2 (MMP2), and 3 (MMP3), tumor necrosis factor-beta (TNF-β), osteopontin, nerve growth factor beta (NGFβ), granulocyte colony-stimulating factor (GCSF), heparin-binding (HB) epithelial growth factor (EGF)-like growth factor (HB-EGF), and IL-13. Overall, our results suggest that the culture conditions might have influenced the secretory profiles of cultured cells and, consequently, the observed effects. Additional studies should further explore the effects that different culture systems have on the secretome potential of PD.This work has been funded by la Caixa Foundation and Portuguese Foundation for Science and Technology (FCT) under the agreement LCF/PR/HP20/52300001; ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122); by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020. CRM was supported by a Ph.D. scholarship from FCT and the company Stemmatters, Biotecnologia e Medicina Regenerativa SA (PD/BDE/127833/2016). Funding received by iBB-Institute for Bioengineering and Biosciences from FCT (UID/BIO/04565/2020) and through the project PTDC/EQU-EQU/31651/2017 is acknowledged. MAF was supported by a Ph.D. scholarship from FCT (SFRH/PD/BD/128328/2017). RC was supported by the EXOpro project (PTDC/EQU-QUE/31651/2017). JPS was supported by a Ph.D. scholarship from FCT and the company Bn’ML—Behavioral & Molecular Lab (PD/BDE/127834/2016). DS was supported by a Ph.D. scholarship from FCT and the company Stemmatters, Biotecnologia e Medicina Regenerativa S.A. (PD/BDE/135567/2018) JC was supported by a Ph.D. scholarship from FCT (SFRH/BD/5813/2020)

Mid-infrared spectroscopy: a groundbreaking tool for monitoring mammalian cells processes

Mammalian cells are extensively used in cell biology studies, e.g. as a model system of human pathologies, or as a major source of very high-value biopharmaceuticals (that can be the cells itself or their products e.g. heterologous proteins). As such, it is highly pertinent to develop monitoring methods for mammalian cultivations capable of delivering detailed bioprocess information in a rapid and economic way. It is relevant to acquire information concerning the conventional critical variables (as cell growth, consumption of nutrients, production and consumption of by-products and the bioproduct production), and the cell metabolism towards a better understanding of the culture process and consequently for more efficient optimization and control procedures. In the present work, Mid-infrared (MIR) spectroscopy was evaluated as a monitoring technique enabling the acquisition of said bioprocess information in a simple (single step of dehydration), rapid (minutes), economic (without reagent consumption), label-free and high-throughput mode (using 96-wells microplates). The new method was evaluated across a highly diverse set of mammalian culture processes: The monitoring of ex vivo expansion of human mesenchymal stem/stromal cells (MSC) conducted under diverse culture strategies, where it was possible to accurately predict glucose, lactate and ammonia concentrations. The monitoring of recombinant human embryonic kidney cells producing green fluorescent protein, which enabled the estimation of transfection efficiency and the metabolic impact of protein production on the host cell metabolism. Finally, the monitoring of infected gastric cell lines with Helicobacter pylori, which enabled to identify spectral biomarkers for defining the status of infection (infected vs non infected) and to characterize the infection conducted by virulent strains, usually associated to severe gastric diseases as peptide ulcer and gastric cancer. In resume, high-throughput MIR spectroscopy enabled to adequately monitor diverse mammalian cell cultures, thus allowing to attain meaningful information concerning said bioprocesses, from traditional critical variables of the process, to the metabolic status of mammalian host cells and even to define disease biomarkers in a groundbreaking way.info:eu-repo/semantics/publishedVersio

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

Umbilical cord matrix derived-mesenchymal stem cell production in microcarrier- based culture systems

Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSC) are promising therapeutic candidates for regenerative medicine settings. UCM MSC have advantages over adult cells as these can be obtained through a noninvasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSC mandatory. In this work, we established two different microcarrier-based culture systems for the scalable production of UCM MSC. In the first approach, we established a spinner flask culture system combining gelatin-based Cultispher®S microcarriers and xeno-free culture medium for the expansion of umbilical UCM MSC. This system enabled the production of 2.4 (±1.1) x105 cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)-fold increase in cell number. The established protocol was then implemented in a stirred-tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. In a second approach, a commercially available human platelet lysate-based culture supplement (UltraGROTM, AventaCell BioMedical, under a collaboration agreement with iBB) (5%(v/v)) was tested to effectively isolate UCM MSC and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSC retained their immunophenotype and multilineage differentiation potential. The UCM MSC cultured under static conditions using UltraGROTM-supplemented medium expanded more rapidly compared to UCM MSC expanded using a previously established protocol [1]. Importantly, UCM MSC were successfully expanded under dynamic conditions on plastic microcarriers using UltraGROTM-supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSC expanded by \u3e13-fold after 5–6 days, maintaining their immunophenotype and multilineage differentiation ability. By using this human platelet lysate supplement we were able to establish an easily scalable integrated culture platform based on for the effective isolation and expansion of UCM MSC in a xenogeneic-free microcarrier-based system. Overall, both strategies suited the intrinsic features of UCM-derived MSC and represent an important advance towards a GMP compliant large-scale production platform for these cells. [1] Carmelo JG, Fernandes-Platzgummer A, Diogo MM, da Silva CL, Cabral JM. “A xeno-free microcarrier-based stirred culture system for the scalable expansion of human mesenchymal stem/stromal cells isolated from bone marrow and adipose tissue.” Biotechnol J. 2015 Aug;10(8):1235-47

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