Mesenchymal Stromal Cells Production Research Articles

Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate

Background aimsThe selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. MethodsIn this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. ResultsThe introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 105 and 1.02 ± 0.005 × 105 cells/mL compared with 0.79 ± 0.05 × 105 and 0.36 ± 0.04 × 105 cells/mL in FBS-containing medium. ConclusionsThis study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up.

Unraveling mechanisms of mesenchymal stromal cell-mediated immunomodulation through patient monitoring and product characterization.

Mesenchymal stromal cells (MSCs) are increasingly used in the treatment of a variety of clinical conditions and to modulate immune responses in conditions related to auto-/alloimmunity, including graft-versus-host disease (GvHD). Although pilot data are promising, treatment responses have been highly variable, and further development of this as a therapeutic modality depends on increased insight into the properties of clinical MSC products and on understanding the mechanisms underlying responses in patients. Here we review the mechanisms that possibly underlie the capacity of MSCs to treat auto-/alloimmunity, and describe how patient monitoring can help to identify the in vivo mechanisms of action in the treatment of GvHD. Since MSCs used in the clinic originate from various donors and from a heterogeneous population of cells, we will also discuss recent insights into MSC heterogeneity and their implications for clinical MSC products. Finally, we describe a framework to improve our understanding of the efficacy and working mechanism of MSCs, which involves patient monitoring and more extensive characterization of the heterogeneity within and between different MSC preparations.

Serum-free process development: improving the yield and consistency of human mesenchymal stromal cell production

Background aimsThe cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving product yield and driving manufacturing consistency. MethodsBone marrow–derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. ResultsExpansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation (R2 = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. ConclusionsWe have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.

Regulatory environment for the development of an allogeneic mesenchymal stromal cell product for use in clinical trials

Regulatory environment for the development of an allogeneic mesenchymal stromal cell product for use in clinical trials

Ex vivo expanded mesenchymal stromal cell minimal quality requirements for clinical application.

Mesenchymal stromal cells (MSCs), as advanced therapy products, must satisfy all the requirements for human use of medicinal products, aiming to maintain the quality and safety of the cells. The MSC manufacturing process for clinical use should comply with the principles of Good Manufacturing Practice (GMP). This ensures that cell preparations are produced and controlled, from the collection and manipulation of raw materials, through the processing of intermediate products, to the quality controls, storage, labeling and packaging, and release. The objective of this document is to provide the minimal quality requirements for the MSC production and its delivery for clinical use, so that the safety of the final cell therapy product will not be compromised. For this purpose, the document evaluates the most important steps of GMP-compliant MSC production: the isolation and expansion process; the validation phase of the process, including all quality controls for the characterization, functionality, potency, and safety of MSCs; and the quality control at the batch release to guarantee the safety of patient infusion.

A Novel Standardized Quantitative Suppression Assay Reveals a Diversity of Human Immune-Regulatory Cell Potency

Immunologic tolerance is a critical homeostatic function to protect self from auto-immunity. Various immune-regulatory cells, including regulatory T cell (Treg), myeloid derived suppressor cell (MDSC), tolerogenic dendritic cell, and mesenchymal stromal cell (MSC) are responsible for orchestrating this tolerance. Immune-regulatory cells play a central role in the pathophysiology of cancer immunity, autoimmune disease, and graft versus host disease and can be used in adoptive cell therapy. There is therefore a need for a standardized method to evaluate the suppressive function of these heterogeneous cell populations. We developed an in vitro standardized quantitative suppression assay utilizing the suppressor cell line KARPAS-299 as a standard by which to compare suppressive potency of human immune-regulatory cell populations.Peripheral CD4 T-cells, used as targets in all assays, were isolated from healthy donors (n=30) using an automated cell separator or by flow sorting. After labeling with Cell Tracer Violet (CTV), CD4 T-cells were co-cultured with immune-regulatory cells such as autologous Treg, autologous or third party MDSC (CD11b+CD14+), third party bone marrow derived MSC, and primary leukemia cells, or with the reference KARPAS-299 cells. After stimulating with CD3/CD28 beads, CD154 activation of CD4 T-cells was measured at 16 hours, and CD4 T-cell proliferation was measured by CTV dilution within the viable cell population at 72 hours. Suppressive capacities of immune-regulatory cell types were represented as KARPAS-299 suppressor units (KSU), calculated using the equation b/a, where b is the percentage of suppression in the presence of a given immune-regulatory cell, and a is the percentage of suppression in the presence of KARPAS-299.KARPAS-299 cells reproducibly suppressed healthy donor CD4 T-cells at suppressor: responder ratio of 4:1 for both CD154 activation (percentage of suppression 46.2±18.8%) and proliferation (51.0±20.7%). Immune-regulatory cells showed diverse suppressive capacities: autologous MDSC (63.2±24.5%), allogeneic MDSC (55.8±21.8%), third party MSC (51.26±23.67%), and autologous Treg, (8.7±10.4%). After standardization with KSU, MDSC and MSC showed significantly higher suppressive capacity compared to Treg (MDSC 1.18±0.6 KSU, MSC 1.3±1.11 KSU, Treg 0.15±0.3 KSU: p<0.0001). This standardized assay was also applicable to other types of proliferating targets, such as flow-sorted conventional T cells (Tcon: CD4+CD127highCD25low: 2.73±1.78 KSU with autologous MDSC, 1.6±0.75 KSU with third party MSC) and CD8 T-cells (1.49±0.96 KSU with autologous MDSC, 1.08±0.77 with third party MSC). We validated the method as a potency assay of MSC products, and showed inter-individual differences in MSC. Finally, we used the assay to demonstrate a modest suppressive capacity of acute myeloid leukemia blasts (0.35±0.28 KSU).This method provides a platform for standardizing suppressor function to facilitate comparison between laboratories and for use in cell product release assay. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A reproducible immunopotency assay to measure mesenchymal stromal cell–mediated T-cell suppression

Background aimsThe T-cell suppressive property of bone marrow–derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T-cell suppression. MethodsAt the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center, we developed an in vitro quality control T-cell suppression immunopotency assay (IPA) that uses anti-CD3 and anti-CD28 antibodies to stimulate T-cell proliferation. We measured MSC-induced suppression of CD4+ T-cell proliferation at various effector-to-target cell ratios with the use of defined peripheral blood mononuclear cells and in parallel compared with a reference standard MSC product. We calculated an IPA value for suppression of CD4+ T cells for each MSC product. ResultsEleven MSC products generated at three independent PACT centers were evaluated for cell surface phenotypic markers and T-cell suppressive properties. Flow cytometry results demonstrated typical MSC cell surface marker profiles. There was significant variability in the level of suppression of T-cell proliferation, with immunopotency assay values ranging from 27% to 88%. However, MSC suppression did not correlate with human leukocyte antigen–DR expression. ConclusionsWe have developed a reproducible immunopotency assay to measure allogeneic MSC-mediated suppression of CD4+ T cells. Additional studies may be warranted to determine how these in vitro assay results may correlate with other immunomodulatory properties of MSCs, in addition to evaluating the ability of this assay to predict in vivo efficacy.

Clinical mesenchymal stromal cell products undergo functional changes in response to freezing

Background aimsCurrent methods of mesenchymal stromal cell (MSC) cryopreservation result in variable post-thaw recovery and phenotypic changes caused by freezing. The objective of this investigation was to determine the influence of ex vivo cell expansion on phenotype of MSCs and the response of resulting phenotypes to freezing and thawing. MethodsHuman bone marrow aspirate was used. MSCs were isolated and cells were assessed for total count, viability, apoptosis and senescence over 6 passages (8–10 doublings/passage) in ex vivo culture. One half of cells harvested at each passage were re-plated for continued culture and the other half were frozen at 1°C/min in a controlled-rate freezer. Frozen samples were stored in liquid nitrogen, thawed and reassessed for total cell count, viability and senescence immediately and 48 h after thaw. ResultsViability did not differ significantly between samples before freeze or after thaw. Senescence increased over time in pre-freeze culture and was significantly higher in one sample that had growth arrest both before freeze and after thaw. Freezing resulted in similar initial post-thaw recovery in all samples, but 48-h post-thaw growth arrest was observed in the sample with high senescence only. ConclusionsHigh pre-freeze senescence appears to correlate with poor post-thaw function in MSC samples, but additional studies are necessary to obtain a sample sizes large enough to quantify results.

Increasing efficiency of human mesenchymal stromal cell culture by optimization of microcarrier concentration and design of medium feed

Background aimsLarge amounts of human mesenchymal stromal cells (MSCs) are needed for clinical cellular therapy. In a previous publication, we described a microcarrier-based process for expansion of MSCs. The present study optimized this process by selecting suitable basal media, microcarrier concentration and feeding regime to achieve higher cell yields and more efficient medium utilization. MethodsMSCs were expanded in stirred cultures on Cytodex 3 microcarriers with media containing 10% fetal bovine serum. Process optimization was carried out in spinner flasks. A 2-L bioreactor with an automated feeding system was used to validate the optimized parameters explored in spinner flask cultures. ResultsMinimum essential medium-α–based medium supported faster MSC growth on microcarriers than did Dulbecco's modified Eagle's medium (doubling time, 31.6 ± 1.4 vs 42 ± 1.7 h) and shortened the process time. At microcarrier concentration of 8 mg/mL, a high cell concentration of 1.08 × 106 cells/mL with confluent cell concentration of 4.7 × 104cells/cm2 was achieved. Instead of 50% medium exchange every 2 days, we have designed a full medium feed that is based on glucose consumption rate. The optimal medium feed that consisted of 1.5 g/L glucose supported MSC growth to full confluency while achieving the low medium usage efficiency of 3.29 mL/106cells. Finally, a controlled bioreactor with the optimized parameters achieved maximal confluent cell concentration with 16-fold expansion and a further improved medium usage efficiency of 1.68 mL/106cells. ConclusionsWe have optimized the microcarrier-based platform for expansion of MSCs that generated high cell yields in a more efficient and cost-effective manner. This study highlighted the critical parameters in the optimization of MSC production process.

Validation and GMP production of mesenchymal stromal cells batches for clinical application in orthopedics and rheumatology

Validation and GMP production of mesenchymal stromal cells batches for clinical application in orthopedics and rheumatology

Soliciting Strategies for Developing Cell-Based Reference Materials to Advance Mesenchymal Stromal Cell Research and Clinical Translation

The mesenchymal stromal cell (MSC) field continues to rapidly progress with a number of clinical trials initiated and completed, with some reported successes in multiple clinical indications, and a growing number of companies established. The field, nevertheless, faces several challenges. Persistent issues include the definition of a MSC and comparability between MSC preparations. This is because of inherent cell heterogeneity, the absence of markers that are unique to MSCs, and the difficulty in precisely defining them by developmental origin. Differences in the properties of MSCs also depend on the site of tissue harvest, phenotypic and genotypic characteristics of the donor and the isolation, and storage and expansion methods used. These differences may be sufficient to ensure that attributes of the final MSC product could differ in potentially significant ways. Since there are currently no gold standards, we propose using a reference material to establish methods of comparability among MSC preparations. We suggest four possible "ruler scenarios" and a method for global distribution. We further suggest that critical to establishing a reference material is the need to define protocols for comparing cells. The main purpose of this article is to solicit input in establishing a consensus-based comparison. A comparative approach will be critical to all stages of translation to better clarify mechanisms of MSC actions, define an optimal cell manufacturing process, ensure best practice clinical investigations, extend the use of an MSC product for new indications, protect an MSC product from imitators, and develop uniform reimbursement policies. Importantly, a reference material may enable a consensus on a practical definition of MSCs.

Beyond genetic stability of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) have been used in the clinic for more than a decade. However, this rapidly emerging area of research (352 clinical trials registered at ClinicalTrials.gov) is associated with much debate and controversy. A major issue is the safety and risk of transformation of MSCs during ex vivo expansion. Previous articles (1,2), now retracted (3,4), or analyses (5) reported the transformation or risk of transformation of MSCs from different sources during expansion. Although such transformation is likely a rare event (6,7), some genetic abnormalities, mainly aneuploidy, can appear during culture but are not related to the transformation per se (8). Consequently, from a regulatory point of view, we need to develop processes avoiding such transformation that use relevant controls to avoid the risk of transformation. A recent review, following a meeting of the European Medicines Agency in London to ensure the safety of MSC production, at least in pre-clinical settings, determined that cytogenetic abnormalities should be assessed (9). In this issue of Cytotherapy, three teams report on the genetic stability of ex vivo-expanded human MSCs. MSCs used in these studies came from three different sources: two from adult tissue [bonemarrow (BMMSCs) (10) and adipose tissue (ASCs) (11)] and one from a fetal source [chorionic villi (CV MSCs) (12)]. The culture processes differed. BMMSCswere expanded in a bioreactor (Quantum Cell Expansion System; Terumo BCT, Lakewood, CO, USA), and ASCs and CV MSCs were expanded in flasks. The media were similar, including fetal bovine serum, with the addition of conditioned medium for CV MSCs. Finally, a wide range of techniques were used to assess genetic stability, including conventional karyotyping,

Serum‐free media for the production of human mesenchymal stromal cells: a review

The regenerative potential of mesenchymal stromal cells (MSC) holds great promise in using them for treatment of a wide range of debilitating diseases. Several types of culture media and systems have been used for large-scale expansion of MSCs in vitro; however, the majority of them rely heavily on using foetal bovine serum (FBS)-supplement for optimal cell proliferation. FBS-based cultures pose the potential threat of spread of transmissible spongiform encephalopathy and bovine spongiform encephalopathy to MSCs and then to their recipients. A recent trend in cell culture is to change from serum-use to serum-free media (SFM). In this context, the current review focuses specifically on employment of various SFM for MSCs and discusses existences of various options with which to substitute FBS. In addition, we analyse MSC population growth kinetic patterns using various SFM for large-scale production of MSCs.

In Vivo Production of Mesenchymal Stromal Cells After Injection of Autologous Platelet-Rich Plasma Activated by Recombinant Human Soluble Tissue Factor in the Bone Marrow of Healthy Volunteers

Autologous mesenchymal stromal cell (MSC)-based therapies offer one of the most promising and safe methods for regeneration or reconstruction of tissues and organs. Routine procedures to obtain adequate amount of autologous stem cells need their expansion through culture, with risks of contamination and cell differentiation, leading to the loss of cell ability for therapies. We suggest the use of human bone marrow (BM) as a physiological bioreactor to produce autologous MSC by injection of autologous platelet-rich plasma activated by recombinant human soluble tissue factor (rhsTF) in iliac crest. A trial on 13 healthy volunteers showed the feasibility and harmlessness of the procedure. The phenotype and cellularity of BM cells were not modified, on day 3 after injection. Endothelial progenitor cells (EPC) were mobilized to the bloodstream, without stimulation of hematopoietic stem cells (HSC). MSC level in BM increased with a specific commitment to preosteoblastic cell population both in vivo and in vitro. This self-stimulation system of BM seems thus to be a promising feasible process 3 days before clinical cell therapy applications.

Mesenchymal Stromal Cells; Role in Tissue Repair, Drug Discovery and Immune Modulation

Mesenchymal stromal cells (MSCs) participate in repair of damaged tissues, possess the potential to serve as a useful tool in the drug discovery field and exert immunosuppressive effects as demonstrated by their ability to modulate the immune response. Herein, the roles played by MSC differentiation and/or production of trophic factors involved in tissue repair are discussed. MSCs offer the opportunity to probe targets that conventional or differentiated cell lines do not express; thus providing a more refined system that allows identification of novel therapeutics. However, there are difficulties associated with drug discovery assays to which MSCs are not exempt. The immunosuppressive potential of MSCs has already been utilised in clinical trials where MSCs have been used to treat patients with graft- versus- host disease (GvHD) and autoimmune diseases. Another possible therapeutic application of MSCs lies in the field of transplantation tolerance. Although the capacity of MSCs to modulate immune responses has received much attention, the role of MSCs in transplantation tolerance is as yet unclear. In this review, we discuss the evidence for MSC induction of a state of tolerance in the transplantation setting.

Impact of oxygen concentration on growth of mesenchymal stromal cells from the marrow of patients with chronic lymphocytic leukemia

AbstractChronic lymphocytic leukemia (CLL) cells interact in the marrow with mesenchymal stromal cells (MSCs), which can enhance CLL-cells' resistance to spontaneous or drug-induced apoptosis. Here we examined the effect of oxygen on the growth and function of MSCs from marrow aspirates of CLL patients. Cultures in ambient oxygen provided for poor recovery and growth of MSCs, which developed features of cell-senescence. However, MSCs were propagated readily from the same cells when they were cultured at a physiologic oxygen concentration of 5%. Such MSCs promoted short-term CLL-cell survival in either 5% or ambient O2. However, longer-term CLL-cell survival was enhanced when the cocultures were maintained in 5% O2 versus 21% O2 because of increased MSC proliferation and production of soluble prosurvival factors, such as CXCL12. This study establishes the importance of physiologic oxygen concentration in the propagation and function of MSCs derived from marrow aspirates of CLL patients in vitro.

Mast Cell-Activated Bone Marrow Mesenchymal Stromal Cells Regulate Proliferation and Lineage Commitment of CD34+ Progenitor Cells

Background: Shortly after allergen exposure, the number of bone marrow (BM) and circulating CD34+ progenitors increases. We aim to analyze the possible mechanism whereby the allergic reaction stimulates BM to release these effector cells in increased numbers. We hypothesize that mast cells (MCs) may play a predominant role in this process.Objective: To examine the effect of IgE-activated MCs on BM mesenchymal stromal cells which regulate proliferation and differentiation of CD34+ progenitors.Methods: Primary MCs were derived from CD34+ precursors and activated with IgE/anti-IgE. BM mesenchymal stromal cells were co-cultured with CD34+ progenitor cells and stimulated with IL-1/TNF or IgE/anti-IgE-activated MCs in Transwell system.Results: BM mesenchymal stromal cells produce low level of thymic stromal lymphopoietin (TSLP) under steady state conditions, which is markedly increased by stimulation with proinflammatory cytokines IL-1 and TNF or IgE-activated MCs. The latter also triggers bone marrow-derived mesenchymal stromal cells production of G-CSF, and GM-CSF while inhibiting SDF-1. MC-activated mesenchymal stromal cells stimulate CD34+ cells to proliferate and to regulate their expression of early allergy-associated genes.Conclusion and Clinical Relevance: This in vitro study indicates that IgE-activated MCs trigger BM mesenchymal stromal cells to release TSLP and hematopoietic growth factors and to regulate the proliferation and lineage commitment of CD34+ precursor cells. The data predict that the effective inhibition of MCs should impair mobilization and accumulation of allergic effector cells and thereby reduce the severity of allergic diseases.

Improved isolation and expansion of bone marrow mesenchymal stromal cells using a novel marrow filter device

Mesenchymal stromal cells (MSCs) have been studied as cell therapy to treat a vast array of diseases. In clinical MSC production, the isolated cells must undergo extensive ex vivo expansion to obtain a sufficient dose of MSCs for the investigational treatment. However, extended tissue culture is fraught with potential hazards, including contamination and malignant transformation. Changes of gene expression with prolonged culture may alter the therapeutic potential of the cells. Increasing the recovery of MSCs from the freshly harvested bone marrow allowing for less ex vivo expansion would represent a major advance in MSC therapy. Human bone marrow cells from eight healthy donors were processed using a marrow filter device and, in parallel, using buoyant density centrifugation by two independent investigators. The initial nucleated cell recovery and the final yield, immunophenotype and trilineage differentiation potential of second-passage MSCs were examined. The marrow filter device generated significantly greater initial cell recovery requiring less investigator time and resulted in approximately 2.5-fold more MSCs after the second passage. The immunophenotype and differentiation potential of MSCs isolated using the two methods were equivalent and consistent with the defining criteria. The two independent investigators generated comparable results. This novel filter device is a fast, efficient and reliable system to isolate MSCs and should greatly expedite pre-clinical and clinical investigations of MSC therapy.

The mesenchymal stromal cells dilemma—does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road?

The use of cryopreserved unmatched allogeneic mesenchymal stromal cells (MSCs) for treatment of steroid-resistant graft-versus-host disease has become medical practice in many European jurisdictions. The enthusiasm for use of MSCs in transplantation medicine builds on compelling phase II clinical trial data published by European collaborative groups in the past few years. Notwithstanding, it was reported in 2009 that a large multicenter phase III clinical trial (NCT00366145) conducted in the USA examining the use of an industrial MSC product (Prochymal; Osiris Therapeutics, Inc., Columbia, MD, USA) failed to meet its primary clinical endpoint of achieving a significant increase of complete response of steroid-resistant graft-versus-host disease lasting at least 28 days compared with placebo. Although peer-reviewed publication of the trial and its results are not in public domain at the time of this writing, it is worthwhile to reflect on the apparent discrepancy between the European experience and this industry-sponsored phase III study. This review presents a heuristic failure analysis focusing on the potential variables affecting MSCs and their utility as a cellular pharmaceutical.

Mesenchymal Stromal Cells Support Follicular Lymphoma-Associated Follicular Helper T-Cells, in Part, Through an IL-6 and IL-21-Dependent Mechanism.

Abstract 2707The biology of follicular lymphoma (FL) is largely dictated by the immune-effector and stromal cells that comprise its tumor microenvironment. Recent studies have demonstrated that various infiltrating T-cell populations can be predictive of clinical outcome, with two such populations, follicular helper T-cells (TFH) and regulatory T-cells (Treg) likely playing critical roles in the biology of this disease. TFH are normally responsible for the activation and maintenance of germinal center B-cells while Tregs suppress effector T-cell priming and function as well as suppress normal B-cell proliferation and differentiation. There is also data to support the notion that both of these cells have direct and/or indirect effects on FL B-cells. These cells and other immune-effector cells within the FL microenvironment have dynamic interactions with mesenchymal stromal cells (MSC), multipotent cells residing in most adult tissues, which can be recruited by FL B-cells into the tumor microenvironment where they inhibit anti-lymphoma T-cell responses and FL B-cell apoptosis. We hypothesized that such MSC play a role in modulating TFH viability, similar to what has been previously reported for Treg, and herein we show such a novel role for MSC, that being the selective support of FL-derived TFH populations.To determine the effect of MSC on the viability of discrete T-cell populations infiltrating FL lymph nodes (FLN), un-separated single cell suspensions (SCS) from FLN (n=13) were cultured for 48 hours alone or with normal tonsillar-derived MSC. The fold change in the proportion of CD4 T-cells that are TFH (CXCR5+PD-1+Bcl-6+), Tregs (CD25+FoxP3+), TH1 (T-bet+), TH2 (GATA-3+) or TH17 (ROR-gamma-t+) cells in SCS incubated with MSC was compared to that of SCS incubated without MSC. A 2.3-fold increase in the percentage of TFH (p=0.006) cells and a 2-fold increase in the percentage of Tregs (p=0.007) were observed in the SCS cultured with MSC compared to those cultured without MSC. The effect of MSC in maintaining TFH and Tregs was selective, as in contrast, the proportion of T-cells expressing the canonical TH1, TH2 and TH17 transcription factors was not increased after culture with MSC. B-cells provide survival signals to TFH and stromal cells provide survival signals to B-cells, therefore we next determined whether the stromal support of TFH was mediated via B-cells. Both purified FL T-cells and B-cell-depleted FL SCS cultured on MSC showed a similar increase in TFH (and Treg) populations as seen with un-separated FL-B SCS, indicating that MSC support of TFH and Tregs was independent of B-cells. MSC only partially supported TFH and Treg populations in transwell experiments suggesting a role for both cell-cell contact and soluble factors. In this regard, IL-6 and IL-21 are known to be secreted by MSC and to signal through gp130 receptors on TFH to induce the transcription of Bcl-6, which is required for TFH differentiation. Blocking IL-6 and IL-21 decreased MSC support of TFH by 42% (p=0.021), while reducing MSC support of Tregs by only 17% (p=0.018) suggesting that IL-6 and IL-21 mediate, in part, the protective effect that MSC have on TFH but that other cytokines are likely to play a role in supporting Tregs. Finally, we have shown that FLN-derived MSC support both FL T-cells and normal lymph node T-cells to a similar extent as the tonsillar MSC used in these experiments. This provides support for the use of tonsillar MSC in these experiments as we were able to generate and expand these more consistently than MSC from FLN, which allowed us to use a consistent MSC product for all experiments.These findings therefore demonstrate a new role for MSC in FL, that being to support FL TFH in addition to Treg populations in the tumor microenvironment. MSCs have been shown to support FL B-cell viability and suppress anti-lymphoma T-cell responses. This finding that MSCs support TFH cells, a population that may provide survival signals to FL B-cells, further supports the potential of targeting MSC as a novel therapeutic strategy for patients with FL. Disclosures:No relevant conflicts of interest to declare.