Clinical-grade Mesenchymal Stromal Cells Research Articles

Immunomodulatory performance of GMP-compliant, clinical-grade mesenchymal stromal cells from four different sources

Inflammatory and autoimmune diseases are among the most challenging disorders for health care professionals that require systemic immune suppression which associates with various side effects. Mesenchymal stromal cells (MSCs) are capable of regulating immune responses, mainly through paracrine effects and cell-cell contact. Since MSCs are advanced therapy medicinal products (ATMPs), they must follow Good Manufacturing Practice (GMP) regulations to ensure their safety and efficacy. In this study, we evaluated the immunomodulatory effects of GMP-compliant clinical grade MSCs obtained from four different sources (bone marrow, adipose tissue, Wharton’s Jelly, and decidua tissue) on allogeneic peripheral blood mononuclear cells (PBMCs). Our results revealed that WJ-MSCs were the most successful group in inhibiting PBMC proliferation as confirmed by BrdU analysis. Moreover, WJ-MSCs were the strongest group in enhancing the regulatory T cell population of PBMCs. WJ-MSCs also had the highest secretory profile of prostaglandin E2 (PGE-2), anti-inflammatory cytokine, while interleukin-10 (IL-10) secretion was highest in the DS-MSC group. DS-MSCs also had the lowest secretion of IL-12 and IL-17 inflammatory cytokines. Transcriptome analysis revealed that WJ-MSCs had the lowest expression of IL-6, while DS-MSCs were the most potent group in the expression of immunomodulatory factors such as hepatocyte growth factor (HGF) and transforming growth factor-β (TGF- β). Taken together, our results indicated that GMP-compliant Wharton’s Jelly and decidua-derived MSCs showed the best immunomodulatory performance considering paracrine factors.

Comparability exercise of critical quality attributes of clinical-grade human mesenchymal stromal cells from the Wharton’s jelly: single-use stirred tank bioreactors versus planar culture systems

Background aimsThe increasing demand of clinical-grade mesenchymal stromal cells (MSCs) for use in advanced therapy medicinal products (ATMPs) require a re-evaluation of manufacturing strategies, ensuring scalability from two-dimensional (2D) surfaces to volumetric (3D) productivities. Herein we describe the design and validation of a Good Manufacturing Practice–compliant 3D culture methodology using microcarriers and 3-L single-use stirred tank bioreactors (STRs) for the expansion of Wharton's jelly (WJ)-derived MSCs in accordance to current regulatory and quality requirements. MethodsMSC,WJ were successfully expanded in 3D and final product characterization was in conformity with Critical Quality Attributes and product specifications previously established for 2D expansion conditions. ResultsAfter 6 days of culture, cell yields in the final product from the 3D cultures (mean 9.48 × 108 ± 1.07 × 107 cells) were slightly lower but comparable with those obtained from 2D surfaces (mean 9.73 × 108 ± 2.36 × 108 cells) after 8 days. In all analyzed batches, viability was >90%. Immunophenotype of MSC,WJ was highly positive for CD90 and CD73 markers and lacked of expression of CD31, CD45 and HLA-DR. Compared with 2D expansions, CD105 was detected at lower levels in 3D cultures due to the harvesting procedure from microcarriers involving trypsin at high concentration, and this had no impact on multipotency. Cells presented normal karyotype and strong immunomodulatory potential in vitro. Sterility, Mycoplasma, endotoxin and adventitious virus were negative in both batches produced. ConclusionsIn summary, we demonstrated the establishment of a feasible and reproducible 3D bioprocess using single-use STR for clinical-grade MSC,WJ production and provide evidence supporting comparability of 3D versus 2D production strategies. This comparability exercise evaluates the direct implementation of using single-use STR for the scale-up production of MSC,WJ and, by extension, other cell types intended for allogeneic therapies.

Engineered clinical-grade mesenchymal stromal cells combating SARS-CoV-2 omicron variants by secreting effective neutralizing antibodies

BackgroundThe emergence of SARS-CoV-2 becomes life-threatening for the older and immunocompromised individuals, whereas limited treatment is available on these populations. Mesenchymal stromal cells (MSCs) have been reported to be useful in SARS-CoV-2 treatment and reduce SARS-CoV-2-related sequelae.ResultsIn this study, we developed an autonomous cellular machine to secret neutralizing antibody in vivo constantly based on the clinical-grade MSCs, to combat SARS-CoV-2 infections. First, various modified recombinant plasmids were constructed and transfected into clinical-grade MSCs by electroporation, for assembly and expression of neutralizing anti-SARS-CoV-2 antibodies. Second, the stable antibody secreting MSCs clones were screened through pseudovirus neutralization assay. Finally, we investigated the pharmacokinetics and biodistribution of neutralizing antibody secreted by engineered MSCs in vivo. The stable clinical-grade MSCs clones, expressing XGv347-10 and LY-CoV1404-5 neutralizing antibodies, exhibited their feasibility and protective efficacy against SARS-CoV-2 infection. Transplanted engineered clinical-grade MSCs effectively delivered the SARS-CoV-2 antibodies to the lung, and the immune hyperresponsiveness caused by COVID-19 was coordinated by MSC clones through inhibiting the differentiation of CD4 + T cells into Th1 and Th17 subpopulations.ConclusionsOur data suggested that engineered clinical-grade MSCs secreting effective neutralizing antibodies as cellular production machines had the potential to combat SARS-CoV-2 infection, which provided a new avenue for effectively treating the older and immunocompromised COVID-19 patients.

Mesenchymal Stromal Cells for Treating Steroid-Resistant Acute and Chronic Graft Versus Host Disease: A Multicenter Compassionate Use Experience.

Graft versus host disease (GVHD) is a severe complication after allogenic hematopoietic cell transplantation (HSCT). Several clinical trials have reported the use of mesenchymal stromal cells (MSCs) for the treatment of GVHD. In March 2008, the Andalusian Health Care System launched a compassionate use program to treat steroid-resistant GVHD with MSC. Clinical-grade MSC were obtained under GMP conditions. MSC therapy was administered intravenously in four separate doses of 1 × 106 cells/kg. Sixty-two patients, 45 males (7 children) and 17 females (2 children), received the treatment. Patients had a median age of 39 years (range: 7–66) at the time of the allogenic HSCT. The overall response was achieved in 58.7% of patients with acute (a)GVHD. Two years’ survival for aGVHD responders was 51.85%. The overall response for patients with chronic (c)GVHD was 65.50% and the 2-year survival rate for responders was 70%. Age at the time of HSCT was the only predictor found to be inversely correlated with survival in aGVHD. Regarding safety, four adverse events were reported, all recovered without sequelae. Thus, analysis of this compassionate use experience shows MSC to be an effective and safe therapeutic option for treating refractory GVHD, resulting in a significant proportion of patients responding to the therapy.

Current good manufacturing practice considerations for mesenchymal stromal cells as therapeutic agents

Producing human mesenchymal stromal cells (MSCs) for clinical use requires adherence to current good manufacturing practice (cGMP) standards. This is necessary for ensuring standardization and reproducibility through the manufacturing process, but also, for product quality and safety. However, the large-scale production of clinical-grade MSCs possesses unique regulatory challenges and hurdles related to the heterogeneous nature of MSC cultures as well as the complex manufacturing process. Following is a compilation of the major issues encountered in the manufacturing of MSCs for clinical use, and our views on the optimal characteristics of the final MSC product.

Finding meaning: Potential relationship between critical quality attributes of MSC's and their Clinical efficacy in GvHD

Background & Aim Mesenchymal stromal cells (MSC) display immunomodulatory properties that make them a very attractive tool to treat diseases such as Acute Graft Versus Host Disease (aGVHD). However, although the use of MSC in aGVHD is feasible and safe, its efficacy is completely unpredictable. MSC were first isolated from bone marrow (BM-MSC) but can also be isolated from different tissues such as Wharton's jelly from Umbilical Cord (WJ-MSC). Indeed WJ-MSC have advantages over BM-MSC for standardization of procedures and large-scale production but equivalence of their immunomodulatory activity is not fully demonstrated. This study seeks to elucidate the relationship between the critical quality attributes of BM-MSC and WJ-MSC and the efficacy in the treatment of aGVHD. Methods, Results & Conclusion To obtain and process the cells, an approved IND was followed in our GMP-compliant production facilities using BM-MSC's (PEI 13-081) or WJ-MSC's (PEI 16-017). Retrospective study of patients of four hospitals of Barcelona area with refractory aGVHD treated with MSC produced at Banc de Sang i Teixits between 2017-2019. We have successfully released 13 clinical-grade batches. Cell viability in all of them was >70% and phenotypic profiling resulted in >95% positivity for the expression of CD73, CD90 and CD105 surface markers. Regarding the HLA-DR- marker, although it was expected to be a negative marker on MSCs, we verified that this expression was highly variable especially in BM-MSC in agreement with our previous observations (Figure 1A). The critical quality attributes for each one of the 13 batches (some of the stock batches have not yet been used in patients) and preliminary results from treated patients are summarized in Figure 1B. Interestingly, no differences were found so far between BM-MSC and WJ-MSC in the clinical response of aGVHD treated patients. Although a comprehensive analysis of the characteristics of MSC may still be needed, it is intriguing that, from our preliminary data, in vitro immunomodulatory capacity of WJ-MSC evaluated by measuring the % of inhibition of PBMNC's proliferation shown in potency assays does not correlate with MSC's clinical efficacy. Further work may be needed to understand the mechanisms of apoptosis in clinical grade MSC in line with recent observations made by other authors who have suggested that the response may be mediated by the receptor's ability to induce MCS's apoptosis.

Developing a flow cytometry-based quantitative ido assay to measure immune potency of mesenchymal stromal cells product for phase i clinical trial

Background & Aim Establishment of a potency assay in the manufacturing of clinical grade mesenchymal stromal cells (MSCs) has been a challenge due to the issues of variability in starting cellular material, complex mechanisms of action, and lack of reference cells or materials. Indoleamine 2, 3-dioxygenase (IDO) is one of the most important factors for human MSC-mediated immunosuppression. IDO is expressed in IFN-γ-licensed MSC, but not in resting MSC in vitro. This intracellular enzyme catalyzes tryptophan degradation toward kynurenine and depletion of tryptophan suppresses T cells proliferation. Methods, Results & Conclusion In this study, we have developed a flow cytometry-based IDO assay for quantitatively measuring the IDO expression level per cell, the IPBV (Intracellular Protein Binding Value) of the cells represents the relative quantity of IDO expressed in the MSC. IPBV is defined as the number of anti-IDO fluorescent antibody molecules bound to intracellular IDO and standardized by fluorescence intensity of beads. Two kinds of in-house quality control reference cells were running in parallel with product to ensure that the assay is performing as expected and the level of IDO expression. Validation of the quantitative IDO assay, including accuracy, precision, specificity, linearity, range and robustness, were presented in this study. Moreover, we presented the application of the quantitative IDO assay for analysis of MSC product stability, comparability, and compatibility in the manufacturing of clinical grade MSC-based product- RegStem® ASC. In this study, we demonstrated the flow cytometry-based quantitative MSC IDO assay correlates with MSC's inhibition of autologous T cells proliferation, as well as the correlation with preliminary clinical effects in the phase I autologous MSC-based clinical trial for the treatment of Osteoarthritis. As the results, we believe that our novel flow cytometry-based quantitative IDO assay can be a potential potency assay candidate for advanced phase clinical trial of MSCs products.

A double‐virally‐inactivated (Intercept–solvent/detergent) human platelet lysate for in vitro expansion of human mesenchymal stromal cells

Pooled human platelet lysate (HPL) can replace fetal bovine serum (FBS) as xeno-free supplement for ex vivo expansion of mesenchymal stromal cells (MSCs). We evaluate here whether a double-virally-inactivated HPL (DVI-HPL) prepared from expired Intercept-treated platelet concentrates (PCs) and treated by solvent/detergent (S/D) can be used for MSC expansion. Expired Intercept-treated PCs in 65% platelet (PLT) additive solution were pooled and subjected to a 1% tri-n-butyl phosphate/1% Triton X-45 treatment followed by soybean oil, hydrophobic interaction chromatography purification, and sterile filtration. Bone marrow-derived MSCs (BM-MSCs) were expanded for four passages in growth medium containing 10% DVI-HPL, I-HPL (from Intercept-PC only), untreated HPL, and FBS. MSC morphology, doubling time, immunophenotype, immunosuppressive activity, and differentiation capacity were compared. Expanded cells had typical spindle morphology and showed higher viability in all HPL conditions than in FBS. The DVI-HPL and FBS-expanded cells were morphologically larger than in I-HPL and HPL supplements. The cumulative population doubling was lower using DVI-HPL than with HPL and I-HPL, but significantly higher than using FBS. Immunophenotype was not affected by the supplements used. Immunosuppressive activity was maintained with all supplements. Differentiation capacity into chondrocytes and osteocytes was more effective in DVI-HPL but less toward adipocytes compared to other supplements. Human PLT lysate made from Intercept-PCs subjected to S/D treatment may be an alternative to untreated HPL and to I-HPL for BM-MSC expansion. This finding reinforces the potential of HPL as a virally safe alternative to FBS for clinical grade MSC expansion protocols.

Manufacturing of primed mesenchymal stromal cells for therapy.

Mesenchymal stromal cells (MSCs) for basic research and clinical applications are manufactured and developed as unique cell products by many different manufacturers and laboratories, often under different conditions. The lack of standardization of MSC identity has limited consensus around which MSC properties are relevant for specific outcomes. In this Review, we examine how the choice of media, cell source, culture environment and storage affects the phenotype and clinical utility of MSC-based products, and discuss the techniques better suited to prime MSCs with specific phenotypes of interest and the need for the continued development of standardized assays that provide quality assurance for clinical-grade MSCs. Bioequivalence between cell products and batches must be investigated rather than assumed, so that the diversity of phenotypes between differing MSC products can be accounted for to identify products with the highest therapeutic potential and to preserve their safety in clinical treatments.

Brief Report: Proteasomal Indoleamine 2,3-Dioxygenase Degradation Reduces the Immunosuppressive Potential of Clinical Grade-Mesenchymal Stromal Cells Undergoing Replicative Senescence.

Owing to their immunosuppressive properties, mesenchymal stromal cells (MSCs) obtained from bone marrow (BM-MSCs) or adipose tissue (ASCs) are considered a promising tool for cell therapy. However, important issues should be considered to ensure the reproducible production of efficient and safe clinical-grade MSCs. In particular, high expansion rate, associated with progressive senescence, was recently proposed as one of the parameters that could alter MSC functionality. In this study, we directly address the consequences of replicative senescence on BM-MSC and ASC immunomodulatory properties. We demonstrate that MSCs produced according to GMP procedures inhibit less efficiently T-cell, but not Natural Killer (NK)- and B-cell, proliferation after reaching senescence. Senescence-related loss-of-function is associated with a decreased indoleamine 2,3-dioxygenase (IDO) activity in response to inflammatory stimuli. In particular, although STAT-1-dependent IDO expression is transcriptionally induced at a similar level in senescent and nonsenescent MSCs, IDO protein is specifically degraded by the proteasome in senescent ASCs and BM-MSCs, a process that could be reversed by the MG132 proteasome inhibitor. These data encourage the use of appropriate quality controls focusing on immunosuppressive mechanisms before translating clinical-grade MSCs in the clinic. Stem Cells 2017;35:1431-1436.

Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production.

BackgroundClinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL).MethodsIn this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors.ResultsWe characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells.ConclusionsOur study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria.Clinical trialsUse of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007. Registered August 26, 2013.MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540. Registered April 23, 2013.Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027. Registered October 31, 2014.Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145. Registered August 17, 2006.A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283. Registered May 18, 2012.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0370-8) contains supplementary material, which is available to authorized users.

Clinical-scale expansion of mesenchymal stromal cells: a large banking experience

BackgroundMesenchymal stromal cells (MSC) are largely investigated in clinical trials aiming to control inappropriate immune reactions (GVHD, Crohn’s disease, solid organ transplantation). As the percentage of MSC precursors in bone marrow is very low, these must be expanded in vitro to obtain therapeutic cell doses. We describe here the constitution of an allogeneic human third-party MSC bank from screened healthy volunteer donors in compliance with quality specifications and ISCT-release criteria and report follow-up of different aspects of this activity since 2007.Methods68 clinical-grade large-scale MSC cultures were completed and analyzed. The whole process was described, including volunteer donor screening, bone marrow collection, mononuclear cell isolation and expansion over 4 weeks, harvesting, cryopreservation, release, administration and quality controls of the cells (including microbiology, phenotype, and potency assays).ResultsFrom 59 validated donors, 68 cultures were completed (mean of final yields: 886 × 106 cells/culture) and a total of 464 MSC aliquots have been produced and stored in liquid nitrogen (mean of 132.8 × 106 cells/bag). Each MSC batch underwent extensive testing to verify its conformity with EBMT and ISCT release criteria and was individually validated. As of June 1 2015, 314 bags have been released and infused to patients included in 6 different clinical protocols. All thawed MSC units satisfied to release criteria and no infusion-related toxicity was reported.ConclusionIn conclusion, despite low passage cultures, we have been able to create an allogeneic “off-the-shelf” MSC bank with a large number of frozen aliquots and report here an efficient clinical-grade MSC banking activity in place for more than 7 years. Our challenge now is to produce MSC in compliance with good manufacturing practices (GMP) as, in the meantime, MSC have become considered as advanced therapy medicinal products (ATMP). Another significant challenge remains the development of relevant potency assay.

Treatment of Chronic GvHD with Mesenchymal Stromal Cells Induces Durable Responses; A Phase II Study

IntroductionChronic graft-versus-host disease (cGvHD) is major cause of late morbidity and mortality after allogeneic hematopoietic stem cell transplantation, with no established second-line treatment. Mesenchymal stromal cells (MSC) have been widely studied in the treatment of acute GvHD, but the clinical reports in cGvHD are scarse. In this study, we aim to evaluate the effects of multiple infusions of MSC on patients with severe refractory cGvHD, including long-term follow-up and immunological evaluations.Patients and MethodsThe study comprises 11 patients (6 female/5 male, with a median age of 49 (range 20-61)) with severe cGvHD from Karolinska University Hospital, Stockholm, Sweden. Inclusion criteria was cGvHD of grade moderate to severe, refractory to or not tolerating 3 months standard treatment of calcineurin inhibitor plus steroids. Median time from cGvHD diagnosis to study inclusion was 18 months (range 6-70), and median number of previous systemic cGvHD treatments was 4 (range 1-10). Median of cGvHD affected organs was 5 (range 3-6), of which NIH organ score >= 2 in a median of 4 (range 1-5). The global severity score median was 8 (range 6-9). Most commonly affected organs were skin (n=10), eyes (9) and joints (8).Clinical grade MSC were harvested from bone marrow of unrelated healthy donors. MSCs were administered intravenously at a dose of 2x10^6 cells/kg at 4-6 weeks intervals. A minimum of 6 doses were given, where response to treatment after 6 doses was observed an additional 1-3 doses were given. All patients continued on their previous immunosuppressive regime to be tapered as clinically indicated.Response evaluation was carried out every three months until the end of treatment. A final formal evaluation was made 12 months after the last dose of MSC, and patients were then followed clinically outside of the study for a median follow-up time of 30 months (range 4-48) from inclusion. Response was graded according to the NIH 2014 consensus criteria with one addition: in case of sclerodermatous disease a reduction in total sclerotic body surface area (BSA) by at least 25% was considered partial response (PR) of the skin.ResultsMain evaluation time point was after 6 infusions. 2 patients discontinued before this point due to death or increasing donor chimerism and one is still receiving treatment.Of the 8 evaluable patients, 5 showed overall PR and were classified as responders and 3 showed mixed response and were classified as non-responders. In the responders, continuing improvement was noted at each follow-up during MSC treatment and at final evaluation at 12 months after finishing MSC treatment. Organ responses were seen in joints (7), skin (4), eyes (3), GI tract (2) and oral cavity, lungs and liver (n=1 each). With a median follow-up time of 29 months (range 21-48), 2 patients have discontinued all systemic immunosuppression and 2 are free of steroids and tapering calcineurin inhibitors. Quality of life was evaluated using the FACT-BMT questionnaire, and responders showed a mean increase in FACT-BMT total score of 6,6 points, or 8%, compared to baseline values, at last follow-up.Clinical response was preceded by reduction in plasma levels of CXCL-9 and CXCL-10, proposed biomarkers for cGvHD, as well as pro-inflammatory cytokines IL-7 and IL-17a.MSC treatment was well tolerated without immediate side effects. 5 events of grade 3 infections were recorded during follow-up. One patient died due to progressive cGvHD after receiving only one MSC infusion. One patient with CLL discontinued the study after 3 infusions due to increasing CD19 donor chimerism, and one year later suffered haematological relapse. One patient with multiple myeloma discontinued after 7 infusions due to reappearence of the M-protein.DiscussionThese promising results show that repeated doses of MSC could be effective in inducing durable responses in severe, refractory cGvHD. Early biomarkers indicative of response would be a useful tool for selecting patients benefiting from prolonged treatment. This study is limited in the number of patients and needs to be followed by larger trials in which the prospective predictive value of biomarker responses could also be evaluated. Close surveillance of patients regarding infectious complications as well as disease recurrence is necessary. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of a cell-banking strategy for the production of clinical grade mesenchymal stromal cells from Wharton's jelly

Background aims. Umbilical cord (UC) has been proposed as a source of mesenchymal stromal cells (MSCs) for use in experimental cell-based therapies provided that its collection does not raise any risk to the donor, and, similar to bone marrow and lipoaspirates, UC-MSCs are multipotent cells with immuno-modulative properties. However, some of the challenges that make a broader use of UC-MSCs difficult include the limited availability of fresh starting tissue, time-consuming processing for successful derivation of cell lines, and the lack of information on identity, potency and genetic stability in extensively expanded UC-MSCs, which are necessary for banking relevant cell numbers for preclinical and clinical studies. Methods. Factors affecting the success of the derivation process (namely, time elapsed from birth to processing and weight of fragments), and methods for establishing a two-tiered system of Master Cell Bank and Working Cell Bank of UC-MSCs were analyzed. Results. Efficient derivation of UC-MSCs was achieved by using UC fragments larger than 7 g that were processed within 80 h from birth. Cells maintained their immunophenotype (being highly positive for CD105, CD90 and CD73 markers), multi-potentiality and immuno-modulative properties beyond 40 cumulative population doublings. No genetic abnormalities were found, as determined by G-banding karyotype, human telomerase reverse transcriptase activity was undetectable and no toxicity was observed in vivo after intravenous administration of UC-MSCs in athymic rats. Discussion. This works demonstrates the feasibility of the derivation and large-scale expansion of UC-MSCs from small and relatively old fragments of UC typically discarded from public cord blood banking programs.

Frequent occurrence of non-malignant genetic alterations in clinical grade mesenchymal stromal cells expanded for cell therapy protocols

Human bone marrow mesenchymal stromal cells (BM-MSC) represent one of the most investigated “advanced therapeutic medicinal products”.[1][1] Recent safety concerns have focused attention on the possible malignant transformation due to mutations acquired during their large-scale in vitro

Inhibition of mesenchymal stromal cells by pre-activated lymphocytes and their culture media

IntroductionDespite having a proven immunosuppressive potential in vitro, human mesenchymal stromal cells (MSCs) are reported to display variable efficacy in vivo and, in fact, their proven benefit in the clinical practice is still limited and controversial.MethodsThe interplay between clinical grade MSCs and pre-activated donor lymphocytes or selected lymphocyte subsets was studied in vitro. The kinetics of MSC growth and viability was evaluated by adhesion-dependent changes of culture plate impedance and biochemically by a colorimetric assay. Activation of natural killer (NK) cells was assessed as well, using a flow cytometry assay.ResultsA strong inhibition of MSC growth was rapidly induced by the addition of pre-activated lymphocytes but not of resting lymphocytes. Inhibition seems not to be attributable to a single cell population, as similar results can be obtained by depleting NK cells or by using either selected CD4+ or CD8+ lymphocytes. In addition, conditioned medium (CM) from activated lymphocytes was able to inhibit MSC growth in a dose-dependent manner. Furthermore, licensing with IFN-γ partially protected MSCs from pre-activated lymphocytes but not from their CM. These results suggest an inhibitory role of lymphocyte-activation-derived substances. However, the identification of a single molecule responsible for MSC inhibition remained elusive, even if preliminary experiments showed that ATP and, to a lesser extent, TNF-α might play a role.ConclusionsThese results suggest that survival of MSCs can be affected by soluble mediators released by activated lymphocytes. Thus it can be hypothesized that MSC immunosuppressive action in vivo could be impaired by ongoing immune activation through the release of inflammatory mediators.

The effect of platelet lysate fibrinogen on the functionality of MSCs in immunotherapy

Human platelet lysate (PL) represents an attractive alternative to fetal bovine serum (FBS) for the ex vivo expansion of human mesenchymal stromal cells (MSCs). However, there is controversy whether MSCs propagated in unfractionated PL retain their immunosuppressive properties. Since fibrinogen can be a major component of PL, we hypothesized that the fibrinogen content in PL negatively affects the suppressor function of MSCs. Pools of outdated plateletpheresis products underwent a double freeze–thaw centrifugation and filtration to produce unfractionated platelet lysates (uPL), followed by a temperature controlled clotting procedure to produce a fibrinogen depleted platelet lysate (fdPL). Fibrinogen depletion affected neither the mitogenic properties of PL or growth factor content, however fdPL was less prone to develop precipitate over time. Functionally, fibrinogen interacted directly with MSCs, dose dependently increased IL-6, IL-8 and MCP-1 protein production, and compromised the ability of MSCs to up-regulate indoleamine dioxygenase (IDO), as well as, mitigate T-cell proliferation. Similarly uPL expanded MSCs showed a reduced capability of inducing IDO and suppressing T-cell proliferation compared to FBS expanded MSCs. Replacing uPL with fdPL largely restored the immune modulating effects of MSCs. Together these data suggest that fibrinogen negatively affects the immunomodulatory functions of MSCs and fdPL can serve as non-xenogenic mitogenic supplement for expansion of clinical grade MSCs for immune modulation.

Immunoregulatory properties of clinical grade mesenchymal stromal cells: evidence, uncertainties, and clinical application

Mesenchymal stromal cell (MSC)-based therapy holds great promise for treating immune disorders and for regenerative medicine in agreement with their paracrine trophic and immunosuppressive activities. Various processes have been developed worldwide to produce clinical grade MSCs but, so far, it is not known if one given MSC is more efficient than another. In addition, while their broad activity on innate and adaptative immune cell subsets is now widely admitted, the precise mechanisms supporting their immunoregulatory capacities are still a matter of debate. Finally, quantitative immunological potency assays correlated to clinical efficacy and clinically relevant immunomonitoring approaches for MSC-treated patients are sorely needed. Multiple parameters could influence the immunomodulatory potential of therapeutic MSCs. The most important challenge is now to differentiate, within a high number of poorly comparable and even contradictory pre-clinical studies, the parameters that could have some clinical impact from those that are only due to uncontrolled experimental variability. Importantly, besides MSC-related differences, primarily linked to production processes, several important variables associated with immune assays themselves, including selection of effector immune cells, activation signals, and read-out techniques, should be carefully considered to obtain solid results with potential therapeutic application. In this review, we establish a core of common and reproducible immunological properties of MSCs, shed light on technical issues concerning immunomodulatory potential assessment, and put them into perspective when considering clinical application.

Manufacturing mesenchymal stromal cells for phase I clinical trials

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells capable of differentiating into adipocytes, osteoblasts and chondroblasts as well as secreting a vast array of soluble mediators. This potentially makes MSCs important mediators of a variety of therapeutic applications. They are actively under evaluation for immunomodulatory purposes such as graft-versus-host disease and Crohn's disease as well as regenerative applications such as stroke and congestive heart failure. We report our method of generating clinical-grade MSCs together with suggestions gathered from manufacturing experience in our Good Manufacturing Practices facility.

Clinical-Grade Mesenchymal Stromal Cells Produced Under Various Good Manufacturing Practice Processes Differ in Their Immunomodulatory Properties: Standardization of Immune Quality Controls

Clinical-grade mesenchymal stromal cells (MSCs) are usually expanded from bone marrow (BMMSCs) or adipose tissue (ADSCs) using processes mainly differing in the use of fetal calf serum (FCS) or human platelet lysate (PL). We aimed to compare immune modulatory properties of clinical-grade MSCs using a combination of fully standardized in vitro assays. BMMSCs expanded with FCS (BMMSC-FCS) or PL (BMMSC-PL), and ADSC-PL were analyzed in quantitative phenotypic and functional experiments, including their capacity to inhibit the proliferation of T, B, and NK cells. The molecular mechanisms supporting T-cell inhibition were investigated. These parameters were also evaluated after pre-stimulation of MSCs with inflammatory cytokines. BMMSC-FCS, BMMSC-PL, and ADSC-PL displayed significant differences in expression of immunosuppressive and adhesion molecules. Standardized functional assays revealed that resting MSCs inhibited proliferation of T and NK cells, but not B cells. ADSC-PL were the most potent in inhibiting T-cell growth, a property ascribed to interferon-γ-dependent indoleamine 2,3-dioxygenase activity. MSCs did not stimulate allogeneic T cell proliferation but were efficiently lysed by activated NK cells. The systematic use of quantitative and reproducible validation techniques highlights differences in immunological properties of MSCs produced using various clinical-grade processes. ADSC-PL emerge as a promising candidate for future clinical trials.