Allogeneic Mesenchymal Stromal Cells Research Articles

ФОРМИРОВАНИЕ СИНДРОМА ПАРКИНСОНИЗМА У ЭКСПЕРИМЕНТАЛЬНЫХ ЖИВОТНЫХ. НЕЙРОВОСПАЛИТЕЛЬНАЯ ПЕНУМБРА

Much valuable information about the development of Parkinson’s disease (PD) has been obtained from studies on the laboratory animals. Objectives. To compare the development of neurotoxic and neuroinflammatory parkinsonism syndrome in laboratory animals. Material and methods. The number of rats in the group of neuroinflammatory model of parkinsonism syndrome (lipopolysaccharide) was 6, and in the group of neurotoxic model (rotenone) - 20. The control group consisted of 5 animals. The study was approved by the independent Ethics Committee. The development dynamics of parkinsonism syndrome of neurotoxic and neuroinflammatory genesis was assessed in the study of the motor activity of animals, as well as in the laboratory study of biomarkers of dopamine metabolism (dopamine and homovanillic acid) in blood serum and cerebrospinal fluid obtained in 7 and 21 days after the first administration of rotenone or lipopolysaccharide, and also after a single intravenous injection of allogeneic (rat) multipotent mesenchymal stromal cells (MMSC) carried out after 9 injections of rotenone. Results. A decrease in the levels of dopamine and homovanillic acid has been shown in laboratory animals on the development of Parkinson’s syndrome. In rats with a neuroinflammatory model of parkinsonism syndrome, a pre-motor stage of motor disorders development has been laboratorially confirmed. During the first weeks after the introduction of MMSC, regression of the motor symptoms of neurotoxic parkinsonism syndrome and a parallel increase in dopamine and homovanillic acid are determined. Conclusions. The effectiveness of MMSC in the early post-transplantation period is associated with the paracrine effect. It is proposed to call activated microglia, a potential therapeutic target in PD, neuroinflammatory penumbra.

Allogeneic mesenchymal stromal cells for cartilage regeneration: A review of in vitro evaluation, clinical experience, and translational opportunities.

The paracrine signaling, immunogenic properties and possible applications of mesenchymal stromal cells (MSCs) for cartilage tissue engineering and regenerative medicine therapies have been investigated through numerous in vitro, animal model and clinical studies. The emerging knowledge largely supports the concept of MSCs as signaling and modulatory cells, exerting their influence through trophic and immune mediation rather than as a cell replacement therapy. The virtues of allogeneic cells as a ready‐to‐use product with well‐defined characteristics of cell surface marker expression, proliferative ability, and differentiation capacity are well established. With clinical applications in mind, a greater focus on allogeneic cell sources is evident, and this review summarizes the latest published and upcoming clinical trials focused on cartilage regeneration adopting allogeneic and autologous cell sources. Moreover, we review the current understanding of immune modulatory mechanisms and the role of trophic factors in articular chondrocyte‐MSC interactions that offer feasible targets for evaluating MSC activity in vivo within the intra‐articular environment. Furthermore, bringing labeling and tracking techniques to the clinical setting, while inherently challenging, will be extremely informative as clinicians and researchers seek to bolster the case for the safety and efficacy of allogeneic MSCs. We therefore review multiple promising approaches for cell tracking and labeling, including both chimerism studies and imaging‐based techniques, that have been widely explored in vitro and in animal models. Understanding the distribution and persistence of transplanted MSCs is necessary to fully realize their potential in cartilage regeneration techniques and tissue engineering applications.

Dichotomic Potency of IFNγ Licensed Allogeneic Mesenchymal Stromal Cells in Animal Models of Acute Radiation Syndrome and Graft Versus Host Disease.

Mesenchymal stromal cells (MSCs) are being tested as a cell therapy in clinical trials for dozens of inflammatory disorders, with varying levels of efficacy reported. Suitable and robust preclinical animal models for testing the safety and efficacy of different types of MSC products before use in clinical trials are rare. We here introduce two highly robust animal models of immune pathology: 1) acute radiation syndrome (ARS) and 2) graft versus host disease (GvHD), in conjunction with studying the immunomodulatory effect of well-characterized Interferon gamma (IFNγ) primed bone marrow derived MSCs. The animal model of ARS is based on clinical grade dosimetry precision and bioluminescence imaging. We found that allogeneic MSCs exhibit lower persistence in naïve compared to irradiated animals, and that intraperitoneal infusion of IFNγ prelicensed allogeneic MSCs protected animals from radiation induced lethality by day 30. In direct comparison, we also investigated the effect of IFNγ prelicensed allogeneic MSCs in modulating acute GvHD in an animal model of MHC major mismatched bone marrow transplantation. Infusion of IFNγ prelicensed allogeneic MSCs failed to mitigate acute GvHD. Altogether our results demonstrate that infused IFNγ prelicensed allogeneic MSCs protect against lethality from ARS, but not GvHD, thus providing important insights on the dichotomy of IFNγ prelicensed allogenic MSCs in well characterized and robust animal models of acute tissue injury.

Alloreactive Immune Response Associated to Human Mesenchymal Stromal Cells Treatment: A Systematic Review.

The well-known immunomodulatory and regenerative properties of mesenchymal stromal cells (MSCs) are the reason why they are being used for the treatment of many diseases. Because they are considered hypoimmunogenic, MSCs treatments are performed without considering histocompatibility barriers and without anticipating possible immune rejections. However, recent preclinical studies describe the generation of alloantibodies and the immune rejection of MSCs. This has led to an increasing number of clinical trials evaluating the immunological profile of patients after treatment with MSCs. The objective of this systematic review was to evaluate the generation of donor specific antibodies (DSA) after allogeneic MSC (allo-MSC) therapy and the impact on safety or tolerability. Data from 555 patients were included in the systematic review, 356 were treated with allo-MSC and the rest were treated with placebo or control drugs. A mean of 11.51% of allo-MSC-treated patients developed DSA. Specifically, 14.95% of these patients developed DSA and 6.33% of them developed cPRA. Neither the production of DSA after treatment nor the presence of DSA at baseline (presensitization) were correlated with safety and/or tolerability of the treatment. The number of doses administrated and human leucocyte antigen (HLA) mismatches between donor and recipient did not affect the production of DSA. The safety of allo-MSC therapy has been proved in all the studies and the generation of alloantibodies might not have clinical relevance. However, there are very few studies in the area. More studies with adequate designs are needed to confirm these results.

Mesenchymal Stromal Cells for Refractory Acute Graft-Versus-Host Disease By Compassionate Use Provide Comparable Results to Those of Phase II Clinical Trials

INTRODUCTIONAllogeneic Mesenchymal stromal cell (MSC) infusion for refractory acute graft-versus-host disease (GVHD) is currently being evaluated in phase II and III clinical trials, in some of which our group has been involved. Nevertheless, some patients are not eligible for clinical trials and may be treated by compassionate use programs. Our objective was to analyze the results of the compassionate program in our institution to ascertain if the safety and efficacy of the treatment in this “real-life” setting is comparable or not to the published results of the phase II trials. METHODSWe conducted a retrospective study of 43 patients who underwent allogeneic hematopoietic stem cell transplant (allo-SCT) and were treated with MSC by compassionate use in our center from June 2009 to date (August 2017). Allogeneic MSC were produced in our good manufacturing practice (GMP) cellular therapy facility and were obtained from third-party healthy donors. Twenty-eight days after the first infusion, the response and the adverse events were evaluated. In addition, the survival was assessed to date. RESULTSPatient characteristics are described in table 1, and GVHD characteristics are shown in table 2. Median time from diagnosis of GVHD to the first MSC infusion was 13 days (2 - 105), with a median number of prior treatment lines of 1 (range 1 to 4). Seventy six percent of infusions followed a sequential pattern (days 1, 4, 11 and 18), similar to the schedule of our clinical trials (Sánchez-Guijo F. Biol Blood Marrow Transplant 2014). The overall response rate (ORR) after 4 weeks was 72%, with complete response (CR) in 48%, and partial response (PR) in 23%. We did not see differences in responses by organ or by GVHD grade. Among responders, around 40% of patients relapsed with a median of time of 63 days (35 - 203). Median time at last follow-up was 164 days (15 - 2638) and in the group who achieved CR after cellular therapy 62% of patients were alive to date. Among non-responders, 83% of patients died due to GVHD. The median time of overall survival was 16 months (range 3 - 28) vs 2 months (range 1 - 3) in responders vs non-responders, respectively (p=0.018), as it is depicted in figure 1. Non-responders were 2.57 times more likely to die (95% CI, 1.14 - 5.83; p=0.023). Regarding safety, there were no adverse events reported such as infusion reactions or related infections. CONCLUSIONSMSC used for the treatment or refractory GVHD in “real-life” compassionate use programs seem to provide comparable results to published phase II trials, showing a safety profile, a high rate of responses and a potential benefit in survival. [Display omitted] DisclosuresSanchez-Guijo:Incyte: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria.

One repeated transplantation of allogeneic umbilical cord mesenchymal stromal cells in type 1 diabetes: an open parallel controlled clinical study

BackgroundThe preservation or restoration of β cell function in type 1 diabetes (T1D) remains as an attractive and challengeable therapeutic target. Mesenchymal stromal cells (MSCs) are multipotent cells with high capacity of immunoregulation, which emerged as a promising cell-based therapy for many immune disorders. The objective of this study was to examine the efficacy and safety of one repeated transplantation of allogeneic MSCs in individuals with T1D.MethodsThis was a nonrandomized, open-label, parallel-armed prospective study. MSCs were isolated from umbilical cord (UC) of healthy donors. Fifty-three participants including 33 adult-onset (≥ 18 years) and 20 juvenile-onset T1D were enrolled. Twenty-seven subjects (MSC-treated group) received an initial systemic infusion of allogeneic UC-MSCs, followed by a repeat course at 3 months, whereas the control group (n = 26) only received standard care based on intensive insulin therapy. Data at 1-year follow-up was reported in this study. The primary endpoint was clinical remission defined as a 10% increase from baseline in the level of fasting and/or postprandial C-peptide. The secondary endpoints included side effects, serum levels of HbA1c, changes in fasting and postprandial C-peptide, and daily insulin doses.ResultsAfter 1-year follow-up, 40.7% subjects in MSC-treated group achieved the primary endpoint, significantly higher than that in the control arm. Three subjects in MSC-treated group, in contrast to none in control group, achieved insulin independence and maintained insulin free for 3 to 12 months. Among the adult-onset T1D, the percent change of postprandial C-peptide was significantly increased in MSC-treated group than in the control group. However, changes in fasting or postprandial C-peptide were not significantly different between groups among the juvenile-onset T1D. Multivariable logistic regression assay indicated that lower fasting C-peptide and higher dose of UC-MSC correlated with achievement of clinical remission after transplantation. No severe side effects were observed.ConclusionOne repeated intravenous dose of allogeneic UC-MSCs is safe in people with recent-onset T1D and may result in better islet β cell preservation during the first year after diagnosis compared to standard treatment alone.Trial registrationChiCTR2100045434. Registered on April 15, 2021—retrospectively registered, http://www.chictr.org.cn/

Fundamental changes in endogenous bone marrow mesenchymal stromal cells during Type I Diabetes is a pre-neuropathy event

Deficiency of angiogenic and neurotrophic factors under long term diabetes is known to lead to Schwann cell degeneration, clinically manifested as Diabetic Neuropathy (DN). While the transplantation of exogenous allogenic Mesenchymal Stromal Cells (MSCs) has shown amelioration of DN through paracrine action, it is not known what functional changes occur in endogenous bone-marrow MSCs under chronic diabetes in terms of homing, migration and/or paracrine signalling with reference to the end-point clinical manifestation of Diabetic Neuropathy. We thus aimed at determining the changes in BM-MSCs under Type 1 Diabetes with respect to survival, self-renewal, oxidative status, paracrine activity, intracellular Ca2+ response and migration in response to pathological cytokine/chemokine, in reference to the time-point of decline in Nerve Conduction Velocity (NCV) in a rat model. Within one week of diabetes induction, BM-MSCs underwent apoptosis, and compromised their self-renewal capacity, antioxidant defence mechanism and migration toward cytokine/chemokine; whereas epineurial blood vessel thickening and demyelination resulting in NCV decline were observed only after three weeks. By two- and three-weeks post diabetes induction, BM-MSC apoptosis reduced and proliferative ability was restored; however, their self-renewal, migration and intracellular Ca2+ response toward pathological cytokine/chemokine remained impaired. These results indicate that T1D induced intrinsic functional impairments in endogenous BM-MSCs occur before neuropathy onset. This timeline of functional alterations in BM-MSCs also suggest that treatment strategies that target the bone marrow niche early on may help to modulate BM-MSC functional impairments and thus slow down the progression of neuropathy.

Ultrapurified Alginate Gel Containing Bone Marrow Aspirate Concentrate Enhances Cartilage and Bone Regeneration on Osteochondral Defects in a Rabbit Model

Background: Ultrapurified alginate (UPAL) gel implantation has been demonstrated as effective in cartilage repair for osteochondral defects; however, cell transplantation within UPAL gels would be required to treat larger defects. Hypothesis: The combination of UPAL gel and bone marrow aspirate concentrate (BMAC) would enhance cartilage repair and subchondral bone repair for large osteochondral defects. Study Design: Controlled laboratory study. Methods: A total of 104 osteochondral defects (1 defect per knee) of 52 rabbits were randomly divided into 4 groups (26 defects per group): defects without any treatment (Defect group), defects treated using UPAL gel alone (UPAL group), defects treated using UPAL gel containing allogenic bone marrow mesenchymal stromal cells (UPAL-MSC group), and defects treated using UPAL gel containing BMAC (UPAL-BMAC group). At 4 and 16 weeks postoperatively, macroscopic and histologic evaluations and measurements of repaired subchondral bone volumes of reparative tissues were performed. Collagen orientation and mechanical properties of the reparative tissue were assessed at 16 weeks. Results: The defects in the UPAL-BMAC group were repaired with hyaline-like cartilage with well-organized collagen structures. The histologic scores at 4 weeks were significantly higher in the UPAL-BMAC group (16.9 ± 2.0) than in the Defect group (4.7 ± 1.9; P < .05), the UPAL group (10.0 ± 3.3; P < .05), and the UPAL-MSC group (12.2 ± 2.9; P < .05). At 16 weeks, the score in the UPAL-BMAC group (24.4 ± 1.7) was significantly higher than those in the Defect group (9.0 ± 3.7; P < .05), the UPAL group (14.2 ± 3.9; P < .05), and the UPAL-MSC group (16.3 ± 3.6; P < .05). At 4 and 16 weeks, the macroscopic evaluations were significantly superior in the UPAL-BMAC group compared with the other groups, and the values of repaired subchondral bone volumes in the UPAL-BMAC group were significantly higher than those in the Defect and UPAL groups. The mechanical properties of the reparative tissues were significantly better in the UPAL-BMAC group than in the other groups. Conclusion: The implantation of UPAL gel containing BMAC-enhanced hyaline-like cartilage repair and subchondral bone repair of osteochondral defects in a rabbit knee model. Clinical Relevance: These data support the potential clinical application of 1-step treatment for large osteochondral defects using biomaterial implantation with cell transplantation.

P363 Locally injected allogeneic bone marrow-derived mesenchymal stromal cells for the treatment of refractory proctitis: clinical results of a phase IIa trial

Abstract Background Ulcerative proctitis (UP) can be refractory to treatment, which calls for development of new (local) therapies. Local injection of mesenchymal stromal cells (MSCs) has shown beneficial effects in patients with fistulizing Crohn’s disease and promising results have been obtained when MSCs were locally injected in the bowel of mice with experimental colitis. Our primary aim was to determine the safety, feasibility and tolerability of endoscopically injected allogeneic bone marrow-derived MSCs (bmMSCs) in UP patients. Methods UP patients with endoscopic Mayo score (EMS) 2 or 3, who failed on both rectal 5-ASA and corticosteroids for at least 4 weeks, were eligible for inclusion (EudraCT number 2017-003524-75). Rectal therapies were stopped 2 weeks prior to baseline, but other medication was kept stable until at least 6 weeks after MSC injection. MSCs were locally injected in 4 quadrants of the inflamed rectal submucosa if the inflammation was limited to 7 cm. If the length of inflammation was &amp;gt;7 cm, MSCs were injected in another 4 quadrants more proximally as well. Patients in the first cohort (n=7) were treated with 5*106 MSCs/spot and in the second cohort (n=6) with 10*106MSCs/spot. Adverse events, full Mayo score, fecal calprotectin (FCP), histologic activity (Geboes score [GS]) and quality of life (sIBDQ), were assessed at week 0, 2, 6, 12, and 24, and evaluated by non-parametric paired statistical analyses. Results All reported adverse events were minor, no patients required interventions and no feasibility issues were reported. Median[interquartile range (IQR)] full Mayo score was 11[9.5-12] at baseline, 9[8-11] at week 2 (p=0.003), 8[6-10] at week 6 (p=0.001), and 4[1.5-7] at week 24 (p&amp;lt;0.001). The FCP improved in 9/13 patients at week 2, in 6/13 patients at week 6 and in 11/13 patients at week 24 compared to baseline. The EMS at baseline was 3 (n=10 patients) and 2 (n=3 patients) and improved in some patients at week 2 and 6 (NS). At week 24 the EMS was 3 (n=2), 2 (n=4), 1 (n=5) and 0 (n=2)(p=0.002). The median[IQR] GS decreased after 6 weeks (7(6.5-12.5);p=0.09) and 24 weeks (4[2-8];p=0.01) compared to baseline (10[6.5-12.5]). Median[IQR] sIBDQ showed improvement during follow-up; week 2 (45[37.5-52];p=0.1), week 6 (47[42.50-55];p=0.02), week 12 (59[39.50-62];p=0.001) and week 24 (56[44.50-63.50];p=0.001) compared to baseline (41[34-49,50]). No dose- response effects were observed in our study when comparing cohort 1 and 2. Conclusion Local administration of allogeneic bmMSCs appears safe, tolerable and feasible for the treatment of refractory UP, and shows encouraging indications of clinical efficacy. Further mechanistic and immunological analyses are currently being performed.

P273 The duration of clinical remission in patients with Crohn’s disease depends on the frequency of administration of mesenchymal stromal cells of the bone marrow

Abstract Background Anti-cytokine therapy with anti-TNF-α drugs contributes to the achievement of persistent remission of Crohn’s disease (CD). Allogeneic mesenchymal stromal cells (MSC) of the bone marrow are also used for the treatment of CD. Objective to evaluate the duration of remission of CD in the context of mesenchymal stromal cells (MSC) therapy of the bone marrow, depending on the frequency of MSC administration. Methods 76 patients with CD with luminal form of CD (terminal ileitis, colitis, and ileocolitis) were divided into two groups. The first group of patients aged 19 to 58 years (Me-29) (n=34) received culture of MCS according to the scheme: 0-1-2-12-26-52 weeks, then annually 2 times a year. The second group of patients with CD (n=42) aged 20 to 62 years (Me-28) received culture of MCS according to the scheme: 0-1-2-12-26-52 weeks to achieve remission within one year. The effectiveness of therapy was evaluated 12, 24, 36, 48 and 60 months after the start of therapy using the Harvey-Bradshaw index. Results During 12 months of follow-up, 4/34 patients (11.76%) relapsed among patients in 1-st group. In 2-nd group, relapse occurred in 5/42 (11.9%) (HR-0.63; 95% CI 0.288-3.397; p=0.84). After 24 months, 6/34 patients (17.6%) relapsed in 1-st group. In the 2-nd group of patients, the disease relapsed in 11/42 (26.2%) (HR-0.674; 95% CI 0.278-1.634; p=0.37). After 36 months, the disease relapsed in 7/34 patients in 1-st group (20.6%). In the 2-nd group, relapse was in 18/42 (42.8%) (HR-0.48; 95% CI 0.228-1.014; p=0.038). After 48 months, in the 1-st group receiving MSCS, a relapse occurred in 9/34 (26.5%). In the 2-nd group, relapse occurred in 22/42 (52.4%) (HR-0.5; 95% CI 0.269-0.949; p=0.021). After 60 months, 10/34 (29.4%) relapsed in 1-st group. In the 2-nd group, relapse occurred in 24/42 (57.1%) (HR-0.463; 95% CI 0.250-0.860; p=0.007). Conclusion The duration of clinical remission in patients with luminal Crohn’s disease depends on the frequency of administration of mesenchymal stromal cells.

DOP64 Endoscopically injected allogeneic mesenchymal stromal cells alter the mucosal immune cell compartment in patients with ulcerative proctitis

Abstract Background Local mesenchymal stromal cell (MSC)-therapy is approved for the treatment of Crohn’s disease-associated perianal fistulas. However, little is known about the working mechanism of local MSC-therapy. For the first time we evaluated engraftment and immunoregulatory effects of local MSC-therapy in patients with refractory proctitis. To do so, we analyzed biopsies and serum from patients with ulcerative proctitis before and after treatment with endoscopically injected MSCs in a phase IIa clinical trial (EudraCT number 2017-003524-75). Methods Thirteen therapy-refractory ulcerative proctitis patients were endoscopically injected bone marrow-derived allogeneic MSCs from healthy donors. Clinical efficacy was evaluated by the endoscopic and full Mayo score. Engraftment of the MSCs was investigated using fluorescence in-situ hybridization (FISH) of sex chromosomes on post-treatment biopsies. The presence of anti-HLA-antibodies against the MSC-donor was determined in the serum. Changes in immune cell subsets were evaluated using cytometry-by-time-of-flight (CyTOF) analysis. Results Thirteen patients with an endoscopic Mayo score of 2 (n=3) or 3 (n=10) of the rectum were treated with local MSC-therapy. Although complete remission was not achieved, full Mayo score was improved at week 6 (median 8 [IQR 6–10]) compared to baseline (median 11 [IQR 9.5–12]) (p=0.001). Preliminary data using FISH on the Y-chromosome, indicated the presence of MSCs in the rectum biopsies of female patients treated with male donor derived-MSCs at week 6. At baseline, HLA-antibodies were present in four patients. Six weeks after local injection of the MSCs, two out of thirteen patients developed new class I and II HLA-antibodies against the MSCs. Interestingly, in two patients pre-existing HLA-antibodies showed increased/boosted levels after local MSC-therapy, while one additionally developed new HLA-antibodies. CyTOF analysis of inflamed rectal biopsies 6 weeks after MSC treatment revealed significantly increased frequencies of several myeloid subsets (i.e. CD11b+CD14+CCR7+/-CD127+CD25+HLADR+ and CD14+HLA-DR-CD123-CCR7+) and a subset of CD4+ memory T cells with a more exhausted/regulated phenotype (PD-1+TIGIT+CD69+CD38+CD69). Conclusion Local MSC-therapy in patients with refractory proctitis changed the rectal immune profile characterised by a significant increase in a subset of effector memory CD4+ cells and several myeloid subsets, which might be associated with immune modulation. These results provide the basis for future studies on the mechanism of action of MSCs on rectal mucosa. New anti-HLA class antibodies developed in 2/13 patients after local administration. Whether these latter results have consequences for MSC-donor selection deserves further study.

Allogenic Use of Human Placenta-Derived Stromal Cells as a Highly Active Subtype of Mesenchymal Stromal Cells for Cell-Based Therapies.

The application of mesenchymal stromal cells (MSCs) from different sources, including bone marrow (BM, bmMSCs), adipose tissue (atMSCs), and human term placenta (hPSCs) has been proposed for various clinical purposes. Accumulated evidence suggests that the activity of the different MSCs is indirect and associated with paracrine release of pro-regenerative and anti-inflammatory factors. A major limitation of bmMSCs-based treatment for autologous application is the limited yield of cells harvested from BM and the invasiveness of the procedure. Similar effects of autologous and allogeneic MSCs isolated from various other tissues were reported. The easily available fresh human placenta seems to represent a preferred source for harvesting abundant numbers of human hPSCs for allogenic use. Cells derived from the neonate tissues of the placenta (f-hPSC) can undergo extended expansion with a low risk of senescence. The low expression of HLA class I and II on f-hPSCs reduces the risk of rejection in allogeneic or xenogeneic applications in normal immunocompetent hosts. The main advantage of hPSCs-based therapies seems to lie in the secretion of a wide range of pro-regenerative and anti-inflammatory factors. This renders hPSCs as a very competent cell for therapy in humans or animal models. This review summarizes the therapeutic potential of allogeneic applications of f-hPSCs, with reference to their indirect pro-regenerative and anti-inflammatory effects and discusses clinical feasibility studies.

The possibility of using biomedical cell product in the treatment of chronic glomerulonephritis

The basic principles of rational treatment of glomerulonephritis are considered, based on etiology, clinical manifestations and pathomorphological variants of its different forms. Today it has been established that glomerulonephritis can be primary (the etiology is usually unknown) and secondary, when the disease occurs against the background of concomitant pathology (systemic lupus erythematosus, vasculitis, hepatitis, oncological diseases, chronic viral and bacterial infections). The participation of various pathological, genetic and systemic factors in the development of various forms of glomerulonephritis has been shown. The characteristic clinical picture of manifestations of different forms of the disease and the degree of involvement of different renal structures in them are described. Against the background of the severity of the course and prevalence of various forms of glomerulonephritis, it is especially important to develop therapeutic approaches aimed at the full restoration of kidney function and cure of patients. One of such approaches is the use of biomedical cell products based on allogenic mesenchymal multipotent stromal cells and hematopoietic stromal cells. A number of studies have confirmed that the best results can be achieved with the use of cell products in complex therapies with standard treatment methods (use of cytostatics and steroid anti-inflammatory drugs) and alternative drug approaches (combination of monoclonal antibodies and polyenzyme drugs). At the same time, the use of standard and alternative techniques does not lead to a complete recovery of patients, but only transfers the course of the disease from the exacerbation phase to remission. The relevance of further development of biomedical cell products of allogeneic mesenchymal multipotent bone marrow stromal cells is shown, verification of their safety and efficacy in preclinical and clinical studies.

Human bone marrow-derived, pooled, allogeneic mesenchymal stromal cells manufactured from multiple donors at different times show comparable biological functions in vitro, and in vivo to repair limb ischemia

BackgroundWe have previously demonstrated that a pooled population of bone marrow-derived, allogeneic mesenchymal stromal cells (BMMSC), Stempeucel®-1, produced under good manufacturing practices (GMP) conditions, showed clinical efficacy and safety in patients suffering from critical limb ischemia (CLI) due to Buerger’s disease. While Stempeucel®-1 is currently used for CLI and other clinical indications, we wanted to ensure that the product’s continuity is addressed by developing and characterizing a second generation of pooled product (Stempeucel®-1A), manufactured identically from second BM aspirates of the same three donors after a 2-year interval.MethodsThe two versions of Stempeucel® were manufactured and subjected to gene and protein expression analysis. The nature of various growth factors/cytokines secreted and immunomodulatory activity of these two cell populations were compared directly by various in vitro assays. The preclinical efficacy of these two cell types was compared in an experimental model of hind limb ischemia (HLI) in BALB/c nude mice. The reversal of ischemia, blood flow, and muscle regeneration were determined by functional scoring, laser Doppler imaging, and immunohistochemical analyses.ResultsQualitative and quantitative analyses of genes and proteins involved in promoting angiogenic activity and immune regulatory functions revealed high levels of correlation between Stempeucel®-1 and Stempeucel®-1A cell populations. Moreover, intramuscular (i.m) administration of these two cell products in the ischemic limbs of BALB/c nude mice showed significant repair (≥ 70%) of toe and foot necrosis, leading to improved ambulatory function and limb salvage. Furthermore, a biodistribution kinetics study showed that Stempeucel®-1 was mostly localized in the ischemic muscles of mice for a significantly longer time compared to normal muscles, thus playing an essential role in modulating and reversing HLI damage.ConclusionsThis study shows that with a reproducible manufacturing procedure, it is possible to generate large numbers of pooled mesenchymal stromal cells from human bone marrow samples to establish product equivalence. We conclude from these results that, for the first time, two pooled, allogeneic BMMSC products can be repeatedly manufactured at different time intervals using a two-tier cell banking process with robust and comparable angiogenic properties to treat ischemic diseases.

Optimization of gmp-compatible biobanking of allogeneic bone marrow-derived clonal mesenchymal stromal cells for cell therapy applications

Background & Aim: Allogeneic mesenchymal stromal cells (MSCs) are valuable therapeutic candidates used extensively in various clinical trials. However, the findings of Phase III clinical trials raise concerns about the efficacy of MSCs, which are rooted mainly in the applied populations’ heterogeneity. Although several factors, one of the most crucial causes of this heterogeneity is the lack of an optimized cell culture technique to manufacture a homogenous cultured MSCs. Moreover, it is necessary to provide a good manufacturing practice (GMP) platform to ensure the safety of final cell therapy products (CTPs) and satisfy the legal requirements for clinical applications. Establishing a tired cell bank system is the final step to support the proper translation of producing MSCs into medicine by ensuring standardization of the entire cell manufacturing process, accredited identity, function and safety assessments, and transparent sharing of standard operating procedures (SOPs) data. Methods, Results & Conclusion: Here, clonal MSCs (cMSCs) were isolated based on the subfractionation culturing method (SCM) protocol under the GMP-compatible condition and through an innovative, cost-effective screening approach. Then, cMSCs were compared with their heterogeneous counterparts in terms of identity and function. The validated clones were stored in a four-tiered cell bank system consisting of an initial, master, working, and end of product cell banks (ICB, MCB, WCB, and EoPCB). Additionally, to further develop towards GMP- compatible cMSCs production, several identities, quality, and safety assessments were performed during all banking phases. Finally, the cells stored in the EoPCB were released as a drug product (DP) for the Phase I/II clinical trial of coronavirus disease-2019 (COVID-19). Regarding senescence assessment and capability to serial passaging, three similar passagable clones were manufactured. Remarkably, the clones’ genomic stability was fully retained after 15 passages and were neither tumorigenic nor immunogenic. Altogether, this study presents a technical and translational overview of GMP-compatible cMSCs manufacturing technology, which could be used as a guide for the development of similar production processes with the therapeutic goals.

Mesenchymal Stromal Cell Therapy in Novel Porcine Model of Diffuse Liver Damage Induced by Repeated Biliary Obstruction.

In liver surgery, biliary obstruction can lead to secondary biliary cirrhosis, a life-threatening disease with liver transplantation as the only curative treatment option. Mesenchymal stromal cells (MSC) have been shown to improve liver function in both acute and chronic liver disease models. This study evaluated the effect of allogenic MSC transplantation in a large animal model of repeated biliary obstruction followed by partial hepatectomy. MSC transplantation supported the growth of regenerated liver tissue after 14 days (MSC group, n = 10: from 1087 ± 108 (0 h) to 1243 ± 92 mL (14 days); control group, n = 11: from 1080 ± 95 (0 h) to 1100 ± 105 mL (14 days), p = 0.016), with a lower volume fraction of hepatocytes in regenerated liver tissue compared to resected liver tissue (59.5 ± 10.2% vs. 70.2 ± 5.6%, p < 0.05). Volume fraction of connective tissue, blood vessels and bile vessels in regenerated liver tissue, serum levels of liver enzymes (AST, ALT, ALP and GGT) and liver metabolites (albumin, bilirubin, urea and creatinine), as well as plasma levels of IL-6, IL-8, TNF-α and TGF-β, were not affected by MSC transplantation. In our novel, large animal (pig) model of repeated biliary obstruction followed by partial hepatectomy, MSC transplantation promoted growth of liver tissue without any effect on liver function. This study underscores the importance of translating results between small and large animal models as well as the careful translation of results from animal model into human medicine.

Subconjunctival administration of low-dose murine allogeneic mesenchymal stromal cells promotes corneal allograft survival in mice

BackgroundSystemic administration of mesenchymal stromal cells (MSCs) has been efficacious in many inflammatory disease settings; however, little data are available on the potential immunomodulatory effects following local MSC administration in the context of corneal transplantation. The purpose of this study was to assess the potential of subconjunctival injection of MSCs to promote corneal allograft survival.MethodsMSCs were isolated from female C57BL/6 (H-2k) or Balb/c (H-2d) mice and extensively characterized. An allogeneic mouse corneal transplant model was used with Balb/c mice as recipients of C57BL/6 grafts. A dose-finding study starting with 5 × 105 MSCs injected subconjunctivally at day − 7 was tested first followed by a more clinically translatable low-dose single or dual injection strategy on day − 1 and day + 1 before/after transplantation. Graft transparency served as the primary indicator of transplant rejection while neovascularization was also recorded. Lymphocytes (from draining lymph nodes) and splenocytes were isolated from treatment groups on day 2 post-transplantation and characterized by flow cytometry and qRT-PCR.ResultsBoth high- and low-dose injection of allogeneic MSCs on day − 7 led to 100% graft survival over the observation period. Moreover, low-dose dual subconjunctival injection of 5 × 104 allogeneic MSCs on day − 1 or day + 1 led to 100% allograft survival in transplant recipients (n = 7). We also demonstrate that single administration of allogeneic MSCs on either day − 1 or day + 1 promotes rejection-free graft survival in 100% (n = 8) and 86% (n = 7) of transplanted mice, respectively. Early time point ex vivo analysis suggests modulation of innate immune responses towards anti-inflammatory, pro-repair responses by local MSC administration.ConclusionThis work demonstrates that low-dose subconjunctival injection of allogeneic MSCs successfully promotes corneal allograft survival and may contribute to refining future MSC immunotherapies for prevention of corneal allograft rejection.

Doppler examination of aorta after therapeutic hypothermia and administering MSCs in experimental myocardial infarction

Background. At present, there are attempts aimed at repairing the myocardium affected by acute myocardial infarction (MI) via biologically sourced drugs, which are able to stimulate reparative regeneration, including the use of mesenchymal stromal cells (MSCs). On the other hand, therapeutic hypothermia initially after the occurrence of impaired blood flow in the coronary arteries aids in reducing the level of metabolism in cardiomyocytes and, accordingly, suppressing the formation of free radicals and inhibiting apoptosis.&#x0D; Purpose – рerforming and analysing Doppler sonography of the ascending aorta in rats with experimental MI in order to assess the systolic function of the left ventricular myocardium influenced by the combined use of therapeutic hypothermia and administering allogeneic MSCs.&#x0D; Materials and methods. The study involved 90 outbred white rats weighing 240–270 g. Myocardial infarction was reproduced by ligating the left coronary artery. Therapeutic hypothermia was performed in a cold chamber, 60 minutes long. The local skin temperature of the neck area was maintained at +4 оС. A suspension of allogeneic cryopreserved MSCs of the placenta was administered once intravenously. Sonography of the ascending aorta on day 7 and day 30 after ligating the coronary artery was carried out by means of «Сономед 500» ultrasound scanner in B-mode with the Doppler (PW-mode).&#x0D; Results. Studying the aortic blood flow showed that on day 7 after ligating the coronary artery, rats had a decrease in max systolic velocity and a tendency to increase diastolic blood flow velocity. Those changes influenced the pulsatility index and systolic-diastolic ratio. A 19 % decrease in the Stewart index confirmed the development of systolic dysfunction in the control group, the severity of which did not decrease until day 30 of the experiment. The impact of allogeneic MSCs on the process of remodeling the heart of rats after ligating the left coronary artery was evident on day 7 (acute stage of myocardial infarction) via abnormally low (below control) blood flow in the aorta with complete hemodynamics restoring on day 30 of the experiment. The combination of therapeutic hypothermia and administering allogeneic MSCs aided in the minimal deviation of hemodynamic parameters from the normal range. On day 7, there was only an increase in the average aortic blood flow rate by 29%, and a decrease in systolic-diastolic ratio by 12% compared to the corresponding normal range, suggesting compensation of the pumping function of the myocardium.&#x0D; Conclusions. Doppler assessment of systolic and diastolic blood flow velocities in the ascending aorta of rats makes it possible to indirectly record the presence and severity of systolic dysfunction of the left ventricle and follow up myocardial contractile function affected by MI. The combination of therapeutic hypothermia and administering allogeneic MSCs after ligating the left coronary artery helps to maintain hemodynamic parameters in the aorta at a level close to normal range during the acute phase of myocardial infarction as well as at the stage of scarring.

Efficiency of mesenchymal stromal cells (MSCs) in the treatment of patients with critical limb-threatening ischemia

Relevance. The relevance of the study is determined by the fact that hopes are placed in the cell therapy for patients with critical limb-threatening (CLI) ischemia as a method of the restoration of blood circulation in the affected limb in patients who cannot undergo surgical or endovascular intervention. Aim. To evaluate the efficiency of allogeneic MSCs for the treatment of critical lower limb ischemia (randomized placebo-controlled study).Materials and methods. The study included 34 patients with critical lower limb ischemia (grade 4 according to Pokrovsky). There were 18 patients in the MSC group, and 16 patients in the placebo group). The groups were comparable concerning age, disease duration, and comorbidities. Allogeneic MSCs (phenotype CD73+, CD90+, CD105+, CD45–, CD34–, CD14–) were injected into the posterior calf muscles. Clinical outcome, ankle pressure, transcutaneous oxygen tension (tcpO2), and pain-free walking distance (PFWD) were evaluated. The patients were followed-up for 12–36 months. According to the clinical outcome in each group, the patients were divided into subgroups with «effect (+)» or «effect (–)». In 2 patients, there was an «uncertain clinical outcome». When analyzing the results, these patients were assigned to one or another subgroup.Results. In the MSC and placebo groups, the clinical outcome assessed as «effect (+)» or «effect (–)» did not differ (OR 1.5; 95 % CI 0.34–6.7). With different variants of group formation and with the assignment of patients with an «uncertain clinical outcome» to a one or another subgroup, the final results neither differed. According to instrumental research methods (PFWD, tcpO2, ankle pressure, angiography), there were no differences in the MSC and placebo groups. Conclusion. With different variants of analysis and group formation, no convincing evidence that allogeneic MSCs can be effective for the treatment of critical lower limb ischemia have been obtained.

The Current Status of Mesenchymal Stromal Cells: Controversies, Unresolved Issues and Some Promising Solutions to Improve Their Therapeutic Efficacy.

Mesenchymal stromal cells (MSCs) currently constitute the most frequently used cell type in advanced therapies with different purposes, most of which are related with inflammatory processes. Although the therapeutic efficacy of these cells has been clearly demonstrated in different disease animal models and in numerous human phase I/II clinical trials, only very few phase III trials using MSCs have demonstrated the expected potential therapeutic benefit. On the other hand, diverse controversial issues on the biology and clinical applications of MSCs, including their specific phenotype, the requirement of an inflammatory environment to induce immunosuppression, the relevance of the cell dose and their administration schedule, the cell delivery route (intravascular/systemic vs. local cell delivery), and the selected cell product (i.e., use of autologous vs. allogeneic MSCs, freshly cultured vs. frozen and thawed MSCs, MSCs vs. MSC-derived extracellular vesicles, etc.) persist. In the current review article, we have addressed these issues with special emphasis in the new approaches to improve the properties and functional capabilities of MSCs after distinct cell bioengineering strategies.