Rationale for the potential use of mesenchymal stromal cells in liver transplantation.

Mesenchymal stromal cells (MSCs) are multipotent and self-renewing cells that reside essentially in the bone marrow as a non-hematopoietic cell population, but may also be isolated from the connective tissues of most organs. MSCs represent a heterogeneous population of adult, fibroblast-like cells characterized by their ability to differentiate into tissues of mesodermal lineages including adipocytes, chondrocytes and osteocytes. For several years now, MSCs have been evaluated for their in vivo and in vitro immunomodulatory and 'tissue reconstruction' properties, which could make them interesting in various clinical settings, and particularly in organ transplantation. This paper aims to review current knowledge on the properties of MSCs and their use in pre-clinical and clinical studies in solid organ transplantation, and particularly in the field of liver transplantation. The first available clinical data seem to show that MSCs are safe to use, at least in the medium-term, but more time is needed to evaluate the potential adverse effects of long-term use. Many issues must be resolved on the correct use of MSCs. Intensive in vitro and pre-clinical research are the keys to a better understanding of the way that MSCs act, and to eventually lead to clinical success

MSC Manufacturing for Academic Clinical Trials: From a Clinical-Grade to a Full GMP-Compliant Process

Following European regulation 1394/2007, mesenchymal stromal cell (MSCs) have become an advanced therapy medicinal product (ATMP) that must be produced following the good manufacturing practice (GMP) standards. We describe the upgrade of our existing clinical-grade MSC manufacturing process to obtain GMP certification. Staff organization, premises/equipment qualification and monitoring, raw materials management, starting materials, technical manufacturing processes, quality controls, and the release, thawing and infusion were substantially reorganized. Numerous studies have been carried out to validate cultures and demonstrate the short-term stability of fresh or thawed products, as well their stability during long-term storage. Detailed results of media simulation tests, validation runs and early MSC batches are presented. We also report the validation of a new variant of the process aiming to prepare fresh MSCs for the treatment of specific lesions of Crohn’s disease by local injection. In conclusion, we have successfully ensured the adaptation of our clinical-grade MSC production process to the GMP requirements. The GMP manufacturing of MSC products is feasible in the academic setting for a limited number of batches with a significant cost increase, but moving to large-scale production necessary for phase III trials would require the involvement of industrial partners

Rapamycin prevents experimental sclerodermatous chronic graft-versus-host disease in mice

Background: The most widely used mice model of chronic graft-versus-host disease (cGvHD) is an MHC-matched bone marrow transplantation model of sclerodermatous cGvHD. A limitation of that model is that mortality is relatively low, making difficult to study the impact of potentially therapeutic compounds. Aims: To develop a more severe model of cGVHD and to assess the impact of Rapamycin administration in that model. Results: Lethally irradiated Balb/C mice were injected with 10x106 bone marrow cells and 70x106 splenocytes from B10.D2 donor mice. Twenty-one days later, all mice developed cGvHD. For the severe model, donor B10.D2 mice were injected with 0.5x106 splenocytes from Balb/C twenty-one days before transplantation. All mice from the severe model (n=8) died a median of 32 days while 3 of 7 mice in the classical model survived beyond day 52. Mean survival was decreased in the severe model compared to the classical model (32 days versus 37 days; p=0.0185). Recipient mice in the severe group experienced higher weight loss, hair loss and skin fi brosis. Numbers of T lymphocytes (231.9 ± 151.4 versus 951 ± 532.8; p=0.0032) and CD4+ T cells (63.25 ± 41.93 versus 135.0 ± 14.39; p=0.0018) per microliter of blood at day 21 were lower in the severe group than in the classical model. Moreover, number of regulatory T cells (Tregs) was decreased in the severe model (1.250 ± 0.8864 versus 8.000 ± 6.753; p=0.0151). We then investigated whether rapamycin administration could prevent GVHD in the severe model. All (n=8) mice treated with PBS (placebo) died a median of 32 days after transplantation, while 6 of 8 mice given 1 mg/kg/day i.p. rapamycin survived beyond day 52 (p=0.0012). Number of Tregs/μl was higher at day 21 in rapamycin-treated mice than in mice given PBS (2.000±1.195 versus 1.250±0.8864; p=0.0796). Moreover, number of naïve CD4+T (10.00±4.192 versus 30.25±5.185; p= 0.0089) and effector memory T cells (EMT) (30.67±3.180 versus 67.33±7.881; p= 0.0125) were higher in rapamycin mice. Finally, proliferation of EMT (assessed by fl ow cytometry using Ki-67) was higher in PBS than in rapamycin mice (45.28%±4.084 versus 31.90%± 2.003; p=0.0474). Conclusion: We have developed a mice model of severe cGVHD. Interestingly, rapamycin prevented death from cGVHD in that model, perhaps through in vivo expansion of Treg

Administration of Third-Party Mesenchymal Stromal Cells at the Time of Kidney Transplantation: Interim Safety Analysis at One-Year Follow-Up

Mesenchymal stromal cells (MSC)-based therapy has been proposed in kidney transplantation (KTx). We report on the 1-year follow-up of an open-label phase I trial using MSC in KTx. On postoperative day 3, third-party MSC (~2.0x106/kg) were administered to 7 non-immunized first-transplant recip- ients from deceased donors, under standard immunosuppression (Basiliximab, Tacrolimus, MMF and steroids). No HLA matching was required for MSC donors. Seven comparable KTx recipients were included as controls. Informed consent was obtained. No side-effect was noted at the time of MSC injection. Still, 1 patient with a history of ischemic heart disease had a NSTEMI ~3h after MSC infusion. Ten months after KTx, 1 MSC patient had type B aortic dissection and STEMI. Four MSC patients had at least 1 opportunistic infection, whereas 3 controls had polyoma-BK viremia. At day 14, eGFR in MSC and control groups was 47.1 ± 6.8 and 39.7 ± 5.9 ml/min, respectively (p, 0.05). At 1 year, eGFR in MSC and control groups was 46.5 ± 18.6 and 54.2 ± 16.3 ml/min, respectively (p, 0.42). Per-cause biopsies evidenced 1 bor- derline and 1 acute rejections in MSC group, whereas no AR was biopsy-proven in controls. Three patients developed anti-HLA antibodies against MSC (n=1) or shared kidney/MSC (n=2) mismatches.MSC infusion was safe in all patients except one. Incidence of opportunist infections was similar in both groups. No difference in eGFR was found at 1-year post KTx. Putative immunization against MSC was observed in 3 patients

Mesenchymal Stromal Cell Therapy in Ischemia/Reperfusion Injury.

Ischemia/reperfusion injury (IRI) represents a worldwide public health issue of increasing incidence. IRI may virtually affect all organs and tissues and is associated with significant morbidity and mortality. Particularly, the duration of blood supply deprivation has been recognized as a critical factor in stroke, hemorrhagic shock, or myocardial infarction, as well as in solid organ transplantation (SOT). Pathophysiologically, IRI causes multiple cellular and tissular metabolic and architectural changes. Furthermore, the reperfusion of ischemic tissues induces both local and systemic inflammation. In the particular field of SOT, IRI is an unavoidable event, which conditions both short- and long-term outcomes of graft function and survival. Clinically, the treatment of patients with IRI mostly relies on supportive maneuvers since no specific target-oriented therapy has been validated thus far. In the present review, we summarize the current literature on mesenchymal stromal cells (MSC) and their potential use as cell therapy in IRI. MSC have demonstrated immunomodulatory, anti-inflammatory, and tissue repair properties in rodent studies and in preliminary clinical trials, which may open novel avenues in the management of IRI and SOT

Infusion of third-party mesenchymal stem cells after liver transplantation: a phase-1, open-label, clinical study

peer reviewedBackground: Mesenchymal stromal cells (MSC) are multipotent bone mar- row progenitors that have demonstrated significant immunosuppressive effects in various in vivo and in vitro studies. This study aimed to be the first evaluation of the safety and tolerability of MSC infusion after liver transplantation in a prospective, controlled phase-1 study. Methods: 10 liver transplant recipients under standard immunosuppression (TAC-MMF-low dose steroids until day 30) received 1.5–3 9 106/kg third party MSC on post-operative day 3 ` 2. These patients were prospectively compared to a group of 10 control liver recipients. Primary endpoints were MSC infusion toxicity, and incidence of cancer and opportunistic infections at month 6. Secondary endpoints were patient and graft survivals and rejection at month 6, as well as the effects of MSC on recipients’ immune function and on immunohistology of at month 6 graft biopsies. Results: No MSC infusional toxicity was observed. Both groups were comparable in terms of donor and recipient characteristics. There was no difference in primary end-points between control and MSC groups. No patient developed de novo cancer. There was no statistical difference in patient and graft survivals or in rejection rates. There was no graft rejection in the MSC group. Month-6 graft biopsies were not different according to Banff and fibrosis scores. Discussion: This phase 1 study showed excellent tolerability and safety of a single infusion of third-party MSC after liver transplantation. There were no graft safety issues and no excess of immunosuppression after MSC injection. Further analyses of consequences of MSC injection on the immune profile are needed. The possibility of avoiding calcineurin-inhibitors with repeated MSC injections as main immunosuppressive therapy and/of tolerance induction by MSC infusion should be investigated by further studies. This study is in part supported by an ESOT Senior Clinical Research Grant and by the University of Liege

Infusion of third-party mesenchymal stem cells after liver transplantation: a phase-1, open-label, clinical study

peer reviewedBackground: Mesenchymal stromal cells (MSC) are multipotent bone mar- row progenitors that have demonstrated significant immunosuppressive effects in various in vivo and in vitro studies. This study aimed to be the first evaluation of the safety and tolerability of MSC infusion after liver transplantation in a prospective, controlled phase-1 study. Methods: 10 liver transplant recipients under standard immunosuppression (TAC-MMF-low dose steroids until day 30) received 1.5–3 9 106/kg third party MSC on post-operative day 3 ` 2. These patients were prospectively compared to a group of 10 control liver recipients. Primary endpoints were MSC infusion toxicity, and incidence of cancer and opportunistic infections at month 6. Secondary endpoints were patient and graft survivals and rejection at month 6, as well as the effects of MSC on recipients’ immune function and on immunohistology of at month 6 graft biopsies. Results: No MSC infusional toxicity was observed. Both groups were comparable in terms of donor and recipient characteristics. There was no difference in primary end-points between control and MSC groups. No patient developed de novo cancer. There was no statistical difference in patient and graft survivals or in rejection rates. There was no graft rejection in the MSC group. Month-6 graft biopsies were not different according to Banff and fibrosis scores. Discussion: This phase 1 study showed excellent tolerability and safety of a single infusion of third-party MSC after liver transplantation. There were no graft safety issues and no excess of immunosuppression after MSC injection. Further analyses of consequences of MSC injection on the immune profile are needed. The possibility of avoiding calcineurin-inhibitors with repeated MSC injections as main immunosuppressive therapy and/of tolerance induction by MSC infusion should be investigated by further studies. This study is in part supported by an ESOT Senior Clinical Research Grant and by the University of Liege

Bone Marrow-Derived Mesenchymal Stromal Cell Therapy in Severe COVID-19: Preliminary Results of a Phase I/II Clinical Trial

peer reviewedBackground: Treatment of acute respiratory distress syndrome (ARDS) associated with COronaVIrus Disease-2019 (COVID-19) currently relies on dexamethasone and supportive mechanical ventilation, and remains associated with high mortality. Given their ability to limit inflammation, induce immune cells into a regulatory phenotype and stimulate tissue repair, mesenchymal stromal cells (MSCs) represent a promising therapy for severe and critical COVID-19 disease, which is associated with an uncontrolled immune-mediated inflammatory response. Methods: In this phase I-II trial, we aimed to evaluate the safety and efficacy of 3 intravenous infusions of bone marrow (BM)-derived MSCs at 3-day intervals in patients with severe COVID-19. All patients also received dexamethasone and standard supportive therapy. Between June 2020 and September 2021, 8 intensive care unit patients requiring supplemental oxygen (high-flow nasal oxygen in 7 patients, invasive mechanical ventilation in 1 patient) were treated with BM-MSCs. We retrospectively compared the outcomes of these MSC-treated patients with those of 24 matched control patients. Groups were compared by paired statistical tests. Results: MSC infusions were well tolerated, and no adverse effect related to MSC infusions were reported (one patient had an ischemic stroke related to aortic endocarditis). Overall, 3 patients required invasive mechanical ventilation, including one who required extracorporeal membrane oxygenation, but all patients ultimately had a favorable outcome. Survival was significantly higher in the MSC group, both at 28 and 60 days (100% vs 79.2%, p = 0.025 and 100% vs 70.8%, p = 0.0082, respectively), while no significant difference was observed in the need for mechanical ventilation nor in the number of invasive ventilation-free days, high flow nasal oxygenation-free days, oxygen support-free days and ICU-free days. MSC-treated patients also had a significantly lower day-7 D-dimer value compared to control patients (median 821.0 µg/L [IQR 362.0-1305.0] vs 3553 µg/L [IQR 1155.0-6433.5], p = 0.0085). Conclusions: BM-MSC therapy is safe and shows very promising efficacy in severe COVID-19, with a higher survival in our MSC cohort compared to matched control patients. These observations need to be confirmed in a randomized controlled trial designed to demonstrate the efficacy of BM-MSCs in COVID-19 ARDS. Clinical Trial Registration (www.ClinicalTrials.gov), identifier NCT0444545

Comparison of Mesenchymal Stromal Cells From Different Origins for the Treatment of Graft-vs.-Host-Disease in a Humanized Mouse Model

Mesenchymal stromal cells (MSCs) have potent immunomodulatory properties that make them an attractive tool against graft- vs.-host disease (GVHD). However, despite promising results in phase I/II studies, bone marrow (BM-) derived MSCs failed to demonstrate their superiority over placebo in the sole phase III trial reported thus far. MSCs from different tissue origins display different characteristics, but their therapeutic benefits have never been directly compared in GVHD. Here, we compared the impact of BM-, umbilical cord (UC-), and adipose-tissue (AT-) derived MSCs on T-cell function in vitro and assessed their efficacy for the treatment of GVHD induced by injection of human peripheral blood mononuclear cells in NOD-scid IL-2Rγnull HLA-A2/HHD mice. In vitro, resting BM- and AT-MSCs were more potent than UC-MSCs to inhibit lymphocyte proliferation, whereas UC- and AT-MSCs induced a higher regulatory T-cell (CD4+CD25+FoxP3+)/T helper 17 ratio. Interestingly, AT-MSCs and UC-MSCs activated the coagulation pathway at a higher level than BM-MSCs. In vivo, AT-MSC infusions were complicated by sudden death in 4 of 16 animals, precluding an analysis of their efficacy. Intravenous MSC infusions (UC- or BM- combined) failed to significantly increase overall survival (OS) in an analysis combining data from 80 mice (hazard ratio [HR] = 0.59, 95% confidence interval [CI] 0.32–1.08, P = 0.087). In a sensitivity analysis we also compared OS in control vs. each MSC group separately. The results for the BM-MSC vs. control comparison was HR = 0.63 (95% CI 0.30–1.34, P = 0.24) while the figures for the UC-MSC vs. control comparison was HR = 0.56 (95% CI 0.28–1.10, P = 0.09). Altogether, these results suggest that MSCs from various origins have different effects on immune cells in vitro and in vivo. However, none significantly prevented death from GVHD. Finally, our data suggest that the safety profile of AT-MSC and UC-MSC need to be closely monitored given their pro-coagulant activities in vitro