Third-party Mesenchymal Stromal Cells Research Articles

Investigation of scapular dyskinesis and trapezius muscle thickness after neck dissection in patients with head and neck cancer

Mesenchymal stromal cells (MSC) hold promise as a novel immune-modulatory therapy in organ transplantation. First clinical studies have used autologous MSCs; however, the use of allogeneic "off-the-shelf" MSCs is more sustainable for broad clinical implementation, although with the risk of causing sensitization. We investigated safety and feasibility of allogeneic MSCs in renal transplantation, using a matching strategy that prevented repeated mismatches. Ten patients received two doses of 1.5 × 106/kg allogeneic MSCs 6 months after transplantation in a single-center nonrandomized phase Ib trial, followed by lowering of tacrolimus (trough level 3 ng/mL) in combination with everolimus and prednisone. Primary end point was safety, measured by biopsy proven acute rejection (BPAR) and graft loss 12 months after transplantation. Immune monitoring was performed before and after infusion. No BPAR or graft loss occurred and renal function remained stable. One patient retrospectively had DSAs against MSCs, formed before infusion. No major alterations in T and B cell populations or plasma cytokines were observed upon MSC infusion. Administration of HLA selected allogeneic MSCs combined with low-dose tacrolimus 6 months after transplantation is safe at least in the first year after renal transplantation. This sets the stage to further explore the efficacy of third-party MSCs in renal transplantation.

Infusion of human umbilical cord tissue mesenchymal stromal cells in children with autism spectrum disorder.

Ongoing neuroinflammation may contribute to symptoms of autism spectrum disorder (ASD) in at least a portion of affected individuals. Mesenchymal stromal cells (MSCs) have demonstrated the capacity to modulate neuroinflammation, but safety and feasibility of MSC administration in children with ASD have not been well established. In this open‐label, phase I study, 12 children with ASD between 4 and 9 years of age were treated with intravenous (IV) infusions of human cord tissue mesenchymal stromal cells (hCT‐MSCs), a third‐party MSC product manufactured from unrelated donor umbilical cord tissue. Children received one, two, or three doses of 2 × 106 cells per kilogram at 2‐month intervals. Clinical and laboratory evaluations were performed in person at baseline and 6 months and remotely at 12 months after the final infusion. Aside from agitation during the IV placement and infusion in some participants, hCT‐MSCs were well tolerated. Five participants developed new class I anti‐human leukocyte antigen (HLA) antibodies, associated with a specific lot of hCT‐MSCs or with a partial HLA match between donor and recipient. These antibodies were clinically silent and not associated with any clinical manifestations to date. Six of 12 participants demonstrated improvement in at least two ASD‐specific measures. Manufacturing and administration of hCT‐MSCs appear to be safe and feasible in young children with ASD. Efficacy will be evaluated in a subsequent phase II randomized, placebo‐controlled clinical trial.

Infusion of third-party mesenchymal stromal cells after kidney transplantation: a phase I-II, open-label, clinical study

Mesenchymal stromal cells (MSCs) exhibit anti-inflammatory and immune-regulatory properties, and preclinical studies suggest a potential benefit in solid organ transplantation. We report on the 1-year follow-up of an open-label phase I-II trial of a single infusion of third-party MSC post-kidney transplantation, in addition to standard immunosuppression. Ten kidney transplant recipients from deceased donors received third-party bone marrow MSCs (∼2× 106/kg) on day 3 ± 2 post-transplant and were compared to 10 concurrent controls. No adverse effects were noted at MSC injection. One participant with a history of cardiac disease had a non-ST-elevation myocardial infarction approximately 3 hours after MSC infusion. Incidences of opportunistic infections and acute rejection were similar. At day 7 post-transplant, estimated glomerular filtration rate (eGFR) in MSC-treated recipients reached 48.6 ml/min/1.73m2, compared to 32.5 ml/min/1.73m2 in controls and 29.3 ml/min/1.73m2 in our overall cohort of kidney transplant recipients. No difference in eGFR was found at 1 year. MSC-treated recipients showed increased frequencies of regulatory T cells at day 30, with no significant change in B cell frequencies compared to concurrent controls. Four MSC-treated participants developed antibodies against MSC or shared kidney-MSC HLA, with only 1 with MFI >1500. A single infusion of third-party MSC following kidney transplantation appears to be safe, with one cardiac event of unclear relationship to the intervention. MSC therapy is associated with increased regulatory T cell proportion and with improved early allograft function. Long-term effects, including potential immunization against MSC, remain to be studied.

Human Umbilical Cord Derived Mesenchymal Stromal Cells to Treat Steroid-Refractory Acute GvHD III/IV or Overlap Syndrome: Interim Analysis of a Multicenter Phase I/II Study

BackgroundTreatment of patients developing steroid-refractory acute GvHD (SR-aGvHD) after allogeneic hematopoietic cell transplantation (HCT) remains an unmet clinical need and a major therapeutic challenge. Mesenchymal stromal cells (MSC) induce immunosuppression and reduce the inflammatory status initiated by aGvHD. We performed an interim analysis of a multicenter, prospective, non-randomized, phase I/II study, planned to test the safety and activity of human umbilical cord (UC), third-party MSC given sequentially after Pentatostatin for the treatment of SR-aGvHD grade III-IV as well as the overlap syndrome (EUDRACT n. 2012-000582-21), NCT02032446).Patients and MethodsThird-party UC MSC were produced under GMP conditions as previously described (Capelli C et al Cytotherapy, 2011, 13, 786-801). The treatment schedule was based on the sequential administration of Pentostatin, given iv at dose of 1 mg/m^2 for 3 consecutive days, followed by 3 MSC infusions given at weekly intervals. Between October 2013 and May 2018 (range 4.6 years) we enrolled 27 allogeneic HCT adult recipients (22 male) with SR-aGvHD (n 25) or overlap syndrome (n 2), median age 47 years (range 19-62) transplanted from HLA-identical sibling donors (6; 22%), haploidentical donors (2; 7%), unrelated donors (18; 67%) or cord blood (1; 4%). Target organ involvement included skin in 11 cases (44%), gastrointestinal tract (GI) in 23 (92%) and liver in 12 (48%). Nine patients had grade III (33%), 16 patients grade IV (60%) and 2 patients had severe overlap syndrome (7%). Most of patients exhibited multiple organ involvement (n 16, 64%). Patients started MSC therapy at median of 22 days (10-2101) after the onset of aGvHD or overlap syndrome.ResultsThe infusions of UC MSC-infusion were always well tolerated without any immediate or late toxic event. Response to MSC therapy, by day 30 after the final MSC dose, was evaluable in 16 of 27 patients: 7 achieved CR (26%), 6 PR (22%) and 3 NR (11%). Ten patients died before reaching the evaluation time of response: 8 patients for aGvHD and 2 for haematological disease. One patient was lost to follow up after 9 days from the last UC-MSC infusion. Remarkably, the subgroup of patients with single organ involvement (8 GI, 1 liver) were all evaluable showing 6 patients in overall response (67%, 4 RC and 2 PR). No response was observed in 2 cases of severe overlap syndrome. Patients achieving CR had improved longer term OS vs patients in PR/NR (80% vs 50% at 1 year). For patients with grade III and IV SR-aGvHD, CR rates at day 30 were 67% and 31% respectively. During tapering of steroid therapy 4 out of 7 patients that obtained CR had aGvHD recurrence, at median time of 87 days. We observed relapse of underlying malignancy in 5 patients and all died with active disease. At last follow-up 8 out of 27 patients were alive with no GVHD in 5 of them; 14 patients died from TRM: aGvHD or overlap syndrome itself (n=10), infections (n= 2), cGvHD (n= 2). Two patients with uncontrolled GvHD had clinical signs of Transplant Associated Microangiopathy (TAM). A total of 17 reported AE and 12 SAE were registered. In 23 out of 27 patients infectious complications were observed, in most cases virus related (n.25; CMV followed by BK virus and EBV), and fungal infections (3 aspergillus pneumoniae). Blood-stream documented infections (5) and pneumoniae (5) were also observed.ConclusionsThe infusion of cord blood derived MSC proved safe in all cases. As expected in patients with steroid refractory aGvHD, a high rate of infectious complications were observed. When considering that most patients (60 %) had grade IV aGvHD, the response rate and the OS are promising and this warrants not only the completion of this Phase I/II study but also planning a future pivotal trial. DisclosuresNo relevant conflicts of interest to declare.

Manufacturing Mesenchymal Stromal Cells for the Treatment of Graft-versus-Host Disease: A Survey among Centers Affiliated with the European Society for Blood and Marrow Transplantation

The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients’ stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results.

Bone Marrow–Derived Mesenchymal Stromal Cells from Patients with Sickle Cell Disease Display Intact Functionality

Hematopoietic cell transplantation (HCT) is the only cure for sickle cell disease (SCD), but engraftment remains challenging in patients lacking matched donors. Infusion of mesenchymal stromal cells (MSCs) at the time of HCT may promote hematopoiesis and ameliorate graft-versus-host disease. Experimental murine models suggest MSC major histocompatibility complex compatibility with recipient impacts their in vivo function, suggesting autologous MSCs could be superior to third-party MSCs for promoting HCT engraftment. Here we tested whether bone marrow (BM)-derived MSCs from SCD subjects have comparable functionality compared with MSCs from healthy volunteers. SCD MSC doubling time and surface marker phenotype did not differ significantly from non-SCD. Third-party and autologous (SCD) T cell proliferation was suppressed in a dose-dependent manner by all MSCs. SCD MSCs comparably expressed indoleamine-2,3-dioxygenase, which based on transwell and blocking experiments appeared to be the dominant immunomodulatory pathway. The expression of key genes involved in hematopoietic stem cell (HSC)–MSC interactions was minimally altered between SCD and non-SCD MSCs. Expression was, however, altered by IFN-γ stimulation, particularly CXCL14, CXCL26, CX3CL1, CKITL, and JAG1, indicating the potential to augment MSC expression by cytokine stimulation. These data demonstrate the feasibility of expanding BM-derived MSCs from SCD patients that phenotypically and functionally do not differ per International Society of Cell Therapy essential criteria from non-SCD MSCs, supporting initial evaluation (primarily for safety) of autologous MSCs to enhance haploidentical HSC engraftment in SCD.

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.

Mesenchymal Stromal Cells for Treatment of Acute Steroid-Refractory Graft Versus Host Disease: Clinical Responses and Long-Term Outcome.

Acute graft-versus-host disease (aGvHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Steroid-resistant aGvHD is associated with poor outcome, and no commonly accepted salvage therapy is available for its treatment. Here, we report 58 adult patients treated with mesenchymal stromal cells (MSCs) as salvage therapy for steroid-refractory aGvHD. Third-party MSCs expanded in platelet lysate-containing medium were transfused at a median dose of 0.99 × 10(6) cells per kg b.wt. A median of two MSC infusions were administered to each patient. Median time between the onset of aGvHD and the first infusion of MSCs was 12 days (range, 6-62 days). Most patients (79%) had grade IV aGvHD. Five patients showed complete response, five showed very good partial response, 17 showed partial response, and 31 showed no response. The estimated probability of survival after 1 year was 19%, and median survival was 69 days. Overall survival was not significantly different from that of a historical cohort of patients receiving alternative salvage therapy and no MSC infusions. In conclusion, MSC treatment on top of conventional immunosuppression was associated with an overall response rate of 47% but improved outcome in terms of survival remains to be shown.

Cell Therapy in Kidney Transplantation

Current immunosuppressants have nonspecific immuosuppressive effects, and are not helpful for tolerance induction. Consequently, transplant patients cannot discontinue using them, and their nonspecific immunosuppressive effects result in many side effects, including infection and malignancy. However, most of cellular immunotherapy can have donor antigen-specific immunsuppressive effects. Therefore, cell therapy could be an alternative or adjunctive to nonspecific immunosuppressants. Polyclonal or antigen-specific Foxp3+ regulatory T cells have been actively tried for prevention of acute rejection, treatment of chronic rejection, or tolerance induction in clinical trials. Regulatory macrophages are also under clinical trials for kidney transplant patients. IL-10-secreting type 1 regulatory T cells and donoror recipient-derived tolerogenic dendritic cells will also be used for immunoregulation in clinical trials of kidney transplantation. These cells have antigen-specific immunoregulatory effects. Mesenchymal stromal cells (MSCs) have good proliferative capacity and immunosuppressive actions independently of major histocompatibility complex; therefore, even third-party MSCs can be stored and used for many patients. Cell therapy using various immunoregulatory cells is now promising for not only reducing side effects of nonspecific immunosuppressants but also induction of immune tolerance, and is expected to contribute to better outcomes in transplant patients.

Mesenchymal stromal cells inhibit proliferation of virus-specific CD8(+) T cells.

Mesenchymal stromal cells (MSCs) possess broad immunomodulatory capacities that are currently investigated for potential clinical application in treating autoimmune disorders. Third-party MSCs suppress alloantigen-induced proliferation of peripheral blood mononuclear cells providing the rationale for clinical use in graft-versus-host disease (GvHD). We confirmed that MSCs strongly inhibited proliferation of CD8(+) T cells in a mixed lymphocyte reaction. However, MSCs also suppressed proliferation of T cells specifically recognizing cytomegalovirus (CMV) and influenza virus. Inhibition was dose dependent, but independent of the culture medium. MSCs inhibited proliferation of specific CD8(+) T cells and the release of IFN-γ by specific CD8(+) T cells for immunodominant HLA-A2- and HLA-B7- restricted antigen epitopes derived from CMV phosphoprotein 65 and influenza matrix protein. This is in contrast to a recently reported scenario where MSCs exert differential effects on alloantigen and virus-specific T cells potentially having an impact on surveillance and prophylaxis of patients treated by MSCs.

Sequential Third-Party Mesenchymal Stromal Cell Therapy for Refractory Acute Graft-versus-Host Disease

We evaluated the feasibility, safety, and efficacy of the administration of 4 sequential doses (intravenously administered on days 1, 4, 11, and 18) of cryopreserved bone marrow–derived mesenchymal stromal cells (MSC) expanded with platelet lysate and obtained from third-party donors as a second-line treatment for steroid-refractory acute graft-versus-host (aGVHD) disease in a series of 25 patients. All patients received at least 2 doses of MSC, whereas 21 received 3 doses and 18 received the initially planned 4 doses. Because of the achievement of partial response, 4 patients received additional doses of MSC. Median single cell dose administered was 1.1 × 106 MSC/kg of recipient body weight. There were no adverse events related to the MSC infusion in the 99 procedures performed, with the exception of a cardiac ischemic event that occurred twice in a patient with prior history of cardiac ischemia. Response to MSC at 60 days after the first dose was evaluable in 24 patients. Seventeen patients (71%) responded (11 complete and 6 partial responses), with a median time to response of 28 days after the first MSC dose, whereas 7 patients did not respond. In summary, we can conclude that sequential cryopreserved third-party MSC therapy administered on days 1, 4, 11, and 18 is a safe procedure for patients with steroid-refractory aGVHD. This strategy may provide a high rate of overall responses of aGVHD with a low toxicity profile.

Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies Using a Reduced-Intensity Conditioning Regimen and Third-Party Mesenchymal Stromal Cells

Allogeneic hematopoietic stem cell transplantation for patients with a hemoglobinopathy can be curative but is limited by donor availability. Although positive results are frequently observed in those with an HLA-matched sibling donor, use of unrelated donors has been complicated by poor engraftment, excessive regimen-related toxicity, and graft-versus-host disease (GVHD). As a potential strategy to address these obstacles, a pilot study was designed that incorporated both a reduced-intensity conditioning and mesenchymal stromal cells (MSCs). Six patients were enrolled, including 4 with high-risk sickle cell disease (SCD) and 2 with transfusion-dependent thalassemia major. Conditioning consisted of fludarabine (150 mg/m2), melphalan (140 mg/m2), and alemtuzumab (60 mg for patients weighing > 30 kg and .9 mg/kg for patients weighing <30 kg). Two patients received HLA 7/8 allele matched bone marrow and 4 received 4-5/6 HLA matched umbilical cord blood as the source of HSCs. MSCs were of bone marrow origin and derived from a parent in 1 patient and from an unrelated third-party donor in the remaining 5 patients. GVHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil. One patient had neutropenic graft failure, 2 had autologous hematopoietic recovery, and 3 had hematopoietic recovery with complete chimerism. The 2 SCD patients with autologous hematopoietic recovery are alive. The remaining 4 died either from opportunistic infection, GVHD, or intracranial hemorrhage. Although no infusion-related toxicity was seen, the cotransplantation of MSCs was not sufficient for reliable engraftment in patients with advanced hemoglobinopathy. Although poor engraftment has been observed in nearly all such trials to date in this patient population, there was no evidence to suggest that MSCs had any positive impact on engraftment. Because of the lack of improved engraftment and unacceptably high transplant-related mortality, the study was prematurely terminated. Further investigations into understanding the mechanisms of graft resistance and development of strategies to overcome this barrier are needed to move this field forward.

Response and Long-Term Outcome After Treatment With Third-Party Mesenchymal Stromal Cells - Updated Results In 58 Patients With Steroid-Refractory Acute Graft-Versus Host Disease -

Introduction Acute Graft-versus-Host Disease (aGvHD) remains the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). So far, corticosteroids are the only first-line treatment proven to be effective. Steroid-resistance is associated with poor outcome and no commonly accepted second-line salvage therapy is available until now. Mesenchymal stromal cells (MSC) have shown promising immunomodulatory effects and have been suggested as cell-based treatment option in patients with steroid-refractory acute GvHD. Here, we report our experience from a large cohort of patients treated with MSC. Patients, Materials and Methods Fifty-eight patients with steroid-refractory acute GvHD after HSCT were treated with MSC at our centre between 2007 and 2012. MSC were obtained from volunteer third-party donors and expanded in platelet-lysate containing medium. Median age at transplantation was 55 years (range 19-71). In 25 patients AML was diagnosed initially. Further diagnoses were CLL (n=9), ALL (n=5), MDS (n=5) and others (n=14). For transplantation, patients received peripheral blood stem cells (n=56) or bone marrow (n=2) from HLA-identical (n=43) or HLA mismatched donors (n=15) after varying reduced-intensity conditioning regimens (n=50) or myeloablative conditioning (n=8). Eight-teen patients received anti-thymocyte globulin as part of their conditioning. GvHD prophylaxis consisted mostly of cyclosporine A (CsA) plus methotrexate, CsA alone or CsA plus mycophenolate mofetile. The majority of patients suffered from aGvHD grade IV (79%), median interval from HSCT to onset of GvHD was 36 days. Involvement of gastrointestinal tract, liver and skin was observed in 91%, 43% and 41% of patients, respectively. Most patients (64%) had involvement of 2 or 3 organs at the same time. Besides corticosteroids, 48 patients (83%) received at least one additional immunosuppressive agent before the first MSC infusion. Response was assessed 28 days after initiation of MSC treatment and overall survival of the MSC treated cohort was compared to a historic patient cohort (n=36) with steroid-refractory aGvHD not receiving MSC. Results Median time between onset of aGvHD and first application of MSC was 12 days (range 6-62). Altogether 139 doses of MSC were transfused at a median dosage of 0.99x106cells/kg bodyweight (range 0.448 -2.077). A median number of 2 MSC infusions were given per patient (range 1-6). During MSC treatment, 39 patients needed further escalation of immunosuppression due to persistence or progression of GvHD. No side-effects directly related to MSC infusions were observed. Four weeks after first MSC application, 9% (n=5) of patients showed a complete response (CR), 9% (n=5) exhibited a very good partial response (VGPR), 29% (n=17) experienced partial response (PR) whereas 53% (n=31) were classified as non-responders. There were no significant differences in organ specific response. One-year and 2-year-survival after onset of aGvHD was 19% [95% CI: 9-29%] and 17% [95% CI: 7-26%], respectively. Median survival was 69 days [95% CI: 38-100 days]. Causes of death were aGvHD (54%), infectious complications (29%), relapse of the underlying disease (4%) and others (13%). Median follow up was 1689 days and at the end of follow-up, 8 patients were still alive (median survival after HSCT 58 months). Of those eight, 6 were initially classified as responders (CR n=4, VGPR n=1, PR n=1). Responders showed higher survival compared to non-responders (hazard ratio 0.38; p=0.004). Overall survival in the MSC treated cohort did not differ significantly to that of the historic cohort not receiving MSC. Conclusions MSC in combination with further immunosuppressive strategies resulted in a response rate of 47% in patients with steroid-refractory aGvHD and should therefore be considered as a valuable treatment option in a difficult clinical situation. However, compared to our historic cohort, treatment with MSC did not lead to an improvement of survival. Future studies need to define which patient subsets are likely to benefit from MSC therapy and whether certain MSC preparations from specific donors may have a more pronounced and long-lasting immunosuppressive effect. Therefore, further insights into MSC biology are urgently needed to optimize the translation into clinical practice. Disclosures: No relevant conflicts of interest to declare.

Alterations in the Cellular Immune Compartment of Patients Treated with Third-Party Mesenchymal Stromal Cells Following Allogeneic Hematopoietic Stem Cell Transplantation

Adoptive transfer of third-party mesenchymal stromal cells (MSCs) has emerged as a promising tool for the treatment of steroid-refractory graft-versus-host disease (GVHD). Despite numerous in vitro studies and preclinical models, little is known about their effects on the patients' immune system. We assessed immune alterations in the T-cell, B-cell, natural killer cell, dendritic cell, and monocytic compartments of steroid-refractory GVHD patients 30, 90, and 180 days after MSC (n = 6) or placebo (n = 5) infusion, respectively. Infused MSCs were bioactive as suggested by the significant reduction in epithelial cell death, which represents a biomarker for acute GVHD. There were several indications that MSCs shift the patients' immune system toward a more tolerogenic profile. Most importantly, infusion of MSCs was associated with increased levels of regulatory (forkhead box P3 (FOXP3)(+) and interleukin (IL)-10(+) ) T-cells, reduced pro-inflammatory IL-17(+) T(Th17)-cells, and skewing toward type-2 T-helper cell responses. Furthermore, IL-2, which has been recently shown to exert a positive immune modulating effect in GVHD patients, was higher in the MSC patients at all evaluated time points during 6 months after MSC-infusion. Overall, our findings will contribute to the refinement of monitoring tools, for assessing MSC treatment-efficacy and increase our understanding regarding the MSCs' in vivo effects.

Treatment of Familial Hemophagocytic Lymphohistiocytosis with Third-Party Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) represent an attractive tool for cellular therapies on grounds of their immunomodulatory and regenerative properties. Here, we report the first case of familial hemophagocytic lymphohistiocytosis (FHL) treated with MSCs. This rare autosomal recessive disorder is characterized by hyperinflammation that results from a failure of natural control mechanisms to terminate immune responses. Crosstalk between innate (macrophages) and adaptive (T cells) immunity is heavily altered. Immunochemotherapy is only temporarily effective in the control of FHL, and the outcome is usually fatal unless the patient undergoes allogeneic stem cell transplantation. Our hypothesis was that the application of MSCs could be effective in the treatment of FHL, since MSCs possess a broad repertoire of immunomodulating mechanisms impacting both innate and adaptive immunity pathways. In fact, the adoptive transfer of third-party MSCs transiently controlled the extreme immunological deterioration in the described patient who was otherwise not responsive to standard treatment, including repetitive chemotherapy. If these transient effects of MSCs can be confirmed in future-controlled clinical trials, adoptive MSC therapy could represent a salvage agent in FHL acting as a bridge to definitive treatment with stem cell transplantation.

Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses

Infusion of human third-party mesenchymal stromal cells (MSCs) appears to be a promising therapy for acute graft-versus-host disease (aGvHD). To date, little is known about how MSCs interact with the body's innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46) and DAF (CD55), but were protected from complement lysis via expression of protectin (CD59). Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells.

Feasibility and Safety of Adoptive Immunotherapy with CIK Cells after Cord Blood Transplantation

Five patients with aggressive acute leukemias who had relapsed after cord blood transplantation were treated with cord blood derived cytokine-induced killer (CIK) cells. These were obtained by ex vivo expansion, using as starting material the washouts of the cord blood units, left over at the end of the transplant. We did not observe any acute or delayed adverse event, and observed 1 partial response in 1 patient concomitantly with the development of acute grade III graft-versus-host disease (GVHD). These observations show the relatively low toxicity of cord blood-derived CIK cells and, more importantly, the feasibility of this immunotherapy program for patients who could not otherwise benefit from donor lymphocyte infusions.

Similar proteomic profiles of human mesenchymal stromal cells from different donors

Bone marrow (BM) stromal cells, also referred to as mesenchymal stromal cells (MSC), can be expanded ex vivo and are able to differentiate along multiple lineages, including chondrocytes, osteoblasts and adipocytes. MSC are known to secrete a number of cytokines and regulatory molecules implicated in different aspects of hematopoiesis, and seem to modulate the immune system. MSC appear to be promising candidates for cellular therapy associated with BM transplantation (BMT). We compared protein expression profiles of MSC cultures derived from different BM donors using two-dimensional (2-D) gel electrophoresis and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) tandem mass spectrometry (MS/MS), and compared mixed lymphocyte reaction (MLR) assays in the absence and presence of third-party human (h) MSC derived from different donors during the same culture passage. In a window of observation (pH 4-7, molecular weight 10-220 kDa), about 172 protein spots were obtained in each 2-D gel, corresponding to 84 distinct proteins. A comparative analysis demonstrated a very similar proteomic profile of cells of the first passage derived from different donors, suggesting that these cells have the same expression pattern. Additionally, cells derived from different donors were equally able to inhibit lymphocyte proliferation. These results encourage the use of third-party MSC in cellular therapies, as cells derived from different individuals seem to have the same proteomic pattern and exhibit functionally similar properties.

Cotransplantation of third-party mesenchymal stromal cells can alleviate single-donor predominance and increase engraftment from double cord transplantation

AbstractAlthough the infusion of umbilical cord blood (UCB) from multiple donors can be a strategy to overcome the cell dose limitation frequently encountered in UCB transplantation, clinical trials have revealed that cells from one donor dominate engraftment. To investigate the origin of and the factors influencing this inequality, we performed mixed transplantation of 2 UCB units with varying degrees of HLA disparities into NOD/SCID mice and determined donor origins by polymerase chain reaction–sequence-specific oligonucleotide probe (PCR-SSOP) or real-time quantitative (RQ)–PCR for human short tandem repeats (STRs). When total mononuclear cells from 2 units were transplanted as a mixture, cells from one donor predominated (ratio, 81:19), despite comparable overall engraftment when infused as single units, and no augmentation in overall engraftment was observed when compared with the single-unit controls. However, lineage depletion or cotransplantation of mesenchymal stromal cells (MSCs) expanded from third-party bone marrow resulted in more balanced coengraftment. Direct comparison of double UCB transplantation in the presence or absence of MSCs showed that the reduced deviation in the donor ratio (1.8:1 vs. 2.8:1) correlated with a higher overall level of engraftment with MSC cotransplantation. These results indicate that third-party MSCs can be used to alleviate donor deviation and to facilitate engraftment of multidonor UCB.