Helene Roelofs

Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study.

BACKGROUNDnSevere graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic transplantation with haemopoietic stem cells. Mesenchymal stem cells modulate immune responses in vitro and in vivo. We aimed to assess whether mesenchymal stem cells could ameliorate GVHD after haemopoietic-stem-cell transplantation.nnnMETHODSnPatients with steroid-resistant, severe, acute GVHD were treated with mesenchymal stem cells, derived with the European Group for Blood and Marrow Transplantation ex-vivo expansion procedure, in a multicentre, phase II experimental study. We recorded response, transplantation-related deaths, and other adverse events for up to 60 months follow-up from infusion of the cells.nnnFINDINGSnBetween October, 2001, and January, 2007, 55 patients were treated. The median dose of bone-marrow derived mesenchymal stem cells was 1.4x10(6) (min-max range 0.4-9x10(6)) cells per kg bodyweight. 27 patients received one dose, 22 received two doses, and six three to five doses of cells obtained from HLA-identical sibling donors (n=5), haploidentical donors (n=18), and third-party HLA-mismatched donors (n=69). 30 patients had a complete response and nine showed improvement. No patients had side-effects during or immediately after infusions of mesenchymal stem cells. Response rate was not related to donor HLA-match. Three patients had recurrent malignant disease and one developed de-novo acute myeloid leukaemia of recipient origin. Complete responders had lower transplantation-related mortality 1 year after infusion than did patients with partial or no response (11 [37%] of 30 vs 18 [72%] of 25; p=0.002) and higher overall survival 2 years after haemopoietic-stem-cell transplantation (16 [53%] of 30 vs four [16%] of 25; p=0.018).nnnINTERPRETATIONnInfusion of mesenchymal stem cells expanded in vitro, irrespective of the donor, might be an effective therapy for patients with steroid-resistant, acute GVHD.

Autologous bone marrow-derived mesenchymal stromal cell treatment for refractory luminal Crohn's disease: results of a phase I study

Background and aim Mesenchymal stromal cells (MSCs) are pluripotent cells that have immunosuppressive effects both in vitro and in experimental colitis. Promising results of MSC therapy have been obtained in patients with severe graft versus host disease of the gut. Our objective was to determine the safety and feasibility of autologous bone marrow derived MSC therapy in patients with refractory Crohns disease. Patients and intervention 10 adult patients with refractory Crohns disease (eight females and two males) underwent bone marrow aspiration under local anaesthesia. Bone marrow MSCs were isolated and expanded ex vivo. MSCs were tested for phenotype and functionality in vitro. 9 patients received two doses of 1–2×106u2005cells/kg body weight, intravenously, 7u2005days apart. During follow-up, possible side effects and changes in patients Crohns disease activity index (CDAI) scores were monitored. Colonoscopies were performed at weeks 0 and 6, and mucosal inflammation was assessed by using the Crohns disease endoscopic index of severity. Results MSCs isolated from patients with Crohns disease showed similar morphology, phenotype and growth potential compared to MSCs from healthy donors. Importantly, immunomodulatory capacity was intact, as Crohns disease MSCs significantly reduced peripheral blood mononuclear cell proliferation in vitro. MSC infusion was without side effects, besides a mild allergic reaction probably due to the cryopreservant DMSO in one patient. Baseline median CDAI was 326 (224–378). Three patients showed clinical response (CDAI decrease ≥70 from baseline) 6u2005weeks post-treatment; conversely three patients required surgery due to disease worsening. Conclusions Administration of autologous bone marrow derived MSCs appears safe and feasible in the treatment of refractory Crohns disease. No serious adverse events were detected during bone marrow harvesting and administration.

Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1

Mesenchymal stem cells are self-renewing cells with the ability to differentiate into osteocytes, chondrocytes and adipocytes. This article describes a subset of mesenchymal stem cells with distinct phenotypic and functional properties. Background Conventionally, mesenchymal stem cells are functionally isolated from primary tissue based on their capacity to adhere to a plastic surface. This isolation procedure is hampered by the unpredictable influence of co-cultured hematopoietic and/or other unrelated cells and/or by the elimination of a late adhering mesenchymal stem cells subset during removal of undesired cells. To circumvent these limitations, several antibodies have been developed to facilitate the prospective isolation of mesenchymal stem cells. Recently, we described a panel of monoclonal antibodies with superior selectivity for mesenchymal stem cells, including the monoclonal antibodies W8B2 against human mesenchymal stem cell antigen-1 (MSCA-1) and 39D5 against a CD56 epitope, which is not expressed on natural killer cells. Design and Methods Bone marrow derived mesenchymal stem cells from healthy donors were analyzed and isolated by flow cytometry using a large panel of antibodies against surface antigens including CD271, MSCA-1, and CD56. The growth of mesenchymal stem cells was monitored by colony formation unit fibroblast (CFU-F) assays. The differentiation of mesenchymal stem cells into defined lineages was induced by culture in appropriate media and verified by immunostaining. Results Multicolor cell sorting and CFU-F assays showed that mesenchymal stem cells were ~90-fold enriched in the MSCA-1+CD56− fraction and ~180-fold in the MSCA-1+CD56+ fraction. Phenotype analysis revealed that the expression of CD10, CD26, CD106, and CD146 was restricted to the MSCA-1+CD56− mesenchymal stem cells subset and CD166 to MSCA-1+CD56± mesenchymal stem cells. Further differentiation of these subsets showed that chondrocytes and pancreatic-like islets were predominantly derived from MSCA-1+CD56± cells whereas adipocytes emerged exclusively from MSCA-1+CD56− cells. The culture of single sorted MSCA-1+CD56+ cells resulted in the appearance of phenotypically heterogeneous clones with distinct proliferation and differentiation capacities. Conclusions Novel mesenchymal stem cells subsets with distinct phenotypic and functional properties were identified. Our data suggest that the MSCA-1+CD56+ subset is an attractive starting population for autologous chondrocyte transplantation.

Autologous Bone Marrow-Derived Mesenchymal Stromal Cells for the Treatment of Allograft Rejection After Renal Transplantation: Results of a Phase I Study

Despite excellent short‐term results, long‐term survival of transplanted kidneys has not improved accordingly. Although alloimmune responses and calcineurin inhibitor‐related nephrotoxicity have been identified as main drivers of fibrosis, no effective treatment options have emerged. In this perspective, mesenchymal stromal cells (MSCs) are an interesting candidate because of their immunosuppressive and regenerative properties. Of importance, no other clinical studies have investigated their effects in allograft rejection and fibrosis. We performed a safety and feasibility study in kidney allograft recipients to whom two intravenous infusions (1 million cells per kilogram) of autologous bone marrow (BM) MSCs were given, when a protocol renal biopsy at 4 weeks or 6 months showed signs of rejection and/or an increase in interstitial fibrosis/tubular atrophy (IF/TA). Six patients received MSC infusions. Clinical and immune monitoring was performed up to 24 weeks after MSC infusions. MSCs fulfilled the release criteria, infusions were well‐tolerated, and no treatment‐related serious adverse events were reported. In two recipients with allograft rejection, we had a clinical indication to perform surveillance biopsies and are able to report on the potential effects of MSCs in rejection. Although maintenance immunosuppression remained unaltered, there was a resolution of tubulitis without IF/TA in both patients. Additionally, three patients developed an opportunistic viral infection, and five of the six patients displayed a donor‐specific downregulation of the peripheral blood mononuclear cell proliferation assay, not reported in patients without MSC treatment. Autologous BM MSC treatment in transplant recipients with subclinical rejection and IF/TA is clinically feasible and safe, and the findings are suggestive of systemic immunosuppression.

Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti‐inflammatory macrophages

Multipotent stromal cells (MSC) have been shown to possess immunomodulatory capacities and are therefore explored as a novel cellular therapy. One of the mechanisms through which MSC modulate immune responses is by the promotion of regulatory T cell (Treg) formation. In this study, we focused on the cellular interactions and secreted factors that are essential in this process. Using an in vitro culture system, we showed that culture‐expanded bone marrow‐derived MSC promote the generation of CD4+CD25hiFoxP3+ T cells in human PBMC populations and that these populations are functionally suppressive. Similar results were obtained with MSC‐conditioned medium, indicating that this process is dependent on soluble factors secreted by the MSC. Antibody neutralization studies showed that TGF‐β1 mediates induction of Tregs. TGF‐β1 is constitutively secreted by MSC, suggesting that the MSC‐induced generation of Tregs by TGF‐β1 was independent of the interaction between MSC and PBMC. Monocyte‐depletion studies showed that monocytes are indispensable for MSC‐induced Treg formation. MSC promote the survival of monocytes and induce differentiation toward macrophage type 2 cells that express CD206 and CD163 and secrete high levels of IL‐10 and CCL‐18, which is mediated by as yet unidentified MSC‐derived soluble factors. CCL18 proved to be responsible for the observed Treg induction. These data indicate that MSC promote the generation of Tregs. Both the direct pathway through the constitutive production of TGF‐β1 and the indirect novel pathway involving the differentiation of monocytes toward CCL18 producing type 2 macrophages are essential for the generation of Tregs induced by MSC. Stem Cells 2013;31:1980‐1991

Pretreatment with interferon-γ enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis

Mesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohns disease. MSCs are pluripotent cells with immunosuppressive properties. Recent data suggest that resting MSCs do not have significant immunomodulatory activity, but that the immunosuppressive function of MSCs has to be elicited by interferon‐γ (IFN‐γ). In this article, we assessed the effects of IFN‐γ prestimulation of MSCs (IMSCs) on their immunosuppressive properties in vitro and in vivo. To this end, we pretreated MSCs with IFN‐γ and assessed their therapeutic effects in dextran sodium sulfate (DSS)‐ and trinitrobenzene sulfonate (TNBS)‐induced colitis in mice. We found that mice treated with IMSCs (but not MSCs) showed a significantly attenuated development of DSS‐induced colitis. Furthermore, IMSCs alleviated symptoms of TNBS‐induced colitis. IMSC‐treated mice displayed an increase in body weight, lower colitis scores, and better survival rates compared with untreated mice. In addition, serum amyloid A protein levels and local proinflammatory cytokine levels in colonic tissues were significantly suppressed after administration of IMSC. We also observed that IMSCs showed greater migration potential than unstimulated MSCs to sites within the inflamed intestine. In conclusion, we show that prestimulation of MSCs with IFN‐γ enhances their capacity to inhibit Th1 inflammatory responses, resulting in diminished mucosal damage in experimental colitis. These data demonstrate that IFN‐γ activation of MSCs increases their immunosuppresive capacities and importantly, their therapeutic efficacy in vivo. STEM CELLS 2011;29:1549–1558

Adipose Tissue-Derived Multipotent Stromal Cells Have a Higher Immunomodulatory Capacity Than Their Bone Marrow-Derived Counterparts

Adipose tissue‐derived multipotent stromal cells (AT‐MSCs) are studied as an alternative to bone marrow‐derived multipotent stromal cells (BM‐MSCs) for immunomodulatory treatment. In this study, we systematically compared the immunomodulatory capacities of BM‐MSCs and AT‐MSCs derived from age‐matched donors. We found that BM‐MSCs and AT‐MSCs share a similar immunophenotype and capacity for in vitro multilineage differentiation. BM‐MSCs and AT‐MSCs showed comparable immunomodulatory effects as they were both able to suppress proliferation of stimulated peripheral blood mononuclear cells and to inhibit differentiation of monocyte‐derived immature dendritic cells. However, at equal cell numbers, the AT‐MSCs showed more potent immunomodulatory effects in both assays as compared with BM‐MSCs. Moreover, AT‐MSCs showed a higher level of secretion of cytokines that have been implicated in the immunomodulatory modes of action of multipotent stromal cells, such as interleukin‐6 and transforming growth factor‐β1. This is correlated with higher metabolic activity of AT‐MSCs compared with BM‐MSCs. We conclude that the immunomodulatory capacities of BM‐MSCs and AT‐MSCs are similar, but that differences in cytokine secretion cause AT‐MSCs to have more potent immunomodulatory effects than BM‐MSCs. Therefore, lower numbers of AT‐MSCs evoke the same level of immunomodulation. These data indicate that AT‐MSCs can be considered as a good alternative to BM‐MSCs for immunomodulatory therapy.

Co-infusion of ex vivo- expanded, parental MSCs prevents life-threatening acute GVHD, but does not reduce the risk of graft failure in pediatric patients undergoing allogeneic umbilical cord blood transplantation

When compared with BMT, umbilical cord blood transplantation (UCBT) is associated with a lower rate of engraftment and delayed hematological/immunological recovery. This leads to increased risk of TRM in the early post transplantation period due to infection. Acute GVHD, although occurring less frequently in UCBT compared with BMT, is also significantly associated with increased rate of early TRM. BM MSCs are known to support normal in vivo hematopoiesis, and co-transplantation of MSCs has been shown to enhance engraftment of human cord blood hematopoietic cells in nonobese diabetic/SCID mice. In 13 children with hematological disorders (median age 2 years) undergoing UCBT, we co-transplanted paternal, HLA-disparate MSCs with the aim of improving hematological recovery and reducing rejection. We observed no differences in hematological recovery or rejection rates compared with 39 matched historical controls, most of whom received G-CSF after UCBT. However, the rate of grade III and IV acute GVHD was significantly decreased in the study cohort when compared with controls (P=0.05), thus resulting in reduced early TRM. Although these data do not support the use of MSCs in UCBT to support hematopoietic engraftment, they suggest that MSCs, possibly because of their immunosuppressive effect, may abrogate life-threatening acute GVHD and reduce early TRM.

Multiple infusions of mesenchymal stromal cells induce sustained remission in children with steroid-refractory, grade III-IV acute graft-versus-host disease.

Mesenchymal stromal cell (MSC) infusions have been reported to be effective in patients with steroid‐refractory, acute graft‐versus‐host disease (aGvHD) but comprehensive data on paediatric patients are limited. We retrospectively analysed a cohort of 37 children (aged 3 months‐17 years) treated with MSCs for steroid‐refractory grade III–IV aGvHD. All patients but three received multiple MSC infusions. Complete response (CR) was observed in 24 children (65%), while 13 children had either partial (n = 8) or no response (n = 5). Cumulative incidence of transplantation‐related mortality (TRM) in patients who did or did not achieve CR was 17% and 69%, respectively (P = 0·001). After a median follow‐up of 2·9 years, overall survival (OS) was 37%; it was 65% vs. 0% in patients who did or did not achieve CR, respectively (P = 0·001). The median time from starting steroids for GvHD treatment to first MSC infusion was 13 d (range 5–85). Children treated between 5 and 12 d after steroid initiation showed a trend for better OS (56%) and lower TRM (17%) as compared with patients receiving MSCs 13–85 d after steroids (25% and 53%, respectively; P = 0·22 and 0·06, respectively). Multiple MSC infusions are safe and effective for children with steroid‐refractory aGvHD, especially when employed early in the disease course.

Modulation of Immune Responses by Mesenchymal Stem Cells

Abstract:u2002 Mesenchymal stem cells (MSCs) are multipotent progenitor cells and interest in MSC therapy has been raised by the observation that MSCs are able to modulate immune responses in vitro and in vivo. Here, we show that MSCs are not intrinsically immune privileged and are capable of inducing memory T cell responses following injection in vivo in immunocompetent hosts. After cotransplantation in recipients that have received sublethal irradiation, allogeneic MSCs can still induce an alloresponse that may result in graft rejection, suggesting that the immunogenicity of allogeneic MSCs are not fully prevented by a nonmyeloablative conditioning regimen. It is still unclear whether the immunogenicity of allogeneic MSCs is also preserved following a fully myeloablative conditioning regimen.