Berit Sundberg

Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells

Adult bone-marrow-derived mesenchymal stem cells are immunosuppressive and prolong the rejection of mismatched skin grafts in animals. We transplanted haploidentical mesenchymal stem cells in a patient with severe treatment-resistant grade IV acute graft-versus-host disease of the gut and liver. Clinical response was striking. The patient is now well after 1 year. We postulate that mesenchymal stem cells have a potent immunosuppressive effect in vivo.

Mesenchymal Stem Cells Inhibit and Stimulate Mixed Lymphocyte Cultures and Mitogenic Responses Independently of the Major Histocompatibility Complex

We aimed to study the effects of mesenchymal stem cells (MSCs) on alloreactivity and effects of T‐cell activation on human peripheral blood lymphocytes (PBLs) in vitro. MSCs were expanded from the bone marrow of healthy subjects. MSCs isolated from second to third passage were positive for CD166, CD105, CD44, CD29, SH‐3 and SH‐4, but negative for CD34 and CD45. MSCs cultured in osteogenic, adipogenic or chondrogenic media differentiated, respectively, into osteocytes, adipocytes or chondrocytes. MSC added to PBL cultures had various effects, ranging from slight inhibition to stimulation of DNA synthesis. The stimulation index (SI = (PBL + MSC)/PBL) varied between 0.2 and 7.3. The SI was not affected by the MSC dose or by the addition of allogeneic or autologous MSCs to the lymphocytes.

Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease.

Background. Mesenchymal stem cells (MSC) have immunomodulatory effects. The aim was to study the effect of MSC infusion on graft-versus-host disease (GVHD). Methods. We gave MSC to eight patients with steroid-refractory grades III-IV GVHD and one who had extensive chronic GVHD. The MSC dose was median 1.0 (range 0.7 to 9)×106/kg. No acute side-effects occurred after the MSC infusions. Six patients were treated once and three patients twice. Two patients received MSC from HLA-identical siblings, six from haplo-identical family donors and four from unrelated mismatched donors. Results. Acute GVHD disappeared completely in six of eight patients. One of these developed cytomegalovirus gastroenteritis. Complete resolution was seen in gut (6), liver (1) and skin (1). Two died soon after MSC treatment with no obvious response. One of them had MSC donor DNA in the colon and a lymph node. Five patients are still alive between 2 months and 3 years after the transplantation. Their survival rate was significantly better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSC during the same period (P=0.03). One patient treated for extensive chronic GVHD showed a transient response in the liver, but not in the skin and he died of Epstein-Barr virus lymphoma. Conclusion. MSC is a very promising treatment for severe steroid-resistant acute GVHD.

Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells.

Background. Mesenchymal stem cells (MSCs) can reduce the incidence of graft-versus-host disease because of their ability to inhibit T-lymphocyte proliferation. There are no publications on the effect that MSCs have on cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, effector cells vital for the graft-versus-leukemia effect. Methods. Cytotoxic T cells were primed in mixed lymphocyte culture (MLC) against irradiated stimulator lymphocytes, and irradiated third-party MSCs were added at different time points. The CTLs were collected, and their cytotoxic potential was analyzed in a chromium-release assay against the same stimulator cells as in the MLC. Purified NK cells were mixed with irradiated MSCs, and the lysis was measured in chromium-release assay against K562 target cells. Results. We found that MSCs inhibited CTL-mediated lysis by 70% if added at the beginning of the 6-day MLC. The lysis was not affected on day 3 or in the cytotoxic phase. Furthermore, MSCs inhibited the formation of cytotoxic lymphocytes when the cells were separated in a transwell system, which indicates that the effect is mediated by a soluble factor. NK cell-mediated lysis of K562 cells was not inhibited by MSCs. MSCs did not induce proliferation of allogeneic lymphocytes, and they were not lysed by allogeneic CTLs or NK cells. Conclusion. Our findings indicate that MSCs escape recognition by CTLs and alloreactive NK cells, and inhibit the formation of cytotoxic T cells by secreting a soluble factor, but that they do not interfere with CTLs and NK cell lysis.

Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells.

Seven patients underwent treatment with mesenchymal stem cells (MSCs), together with allogeneic hematopoietic stem cell transplantation (HSCT). MSCs were given to three patients for graft failure and four patients were included in a pilot study. HSCT donors were three human leukocyte antigen (HLA)-identical siblings, three unrelated donors and one cord blood unit. The conditioning was myeloablative in four patients and reduced in three patients. MSC donors were HLA-identical siblings in three cases and haploidentical in four cases. Neutrophil counts >0.5 × 109/l was reached at a median of 12 (range 10–28) days. Platelet counts >30 × 109/l was achieved at a median of 12 (8–36) days. Acute graft-versus-host disease (GVHD) grade 0–I was seen in five patients. Two patients developed grade II, which in one patient evolved into chronic GVHD. One severe combined immunodeficiency (SCID) patient died of aspergillosis, the others are alive and well. One patient, diagnosed with aplastic anemia had graft failure after her first transplantation and severe Henoch–Schönlein Purpura (HSP). After retransplantation of MSCs and HSCs, she recovered from both the HSP and aplasia. Thus, co-transplantation of MSC resulted in fast engraftment of absolute neutrophil count (ANC) and platelets and 100% donor chimerism, even in three patients regrafted for graft failure/rejection.

Mesenchymal Stem Cells Inhibit the Expression of CD25 (Interleukin-2 Receptor) and CD38 on Phytohaemagglutinin-Activated Lymphocytes

Mesenchymal stem cells (MSC) are immunomodulatory and inhibit lymphocyte proliferation. We studied surface expression of lymphocyte activation markers and secreted cytokines, when lymphocytes were activated in the presence of MSC. MSC suppressed the proliferation of phytohaemagglutinin (PHA)‐stimulated CD3+, CD4+ and CD8+ lymphocytes. MSC significantly reduced the expression of activation markers CD25, CD38 and CD69 on PHA‐stimulated lymphocytes. Mixed lymphocyte culture (MLC) supernatants containing MSC suppressed proliferation of MLC and PHA‐stimulated lymphocytes dose‐dependently. MSC secrete osteoprotegerin (OPG), but not hepatocyte growth factor (HGF) or transforming growth factor‐β (TGF‐β). Stromal‐cell‐derived factor‐1 (SDF‐1) is not expressed on the cell surface. A recent report suggested that T‐cell suppression by MSC is mediated by HGF and TGF‐β. MSC suppression was not restored by the addition of neutralizing antibodies against SDF‐1, OPG, HGF or TGF‐β, alone or in combination. Addition of guanosine to PHA‐stimulated lymphocyte cultures containing MSC did not affect lymphocyte proliferation. The immunosuppressive effects of cyclosporine and MSC did not interfere, when present in the cultures of PHA‐activated lymphocytes. In summary, human MSC suppress proliferation of both CD4+ and CD8+ lymphocyte and decrease the expression of activation markers.

No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients

Background and Objectives Mesenchymal stromal cells (MSC) may be used in cellular therapy to treat graft-versus-host-disease and autoimmune disorders, and in regenerative medicine. Preliminary data suggest limited cellular allogeneic rejection, but less is known about humoral responses. The objective of this study was to investigate whether antibodies against MSC were present after hematopoietic stem cell transplantation (HSCT) including treatment with HLA matched or mismatched allogeneic MSC. Design and Methods Twelve patients were evaluated using flow cytometric cross matches (FCXM) and enzyme-linked immunosorbent assays. Expression of blood group antigens, regarded as alloantigens giving rise to humoral alloimmunity, on MSC were explored using flow cytometry and immunofluorescence. Results Three of 12 patients exhibited late positivity in the FCXM. In absorption studies, antibodies directed against fetal calf serum (FCS), a component of the MSC culture medium, were identified. Healthy individuals expressed varying levels of anti-FCS antibodies and the same pattern was seen in immunosuppressed HSCT patients. MSC did not express blood group antigens. The patients with positive FCXM are alive and well. Interpretation and Conclusions We have shown that immunosuppressed patients can exhibit anti-FCS antibodies, but no alloantibodies, which may bind to MSC. These antibodies seem clinically insignificant.

Analysis of Tissues Following Mesenchymal Stromal Cell Therapy in Humans Indicates Limited Long-Term Engraftment and No Ectopic Tissue Formation†‡§

Mesenchymal stromal cells (MSCs) are explored as a novel treatment for a variety of medical conditions. Their fate after infusion is unclear, and long‐term safety regarding malignant transformation and ectopic tissue formation has not been addressed in patients. We examined autopsy material from 18 patients who had received human leukocyte antigen (HLA)‐mismatched MSCs, and 108 tissue samples from 15 patients were examined by PCR. No signs of ectopic tissue formation or malignant tumors of MSC‐donor origin were found on macroscopic or histological examination. MSC donor DNA was detected in one or several tissues including lungs, lymph nodes, and intestine in eight patients at levels from 1/100 to <1/1,000. Detection of MSC donor DNA was negatively correlated with time from infusion to sample collection, as DNA was detected from nine of 13 MSC infusions given within 50 days before sampling but from only two of eight infusions given earlier. There was no correlation between MSC engraftment and treatment response. We conclude that MSCs appear to mediate their function through a “hit and run” mechanism. The lack of sustained engraftment limits the long‐term risks of MSC therapy. STEM CELLS2012;30:1575–1578

Mesenchymal stem cells stimulate antibody secretion in human B cells.

Mesenchymal stem cells (MSC) have immunomodulatory effects and inhibit T‐cell responses to alloantigens and mitogens in vitro and in vivo. We wanted to examine the effect of MSC on human B cells. MSC stimulated IgG production, measured in an enzyme‐linked immunospot (ELIspot) assay in blood and spleen lymphocytes. MSC only induced a low proliferation. When a semipermeable membrane separated MSC and mononuclear cells, the IgG production was stimulated in unfractionated lymphocytes. In contrast, enriched B cells required cell contact with MSC to produce IgG. Co‐cultures of MSC and lymphocytes increased IFN‐γ production. MSC produce IL‐6, and addition of MSC to spleen cells dramatically increased IL‐6 levels. After lymphocyte stimulation with lipopolysaccharide (LPS), cytomegalovirus or varicella zoster virus, MSC either stimulated or inhibited IgG response, depending on the level of stimulation by LPS or the viral antigens. Similar results were obtained for enriched B cells. To conclude, MSC stimulate B‐cell antibody secretion. The IgG secretion by activated B cells may be stimulated or inhibited by the addition of MSC, depending on the level of stimulation.

Damage To Porcine Islets Of Langerhans After Exposure To Human Blood In Vitro, Or After Intraportal Transplantation To Cynomologus Monkeys: Protective Effects of scr1 and Heparin1

BACKGROUND Porcine islets offer an attractive alternative to human islets in clinical islet transplantation. The preferred method of islet transplantation is intra-portal injection into the liver. We have recently shown, both in vitro with human islets and in vivo with porcine islets, that islets exposed to allogeneic blood trigger an injurious inflammatory reaction characterized by activation of both coagulation and the complement systems. We have now tested whether a similar reaction is triggered when xenogeneic porcine islets are exposed to human blood in vitro and after intraportal transplantation into primates. Furthermore, we investigated the effect of inhibiting the complement and coagulation systems. METHOD Islets isolated from adult and fetal porcine pancreas were perfused with fresh human blood in surface heparinized PVC tubings for 5-60 min. Blood cell counts and parameters related to coagulation and the complement system were analyzed, and islets were retrieved after the perifusion was examined by immunohistochemical method. Heparin and soluble complement receptor 1 (sCR1; TP10, 100 microg/ml) were added to the system in some experiments. Furthermore, adult porcine islets were transplanted intraportally into untreated and sCR1- (40 mg/kg BW i.v.) treated cynomolgus monkeys, and plasma insulin concentration was monitored during 60 min after transplantation. RESULTS Porcine islets perifused with human blood triggered an immediate inflammatory reaction, characterized by a rapid consumption and activation of platelets, consumption of neutrophils and monocytes, activation of the coagulation and complement systems, and release of large amounts of insulin. Islet morphologic analysis revealed damaged islets embedded in clots and infiltrated with CD11+ leukocytes. C3a and C5b-9 was deposited on the islet surface, but human immunoglobulin was not. Complement inhibition with sCR1 reduced insulin release significantly. Intraportal islet transplantation into untreated cynomolgus monkeys resulted in a marked and rapid increase in plasma insulin concentration indicative of islet damage. Pretreatment of the monkeys with sCR1 resulted in significantly less insulin release than in untreated control monkeys. CONCLUSION Exposure of isolated xenogeneic islets of Langerhans to blood, both in vitro and in vivo, resulted in acute islet damage. Complement and platelets seem to have a central role in the reactions described. Strategies to efficiently inhibit these reactions will be crucial for clinical intraportal islet xenotransplantation to be successful.