Katarina Le Blanc

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.

HLA expression and immunologic propertiesof differentiated and undifferentiated mesenchymal stem cells

OBJECTIVE Mesenchymal stem cells (MSC) do not elicit alloreactive lymphocyte responses due to immune modulations. We investigated the immunologic properties of MSC after differentiation along three lineages: bone, cartilage, and adipose. METHODS AND RESULTS Flow cytometry showed that undifferentiated MSC express HLA class I but not class II, although HLA class II was present intracellularly as detected by Western blot. Addition of interferon gamma (IFN-gamma) for 48 hours induced greater than 90% of cells to express HLA class II. No lymphocyte response was induced by allogeneic irradiated MSC as stimulators. Results were similar using MSC pretreated with IFN-gamma. After growth of cells in medium to induce differentiation to bone, cartilage, or adipose for 6 or 12 days, the expression of HLA class I increased but no class II was detected on the cell surface. The ability to upregulate HLA class II on the cell surface after exposure to IFN-gamma for 48 hours was clearly diminished after the cells had been cultured in differentiation medium for 6 or 12 days, with only 10% of cells expressing HLA class II. Using MSC grown in osteogenic, chondrogenic, or adipogenic medium as stimulator cells, no lymphocyte alloreactivity was seen, even if differentiated MSC had been pretreated with IFN-gamma. MSC inhibit mixed lymphocyte cultures, particularly after osteogenic differentiation. This suppression was further enhanced by IFN-gamma. CONCLUSIONS Undifferentiated and differentiated MSC do not elicit alloreactive lymphocyte proliferative responses and modulate immune responses. The findings support that MSC can be transplantable between HLA-incompatible individuals.

Multipotent mesenchymal stromal cells and the innate immune system

Multipotent mesenchymal stromal cells (MSCs) have unique immunoregulatory and regenerative properties that make them an attractive tool for the cellular treatment of autoimmunity and inflammation. Their underlying molecular mechanisms of action together with their clinical benefit — for example, in autoimmunity — are being revealed by an increasing number of clinical trials and preclinical studies of MSCs. However, autoimmunity and therapy-related alloimmunity are not only triggered and sustained by responses of the adaptive immune system; there is growing evidence that components of the innate immune system also have a key role. It is therefore important to study the crosstalk between MSCs and innate immunity, which ranges from the bone marrow niche to injured tissue.

Fetal mesenchymal stem-cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta

Background. Mesenchymal stem cells (MSC) are progenitors of mesenchymal tissues such as bone, cartilage, and adipose. Adult human leukocyte antigen (HLA)-matched MSC have been used in cellular therapies of bone disorders such as osteogenesis imperfecta, with promising results. Methods. A female fetus with multiple intrauterine fractures, diagnosed as severe osteogenesis imperfecta, underwent transplantation with allogeneic HLA-mismatched male fetal MSC in the 32nd week of gestation. Engraftment analyses of donor cells, immunologic reaction against donor cells, and the well-being of the patient were assessed. Results. At 9 months of age, on slides stained for osteocalcin or osteopontin, a centromeric XY-specific probe revealed 0.3% of XY-positive cells in a bone biopsy specimen. Whole Y genome fluorescent in situ hybridization staining showed a median of 7.4% Y-positive cells (range, 6.8%–16.6%). Bone histology showed regularly arranged and configurated bone trabeculae. Patient lymphocyte proliferation against donor MSC was not observed in co-culture experiments performed in vitro after MSC injection. Complementary bisphosphonate treatment was begun at 4 months. During the first 2 years of life, three fractures were noted. At 2 years of corrected age, psychomotor development was normal and growth followed the same channel, −5 SD. Conclusions. The authors’ findings show that allogeneic fetal MSC can engraft and differentiate into bone in a human fetus even when the recipient is immunocompetent and HLA-incompatible.

Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study

BACKGROUND Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 μg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business.

Concise Review: Hitting the Right Spot with Mesenchymal Stromal Cells

Mesenchymal stromal cells or mesenchymal stem cells (MSCs) have captured considerable scientific and public interest because of their potential to limit physical and immune injury, to produce bioactive molecules and to regenerate tissues. MSCs are phenotypically heterogeneous and distinct subpopulations within MSC cultures are presumed to contribute to tissue repair and the modulation of allogeneic immune responses. As the first example of efficacy, clinical trials for prevention and treatment of graft‐versus‐host disease after hematopoietic cell transplantation show that MSCs can effectively treat human disease. The view of the mechanisms whereby MSCs function as immunomodulatory and reparative cells has evolved simultaneously. Initially, donor MSCs were thought to replace damaged cells in injured tissues of the recipient. More recently, however, it has become increasingly clear that even transient MSC engraftment may exert favorable effects through the secretion of cytokines and other paracrine factors, which engage and recruit recipient cells in productive tissue repair. Thus, an important reason to investigate MSCs in mechanistic preclinical models and in clinical trials with well‐defined end points and controls is to better understand the therapeutic potential of these multifunctional cells. Here, we review the controversies and recent insights into MSC biology, the regulation of alloresponses by MSCs in preclinical models, as well as clinical experience with MSC infusions (Table 1 ) and the challenges of manufacturing a ready supply of highly defined transplantable MSCs. STEM CELLS 2010;28:1446–1455

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.

Defining the risks of mesenchymal stromal cell therapy

Abstract We address the issue of the potential for malignant transformation of cultured mesenchymal stromal cells (MSC) commonly used in clinical cell-therapy protocols and describe the culture conditions under which tumorigenesis is likely to be an extremely uncommon event.

Long-Term Complications, Immunologic Effects, and Role of Passage for Outcome in Mesenchymal Stromal Cell Therapy

Thirty-one patients treated with mesenchymal stromal cells (MSCs) for acute graft-versus-host disease (aGVHD) or hemorrhagic cystitis between 2002 and 2007 were followed to investigate predictors of outcome, immunologic effects in vivo, and long-term survival. There was no correlation between in vitro suppression by MSCs in mixed lymphocyte cultures and outcome. Soluble IL-2 receptors were measured in blood before and after MSC infusion and declined significantly during the first week after MSC infusion (P = .03). Levels of interleukin-6 and HLA-G were unaffected. Infectious complications occurred several years after recovery from aGVHD. Cytomegalovirus viral load was high, and cytomegalovirus disease was common. Among patients recovering from aGVHD, 54% died of late infections, between 4 months and 2 years after MSC treatment. No increase in leukemia relapse or graft rejection was found. Children had a better survival rate than adults (P = .005). In GVHD patients, 1-year survival was 75% in patients who received early-passage MSCs (from passages 1-2) in contrast to 21% using later passage MSCs (from passages 3-4) (P < .01). We conclude that treatment with early-passage MSCs improved survival in patients with therapy-resistant GVHD. Death from infection was common in MSC-treated patients, but there was no increase in leukemia relapse.

Treatment with granulocyte colony-stimulating factor after allogeneic bone Marrow transplantation for acute leukemia increases the risk of graft-versus-host disease and death: A study from the acute leukemia working party of the European Group for Blood and Marrow Transplantation

PURPOSE Granulocyte colony-stimulating factor (G-CSF) is given after bone marrow transplantation (BMT) to shorten the neutropenic phase. Its effects have not been evaluated in a large patient population. PATIENTS AND METHODS We studied 1,789 patients with acute leukemia receiving BMT and 434 patients receiving peripheral-blood stem cells (PBSCs) from HLA-identical siblings from 1992 to 2002 and reported the findings to the European Group for Blood and Marrow Transplantation. Among the BMT and PBSC patients, 501 (28%) and 175 (40%), respectively, were treated with G-CSF during the first 14 days after the transplantation. The outcome variables were entered into a Cox proportional hazards model. RESULTS BMT and PBSC patients treated with G-CSF had a faster engraftment of absolute neutrophils greater than 0.5 x 10(9)/L (P <.01), but platelet engraftment ( > 50 x 10(9)/L) was slower (P <.001). In the BMT patients, acute graft-versus-host disease (GVHD) grades II to IV was 50% +/- 5% (+/- 95% CI) in the G-CSF group versus 39% +/- 3% in the controls (relative risk [RR], 1.33; P =.007, in the multivariate analysis). The incidence of chronic GVHD was also increased (RR, 1.29; P =.03). G-CSF was associated with an increase in transplantation-related mortality (TRM; RR, 1.73; P =.00016) and had no effect on relapse but reduced survival (RR, 0.59; P <.0001) and leukemia-free survival rates (LFS; RR, 0.64; P =.0003). No such effects of G-CSF were seen in patients receiving PBSC. CONCLUSION After BMT, platelet engraftment was delayed, and GVHD and TRM were increased. Survival and LFS were reduced. This suggests that G-CSF should not be given shortly after BMT.