Michel Toungouz

Treatment of osteonecrosis of the femoral head with implantation of autologous bone-marrow cells. A pilot study.

BACKGROUND Aseptic nontraumatic osteonecrosis of the femoral head is a disorder that can lead to femoral head collapse and the need for total hip replacement. Since osteonecrosis may be a disease of mesenchymal cells or bone cells, the possibility has been raised that bone marrow containing osteogenic precursors implanted into a necrotic lesion of the femoral head may be of benefit in the treatment of this condition. For this reason, we studied the implantation of autologous bone-marrow mononuclear cells in a necrotic lesion of the femoral head to determine the effect on the clinical symptoms and the stage and volume of osteonecrosis. METHODS We studied thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head, according to the system of the Association Research Circulation Osseous. The hips were allocated to a program of either core decompression (the control group) or core decompression and implantation of autologous bone-marrow mononuclear cells (the bone-marrow-graft group). Both patients and assessors were blind with respect to treatment-group assignment. The primary outcomes studied were safety, clinical symptoms, and disease progression. RESULTS After twenty-four months, there was a significant reduction in pain (p = 0.021) and in joint symptoms measured with the Lequesne index (p = 0.001) and the WOMAC index (p = 0.013) within the bone-marrow-graft group. At twenty-four months, five of the eight hips in the control group had deteriorated to stage III, whereas only one of the ten hips in the bone-marrow-graft group had progressed to this stage. Survival analysis showed a significant difference in the time to collapse between the two groups (p = 0.016). Implantation of bone-marrow mononuclear cells was associated with only minor side effects. CONCLUSIONS Implantation of autologous bone-marrow mononuclear cells appears to be a safe and effective treatment for early stages of osteonecrosis of the femoral head. Although the findings of this study are promising, their interpretation is limited because of the small number of patients and the short duration of follow-up. Further study is needed to confirm the results.

Mesenchymal stromal cells use PGE2 to modulate activation and proliferation of lymphocyte subsets: Combined comparison of adipose tissue, Wharton’s Jelly and bone marrow sources

Due to their immunomodulatory properties, adipose tissue (AT) and Whartons Jelly (WJ) constitute valuable alternatives to BM as sources of MSCs for managing graft-versus-host disease. To ensure the efficiency of AT- and WJ-MSCs implies the characterization of their immunomodulatory functions in comparison to those of BM. In this study, we investigated the capacity of AT- and WJ-MSCs to modulate lymphocyte reactions in response to different stimuli as well as the specificity of this immunomodulation. AT- and WJ-MSC displayed potent immunosuppressive effects on lymphocyte responses in a dose-dependent manner. These effects included the prevention of lymphocyte activation as well as the suppression of T-cell proliferation regardless of the stimuli used to activate lymphocytes. These effects were mediated through the expression of COX1/COX2 enzymes and by the production of PGE2. CD4(+) and CD8(+) T-lymphocytes were equally targeted by MSCs demonstrating that the immunomodulation was not restricted to a specific T-cell subpopulation.

Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6

BACKGROUND AIMS Mesenchymal stromal cells (MSC) have been shown to possess immunomodulatory functions and proposed as a tool for managing or preventing graft-versus-host disease (GvHD) as well as promoting clinical transplantation tolerance. We investigated the capacity of human bone marrow (BM) MSC to modulate the proliferation of T cells obtained from peripheral blood (PB) and umbilical cord blood (CB). We addressed the importance of the MSC:T-cell ratio, requirement for cell contact and impact of soluble factors on the MSC-mediated effects. We also analyzed whether regulatory T cells could be modulated by MSC in co-cultures. METHODS The effect of different MSC concentrations on T-cell proliferation induced by allogeneic, mitogenic or CD3/CD28 stimulation was analyzed using bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE) labeling. The level of regulatory T cells was assessed using quantitative real-time polymerase chain reaction (PCR) and flow cytometry analysis. RESULTS MSC induced a dose- and contact-dependent inhibition of T-cell proliferation but lymphocytes from CB and PB were differentially affected. At low concentrations, MSC supported both CB and PB T-cell proliferation, rather than inhibiting their proliferation. This supportive effect was contact independent and soluble factors such interleukin-6 (IL-6) appeared to be involved. Interestingly, among the expanded T-cell population in both CB and PB, regulatory T cells were increased and were a part of the new cells promoted by MSC at low doses. CONCLUSIONS MSC represent an attractive tool for reducing the lymphocyte response by inhibiting T-cell activation and proliferation as well as promoting tolerance by maintaining and promoting the expansion of regulatory cells. Nevertheless, the dual ability of MSC to either sustain or suppress T-cell proliferation according to conditions should be considered in the context of clinical applications.

Myocardial Homing of Nonmobilized Peripheral‐Blood CD34+ Cells After Intracoronary Injection

Granulocyte– colony‐stimulating factor administered for autologous hematopoietic stem cell isolation from blood may favor restenosis in patients implanted after acute myocardial infarction (AMI). We therefore tested the isolation of peripheral‐blood CD34+ cells without mobilization in six patients with AMI. After large‐volume cytapheresis and positive CD34+ cell selection, 3.6 to 27.6 million CD34+ cells were obtained. We performed intra‐coronary implantation of these cells and recorded no restenosis or arrhythmia. We used positron emission tomography (PET) to assess myocardial‐labeled CD34+ cell homing, which accounted for 5.5% of injected cells 1 hour after implantation. In conclusion, large amounts of CD34+ cells, in the range reported in previous studies, can be obtained from nonmobilized peripheral blood. PET with [18F]‐fluorodeoxyglucose cell labeling is an efficient imaging method for homing assessment.

Stem cell therapy for osteonecrosis of the femoral head

Aseptic non-traumatic osteonecrosis of the femoral head is a painful disorder of the hip that can lead to femoral head collapse and the need for total hip replacement. As osteonecrosis may be a disease of mesenchymal cells or bone cells, the possibility has been raised that bone marrow containing osteogenic precursors implanted into the necrotic lesion could be of benefit in this condition. Indeed, bone marrow contains adult stem cells, such as haematopoietic stem cells, mesenchymal stem cells and multipotent stem cells, that might have osteogenic properties. The efficacy of bone marrow implantation into the osteonecrotic zone was studied in two prospective trials. This treatment avoided the progression of the disease to the stage of the subchondral fracture (stage III) and reduced the need for total hip replacement. The mechanisms involved might include improved osteogenesis and angiogenesis. This new therapeutic approach should modify the treatment of early-stage osteonecrosis of the femoral head.

The source of human mesenchymal stromal cells influences their TLR profile as well as their functional properties

Mesenchymal stromal cells (MSC) can be expanded from different sources. We compared the influence of inflammation and TLR ligation on the phenotype and function of MSC derived from bone marrow (BM), adipose tissue (AT), and Whartons jelly (WJ). WJ-MSC were featured by a lack of TLR4 expression. While inflammation upregulated TLR3 in all three MSC types, TLR4 upregulation was observed only on BM-MSC. TLR ligation increased the production of inflammatory cytokines in BM- and AT-MSC but not in WJ-MSC and augmented anti-inflammatory cytokines in AT-MSC. Although inflammation increased in all MSC types the secretion of inflammatory cytokines, additional TLR triggering did not have further effect on WJ-MSC. The immunosuppressive potential of WJ-MSC on MLR was affected neither by inflammation nor by TLR triggering. This resistance was related to an overproduction of HGF. These data indicate that MSC source could be of importance while designing immunomodulating cell therapy in transplantation.

Immune-related antigens, surface molecules and regulatory factors in human-derived mesenchymal stromal cells: the expression and impact of inflammatory priming.

Based on their ability to regulate immune responses, MSCs are considered to be potential candidates for managing immune-mediated diseases in the context of immune therapy. AT and WJ are considered valuable alternatives for BM as a source of MSCs. A detailed and comparative characterization of the immunological profile of MSCs derived from different sources, as well as an understanding of their responsiveness under certain circumstances, such as inflammation, is required to facilitate efficient and well-designed clinical studies. Flow cytometric analyses revealed clear differences among MSC types concerning the expression of the endothelial (e.g., CD31, CD34, CD144 and CD309) and stromal (e.g., CD90 and CD105) associated markers. Regardless of their source, MSCs did not express any of the known hematopoietic markers. All MSCs were uniformly positive for HLA-ABC and lacked the expression of HLA-DR and the co-stimulatory molecules (e.g., CD40, CD80, CD86, CD134 and CD252) required for full T-cell activation. Tissue-specific MSCs presented a modulated expression of cell adhesion molecules that is important for their cellular interactions. MSCs exhibited several surface (e.g., CD73, HLA-G, HO-1 and CD274) and soluble (e.g., HGF, PGE2 and IGFBP-3) immunoregulatory molecules. According to these immunological profiles, the present work provides evidence that the source from which MSCs are derived is important for the design of MSC-based immunointervention approaches. In light of these observations, we may suggest that WJ-MSCs appear to be the most attractive cell population to use in immune cellular therapy when immunosuppressive action is required.

Transient expansion of peptide‐specific lymphocytes producing IFN‐γ after vaccination with dendritic cells pulsed with MAGE peptides in patients with mage‐A1/A3‐positive tumors

Assessment of T‐cell activation is pivotal for evaluation of cancerimmunotherapy. We initiated a clinical trial in patients with MAGE‐A1and/or ‐A3 tumors using autologous DC pulsed with MAGE peptides aimedat analyzing T‐cell‐derived, IFN‐γ secretion by cytokine flowcytometry and ELISPOT. We also tested whether further KLH additioncould influence this response favorably. Monocyte‐derived DC weregenerated from leukapheresis products. They were pulsed with therelevant MAGE peptide(s) alone in group A (n=10 pts) andadditionally with KLH in group B (n=16 pts). A specific buttransient increase in the number of peripheral blood T lymphocytessecreting IFN‐γ in response to the vaccine peptide(s) was observed in6/8 patients of group A and in 6/16 patients of group B. We concludethat anti‐tumor vaccination using DC pulsed with MAGE peptides inducesa potent but transient anti‐MAGE, IFN‐γ secretion that is notinfluenced by the additional delivery of a nonspecific, T‐cellhelp.

Mesenchymal stromal cells and immunomodulation: A gathering of regulatory immune cells.

Because of their well-recognized immunomodulatory properties, mesenchymal stromal cells (MSCs) represent an attractive cell population for therapeutic purposes. In particular, there is growing interest in the use of MSCs as cellular immunotherapeutics for tolerance induction in allogeneic transplantations and the treatment of autoimmune diseases. However, multiple mechanisms have been identified to mediate the immunomodulatory effects of MSCs, sometimes with several ambiguities and inconsistencies. Although published studies have mainly reported the role of soluble factors, we believe that a sizeable cellular component plays a critical role in MSC immunomodulation. We refer to these cells as regulatory immune cells, which are generated from both the innate and adaptive responses after co-culture with MSCs. In this review, we discuss the nature and role of these immune regulatory cells as well as the role of different mediators, and, in particular, regulatory immune cell induction by MSCs through interleukin-10. Once induced, immune regulatory cells accumulate and converge their regulatory pathways to create a tolerogenic environment conducive for immunomodulation. Thus, a better understanding of these regulatory immune cells, in terms of how they can be optimally manipulated and induced, would be suitable for improving MSC-based immunomodulatory therapeutic strategies.

Early immunosuppression withdrawal after living donor liver transplantation and donor stem cell infusion

Long‐term results of organ transplantation are still limited by serious side effects of immunosuppressive drugs. A major issue, therefore, is to elaborate novel therapeutic protocols allowing withdrawal or minimization of immunosuppressive therapy after transplantation. We report on 3 patients prospectively enrolled in an original protocol designed to promote graft acceptance in living donor liver transplantation, using posttransplant conditioning with high doses of antithymocyte globulin followed by injection of donor‐derived stem cells. In 2 patients, early immunosuppression withdrawal was possible, without subsequent graft deterioration. In these 2 cases, in vitro studies showed indices of immunological tolerance as assessed by specific hyporesponsiveness to donor alloantigens in mixed lymphocytes culture. In the third patient, acute rejection rapidly occurred after discontinuation of immunosuppression, and minimal immunosuppression has to be maintained during long‐term follow‐up. In this case, a clearly distinct immunoreactive profile was observed as compared to tolerant patients, as no specific modulation of the antidonor response was observed in vitro. Of note, no macrochimerism could be detected in any of the 3 patients during the follow‐up. In conclusion, these clinical observations demonstrated that, despite the absence of macrochimerism, donor stem cells infusion combined with recipient conditioning may allow early immunosuppression withdrawal or minimization after liver transplantation. Liver Transpt 12:1523–1528, 2006.