Human hepatic stellate cells and inflammation: A regulated cytokine network balance.

AIM: Uncertainty about the safety of cell therapy continues to be a major challenge to the medical community. Inflammation and the associated immune response represent a major safety concern hampering the development of long-term clinical therapy. In vivo interactions between the cell graft and the host immune system are mediated by functional environmental sensors and stressors that play significant roles in the immunobiology of the graft. Within this context, human liver stellate cells (HSC) demonstrated marked immunological plasticity that has main importance for future liver cell therapy application. METHODS: By using qPCR technique, we established the cytokine gene expression profile of HSCs and investigated the effect of an inflammatory environment on the immunobiology of HSCs. RESULTS AND DISCUSSION: HSCs present a specific immunological profile as demonstrated by the expression and modulation of major immunological cytokines. Under constitutive conditions, the cytokine pattern expressed by HSCs was characterized by the high expression of IL-6. Inflammation critically modulated the expression of major immunological cytokines. As evidenced by the induction of the expression of several inflammatory genes, HSCs acquire a pro-inflammatory profile that ultimately might have critical implications for their immunological shape. CONCLUSION: These new observations have to be taken into account in any future liver cell therapy application based on the use of HSCs.

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.

Mesenchymal stromal cells from the foreskin: Tissue isolation, cell characterization and immunobiological properties.

BACKGROUND AIMS: Because of their self-renewal capacity, multilineage potential and immunomodulatory properties, MSCs are an attractive tool for cell-based immunotherapy strategies. Foreskin, considered as a biological waste material, has been shown to be a reservoir of therapeutic cells. METHODS: MSCs were isolated from different foreskin samples, maintained under in vitro culture and defined according to the International Society for Cellular Therapy (ISCT) criteria. We subsequently determined their main cell characteristics as well as their immunobiological properties. The following parameters were determined: (i) morphology and phenotype, (ii) proliferative and clonogenic potentials, (iii) tri-lineage differentiation ability, (iv) immunological profile, (v) immunomodulatory properties and (vi) protein and messenger RNA expression/secretion profile of immunoregulatory cytokines/factors as well as the pattern of toll-like receptors (TLRs). By using a pro-inflammatory cytokine cocktail, we also evaluated the influence of an inflammatory environment on their biology. RESULTS: With a typical fibroblast-like morphology and an ISCT-compliant phenotype, foreskin-MSCs (FSK-MSCs) were highly proliferative and had a great clonogenic potential. They displayed multilineage capacities and interesting immunomodulatory properties. Of importance, FSK-MSCs were not immunogenetic and were further able to inhibit T-cell proliferation. We showed that several immunoregulatory cytokines and factors might be potentially involved in FSK-MSC immunomodulation with particular attention to hepatocyte growth factor and interleukin-11. Moreover, FSK-MSCs expressed several TLRs and were sensitive to the inflammatory environment by properly adjusting their profile and fate. CONCLUSIONS: Foreskin represents a new alternative source for MSCs that is compliant with ISCT criteria. Their unique immunobiological properties allow consideration of FSK-MSCs as a valuable tolerogenic product for cell-based immunotherapy.

Influence of inflammation on the immunological profile of adult-derived human liver mesenchymal stromal cells and stellate cells.

BACKGROUND AIMS: Stem cell therapy for liver diseases has recently emerged as a promising alternative to liver transplantation. Eligible cells should have an appropriate immunophenotype. The aim of the present study was to define the immunological profile of two human liver-derived mesenchymal stromal cell populations, namely, stem cells (ADHLSC) and hepatic stellate cells (HSC). METHODS: The study was conducted under normal and inflammatory conditions with the use of human bone marrow mesenchymal stromal cells (BM-MSC) as reference. RESULTS: Like BM-MSC and ADHLSC, HSC were negative for hematopoietic (CD45) and endothelial (CD34) markers but positive for stromal markers. All cell types were constitutively positive for HLA class I and negative for human leukocyte antigen (HLA) class II and co-stimulatory molecules (CD80, CD86, CD134 and CD252). Inflammation induced the expression of CD40 in all cell types, but the highest values were observed on HSCs; high CD252 expression was only observed on HSC as compared with ADHLSC and BM-MSC. The expression of various adhesion molecules (CD54, CD58, CD106 and CD166) was dissimilar in these three cell types and was differentially influenced by inflammation as well. ADHLSC and HSC constitutively expressed the immunosuppressive molecule HLA-G, whereas CD274 expression was induced by inflammation, as in the case of BM-MSC. Moreover, all cell types expressed the two major natural killer ligands CD112 and CD115. CONCLUSIONS: Toll-like receptors (TLR) 1, 3, 4 and 6 messenger RNA was expressed by both cell types, whereas TLR 2, 5, 7, 9 and 10 were only expressed by ADHLSC. Inflammation increased the expression of TLR 2 and 3 by ADHLSC and HSC. Finally, both liver-derived cell types were immunosuppressive because they inhibited the proliferation of mitogen-activated T cells.

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 Wharton's 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.

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 Wharton's 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.

Bortezomib: a new player in pre- and post-transplant desensitization?

Several desensitization strategies have been investigated for the reversal of acute antibody-mediated rejection or for the removal of preformed anti-HLA antibodies, with the aim to promote access to renal transplantation. Today, their success appears limited or incomplete. Bortezomib, a selective inhibitor of the 26S proteasome, which is largely used in the treatment of multiple myeloma, could be a novel promising desensitizing agent. Its mechanism of action and preliminary clinical use in renal transplantation is reviewed here.

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 and coronary endothelial function after autologous blood CD34+ progenitor cells intracoronary injection in the chronic phase of myocardial infarction.

Transcoronary transplantation of progenitor cells has been proposed as a novel therapy for ischemic heart failure. The primary aims were to assess the feasibility of obtaining CD34+ cells from blood without mobilization in chronic conditions and to compare homing with results reported in acute conditions. We also evaluated the effect of CD34+ on endothelial function. In 7 patients with a history of an anterior myocardial infarction (20 +/- 2 months), a large amount of CD34 (18.2 +/- 3.0 x 10(6)) were obtained and an intracoronary infusion into the left anterior descending artery via an over-the-wire balloon catheter was performed. Myocardial homing involved 3.2% +/- 0.6% of injected cells. Endothelial function studied with increasing doses of bradykinin was not significantly modified after 3 months. In the treated group, compared with 5 nonrandomized control patients with a similar clinical history, the only echocardiographic significant change (2-way analysis of variance) was a decrease in end-systolic volume (P < 0.03). In conclusion, large amounts of CD34+ cells can be obtained from blood, without mobilization, in the chronic phase of myocardial infarction. As reported in the acute situation 1 hour after treatment, intracoronary infusion of CD34+ cells results in myocardial homing of a few percents of the cells. In this small group of patients, no effect of this therapy is detected on the endothelial function and only marginal changes are observed on echocardiographic parameters.

Human leucocyte antigen (HLA) class I and II typing in Belgian multiple sclerosis patients.

This is one of the first studies to compare the frequencies of different human leucocyte antigen (HLA) class I and II alleles and haplotype HLA-DRB1*15-DQB1*06 in a cohort of 119 patients with multiple sclerosis (MS) and a cohort of 124 healthy controls in Belgium. An association with MS was found for the HLA-DRB1*15 (odds ratio [OR] 2.60 [95% confidence interval (CI) 1.51-4.50]) and HLA-DQB1*06 (OR 1.97 [95% CI 1.18-3.29]) alleles, and for haplotype DRB1*15-DQB1*06 (OR 2.63 [95% CI 1.52-4.56]). The HLA-B*07 allele also tended to be more frequent in MS patients (OR 1.46 [95% CI 0.80-2.65]) and more frequent among MS patients with than in those without the HLA-DRB1*15 allele (26/54 [48.1%] versus 6/65 [9.2%]; p value <0.0001). Other alleles were underrepresented in MS patients, such as the HLA-DRB1*07 (OR 0.39 [95% CI 0.21-0.73]) and HLA-A*02 (OR 0.56 [95% CI 0.34-0.94]), showing a protective role against the disease. The HLA-B*44 (OR 0.58 [95% CI 0.31-1.09]) and HLA-DRB1*04 (OR 0.75 [95% CI 0.42-1.34]) alleles tended to be less frequent in MS patients. Altogether, the significant results observed in this population are in line with those from other countries and confirm that propensity to MS can be due to a complex presence of various HLA class I and class II alleles.

Comparison and immunobiological characterization of retinoic acid inducible gene-I-like receptor expression in mesenchymal stromal cells.

Due to their immunomodulatory and regenerative properties, Mesenchymal stromal cells (MSC) have generated major interests in several clinical settings including transplantation and inflammatory diseases. MSC functions can be influenced by their tissue origin. Their microenvironment strongly affects their biology notably through TLR sensing. In this study, we show that MSC isolated from four different sources express another type of cytosolic pathogen recognition receptors known as retinoic acid inducible gene-I (RIG-I)-like receptors (RLR). RLR activation in MSC induces the production of Type I IFN (IFN-ß) and Type III IFN (IFN-λ1). The highest producers are adipose tissue(AT)-MSC. We further show that Interferon production is induced through TBK1/IKK-ε signaling and IRF7 phosphorylation. Depending on MSC source, the knockdown of TLR3 and/or RIG-I decreases the MSC response to RLR ligand poly(I:C)/Lyovec. Among the different MSC types, AT-MSCs display the highest sensitivity to viral stimuli as shown by the alteration of their viability after prolonged stimulation. Our work indicates that this could be linked to an increase of pro-apoptotic Noxa expression. Finally, the expression of IDO1 and LIF upon RLR activation indicate the increase of MSC immunomodulatory potential, especially in AT-MSCs. Altogether, these data should be considered when designing MSC-based therapy in clinical settings where inflammation or infection are present.

The Immunomodulatory Potential of Mesenchymal Stromal Cells: A Story of a Regulatory Network.

Mesenchymal stromal cells (MSCs) have recently been the subject of great interest in the fields of regenerative medicine and immunotherapy due to their unique biological properties. In particular, MSCs possess immunoregulatory properties that can modulate immune as well as inflammatory responses. Although there are many studies about MSC immunomodulation, several complex and conflicting mechanisms have been reported. Herein, we aim to review these mechanisms and identify a link between these pathways. We focus on human studies in which bone marrow-derived MSCs and T cells were investigated. We propose that MSC-induced immunomodulation exists as a network where converging regulatory pathways compete to establish a tolerogenic state. As interleukin-10 seems to play a central role in this network, we also discuss the relationship between this cytokine and other regulatory factors in the context of immunomodulation.

Immune characterization of clinical grade-dendritic cells generated from cancer patients and genetically modified by an ALVAC vector carrying MAGE minigenes.

Gene delivery into dendritic cells (DC) is most efficiently achieved by viral vectors. Recombinant canarypox viruses (ALVAC) were validated safe and efficient in humans. We aimed firstly to evaluate DC transduction by ALVAC vectors, then to investigate if such infection induced or not the maturation of the DC, and finally to assess the efficiency of ALVAC-MAGE-transduced DC to activate specific CTL clones. Clinical grade DC from melanoma patients were generated from blood monocytes and infected with a recombinant ALVAC virus encoding either a marker gene (EGFP) or the MAGE-1-MAGE-3 minigenes. According to the patient-donor, 22+/-16% of immature DC were successfully transduced. Flow cytometry analysis of surface markers expressed on DC after ALVAC infection did not reveal a mature phenotype. Moreover, ALVAC transduction did not interfere with the capacity of the DC to further mature under poly:IC stimulation. But most importantly, our results demonstrated that DC from HLA-A1 patient-donors infected with the recombinant ALVAC MAGE-1-MAGE-3 minigenes virus were capable of activating a MAGE 3/A1 CTL clone more efficiently than same DC loaded with MAGE 3/A1 peptide, as shown by increased IFN-gamma secretion. These results could be the basis for the development of a new clinical strategy in melanoma patient's immunotherapy.

A rapid test to monitor alloreactive responses in whole blood using real-time polymerase chain reaction.

Rapid, simple, and reliable assays to monitor allogeneic responses are essential for the safe development of novel protocols of tailored immunosuppression. Herein, we describe a real-time polymerase chain reaction method based on interleukin-2 and interferon-gamma mRNA quantification upon stimulation of whole blood with allogeneic T cell-depleted peripheral blood mononuclear cells. The technique requires only small blood volumes and results can be obtained within 48 hours. Data obtained in a liver transplant patient receiving a tolerance induction protocol based on the infusion of donor-type hematopoietic stem cells suggest that this rapid whole blood mixed lymphocyte reaction assay could be valuable for the monitoring of patients undergoing solid organ or hematopoietic stem cell transplantation.

IL-6 produced by type I IFN DC controls IFN-gamma production by regulating the suppressive effect of CD4+ CD25+ regulatory T cells.

The dendritic cell family is composed of different subsets differentially governing the immune response. Type I interferon (IFN) dendritic cells (DC) are endowed with the ability to trigger both Th1 and Th2 type responses. In view of the pivotal role of regulatory T cells in limiting the effectiveness of effector cells, we analyzed the interactions between these cells and type I IFN DC. DC were generated from monocytes in the presence of IFN-beta and interleukin (IL)-3 (DCI3) or granulocyte macrophage-colony-stimulating factor and IL-4 (DCG4) and activated by poly(I:C). Despite the release of lower amounts of IL-12 after maturation, DCI3 were able to induce a higher IFN-gamma production by T lymphocytes during the mixed leucocyte reaction (MLR) as compared with DCG4. mRNA analysis disclosed that DCI3 overtranscribed the IL-6 gene and secreted high amounts of the protein. Neutralization of IL-6 revealed that this cytokine specifically contributed to the IFN-gamma release induced by DCI3. Finally, depletion of CD25+ T cells before the MLR identified these cells as a target for IL-6. We conclude that DCI3 are endowed with the property of regulating the suppressive effect of regulatory T cells through high IL-6 production. This novel mechanism of T cell control is relevant for the use of DCI3 in vaccination strategies.

Bone Marrow Mesenchymal Stromal Cells Induce Proliferative, Cytokinic and Molecular Changes During the T Cell Response: The Importance of the IL-10/CD210 Axis.

BACKGROUND: Bone marrow mesenchymal stromal cells (BM-MSCs) display immunomodulatory features, representing a promising tool for cell-based therapies. However, the mechanisms used by MSCs to regulate T cell fate remain unclear. AIMS: We investigated the potential of BM-MSCs to modulate T cell activation, proliferation, cytokine secretion and immunophenotype. MATERIALS AND METHODS: T cells were co-cultured with BM-MSCs to assess their immunomodulatory impact. T cell characterization was performed using cell tracing, ELISA, intracellular and surface staining, flow cytometry analysis and qPCR. RESULTS: The activation and proliferation of T cells were downregulated during coculture with BM-MSCs. We also observed that BM-MSCs upregulated IL-10 secretion as well as the expression of its receptor CD210 on T cells, thus creating a loop favoring the expansion of IL-10-producing T cells. IL-10 neutralization restored T cell proliferation, demonstrating that IL-10 is functionally relevant during immunomodulation. Moreover, BM-MSCs differently modulated CD4 and CD8 T-cell immunophenotype by inducing broad changes in their molecular pattern. CONCLUSIONS: We provide a comprehensive functional and molecular characterization of T cells that are immunomodulated by BM-MSCs. Indeed, a better understanding of the immunological interplay between T cells and MSCs will facilitate the development of new efficient approaches to improve cell-based immune therapies.

Tolerance induction in clinical transplantation: the pending questions.

Despite the dramatic improvement of early graft survival due to the introduction of cyclosporin, late graft loss caused by chronic rejection and the lethal consequences of long-term immunosuppression, such as infection and cancer, remain major concerns for the transplantation community. Tolerance induction would avoid these complications. The ways to go are controversial, reflecting the redundancy of rejection pathways. They include the induction of central tolerance by establishment of mixed chimerism through hematopoietic stem cell transplantation and the induction of "operational tolerance" through immunodeviation involving dendritic or regulatory T cells. Major advances have been made in animal models exploring these strategies and, some preliminary data are even now available in humans, allowing the initiation of pilot clinical trials. In this article, we discuss the key questions that these trials will have to address.

Infusion of donor-derived hematopoietic stem cells in organ transplantation: clinical data.

Many strategies for tolerance induction have been developed, because this is the major goal of clinical transplantation. One of the most effective and best-studied approaches has been based on the injection of hematopoietic cells derived from the donor bone marrow, to establish a state of microchimerism in the recipient. A subset of hematopoietic stem cells might be responsible for the tolerogenic properties. These CD34+ bone marrow stem cells can be isolated and safely injected into kidney transplant recipients. In the authors' clinical trial, no adverse effects were observed, and the infusion of donor CD34+ cells was well tolerated.

Donor stem cell infusion after non-myeloablative conditioning for tolerance induction to HLA mismatched adult living-donor liver graft.

BACKGROUND AND AIM OF THE STUDY: The induction of transplantation tolerance, defined as the survival of a functioning allograft in the absence of continuing immunosuppressive therapy, would be a major advance. Clinical and experimental data have shown that transplantation tolerance could be induced by pre-transplant myeloconditioning and infusion of donor hematopoietic cells. We investigated the feasibility and safety of a protocol to induce tolerance to HLA mismatched living-donor liver graft by pre-transplant non-myeloablative conditioning followed by donor stem cells (SC) infusion, in patients with advanced liver cancers. PATIENTS AND METHODS: Two patients with intrahepatic cancers who did not fulfill criteria for cadaver liver transplantation were included in the study. Preparative regimen consisted in cyclophosphamide and anti-thymocyte globulin, followed by infusion of purified donor CD34(+) stem cells. Living-donor liver transplantation (LDLT) using the liver right lobe was performed after hematological reconstitution, respectively 40 and 55 days after donor stem cell infusion. Immunosuppressive therapies were discontinued when liver graft function returned to normal. RESULTS: The procedure could be completed in the two patients. No severe toxicity of the preparative regimen was observed. Neither patient presented graft versus host reaction after donor stem cell infusion. A transient macrochimerism was observed in the first case, while no chimerism could be detected in the second. Immunosuppression was discontinued, respectively 90 and 28 days, after liver transplantation, without subsequent rejection episode. In the two cases, liver function remained normal for the study period. In both patients, the period of immune reconstitution was prolonged, as illustrated by persisting low CD4(+) cell counts. Mixed lymphocyte cultures, performed after immunosuppression withdrawal, demonstrated donor specific hyporesponsiveness in the first case, but in a context of global hyporeactivity in the two patients. The first patient died from tumor recurrence 370 days after liver transplantation. The second patient is alive, 270 days after liver transplantation, but with a suspicion of tumor relapse as indicated by the reappearance of tumor marker in blood. CONCLUSION: In the two cases, acceptance of HLA mismatched living-donor liver graft was obtained after non-myeloablative conditioning and donor stem cell infusion. Improving the rate of immune reconstitution appears as a priority to reduce the risk of tumor recurrence in such patients.

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.