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Maury S.,Assistance Publique Hopitaux de Paris | Maury S.,University Paris Est Creteil | Lemoine F.M.,Assistance Publique Hopitaux de Paris | Lemoine F.M.,University Pierre and Marie Curie | And 25 more authors.
Science Translational Medicine | Year: 2010

Donor T cells play a pivotal role in the graft-versus-tumor effect after allogeneic hematopoietic stem cell transplantation. Regulatory T cells (T regs) may reduce alloreactivity, the major component of the graft-versus-tumor effect. In the setting of donor lymphocyte infusion after hematopoietic stem cell transplantation, we postulated that Treg depletion could improve alloreactivity and likewise the graft-versus-tumor effect of donor T cells. The safety and efficacy of Treg-depleted donor lymphocyte infusion was studied in 17 adult patients with malignancy relapse after hematopoietic stem cell transplantation. All but one had previously failed to respond to at least one standard donor lymphocyte infusion, and none had experienced graft-versus-host disease. Two of the 17 patients developed graft-versus-host disease after their first Treg-depleted donor lymphocyte infusion and experienced a long-term remission of their malignancy. Four of the 15 patients who did not respond after a first T reg-depleted donor lymphocyte infusion received a second T reg-depleted donor lymphocyte infusion combined with lymphodepleting chemotherapy aimed to also eliminate recipient Tregs. All four developed acute-like graft-versus-host disease that was associated with a partial or complete and durable remission. In the whole cohort, graft-versus-host disease induction through Treg depletion was associated with improved survival. These results suggest that T reg-depleted donor lymphocyte infusion is a safe, feasible approach that induces graft-versus-host or graft-versus-tumor effects in alloreactivity-resistant patients. In patients not responding to this approach, the combination of chemotherapy-induced lymphodepletion of the recipient synergizes with the effect of Treg-depleted donor lymphocyte infusion. These findings offer a rational therapeutic approach for cancer cellular immunotherapy.


Roux S.,University Paris Est Creteil | Roux S.,Service Hospitalier | Bodivit G.,University Paris Est Creteil | Bartis W.,University Paris Est Creteil | And 8 more authors.
Tissue Engineering - Part A | Year: 2015

Stem cells may represent an excellent strategy to improve the healing of skin ulcers. Today the administration mode of stem cells to skin defects remains unsatisfactory. Delivering stem cells with topical treatments represents a new strategy and answering the patients' need. Mesenchymal stromal cells (MSC) have been shown to improve wound healing of cutaneous lesions and amniotic membrane (AM) is known to represent a natural scaffold for cells. The aim of this study is to develop a tissue-engineered product combining MSC and AM for clinical use. In this work we investigated whether the stromal matrix of intact human AM could constitute a scaffold for human MSC derived from either bone marrow (BM) or adipose tissue (AT). For this purpose, clinical-grade AM, MSC, and culture medium were used. We performed experiments of short-term adherence and proliferation for 15 days after the seeding of the cells. Morphological aspects and secretion profiles of MSC onto AM were studied, respectively, by scanning electron microscopy and Luminex analysis. Results demonstrated that the stromal matrix allow the adherence in much greater amount of MSC from BM or AT compared to 2D material. Experiments of proliferation showed that both kinds of MSC could proliferate on the stromal matrix and remain viable 15 days after the seeding of the cells. The 3D analysis of MSC culture demonstrated that both types of MSC invaded the stromal matrix and grew in multiple layers while retaining their fibroblastic morphology. By studying the secretion profile of MSC onto the stromal matrix, we found that both kinds of MSC secrete important cytokines and growth factors for wound healing of cutaneous lesions, such as vascular endothelial growth factor, hepatocyte growth factor, and basic fibroblast growth factor. In conclusion, these results suggest that the stromal matrix of AM seeded with MSC represents a bioactive scaffold that should be evaluated in patients with a nonhealing cutaneous wound. © Mary Ann Liebert, Inc. 2015.


Leotot J.,University Paris Est Creteil | Lebouvier A.,University Paris Est Creteil | Hernigou P.,University Paris Est Creteil | Bierling P.,Etablissement Francais du Sang d'Ile de France | And 4 more authors.
Cell Transplantation | Year: 2015

In the context of clinical applications of bone regeneration, cell seeding into scaffolds needs to be safe and easy. Moreover, cell density also plays a crucial role in the development of efficient bone tissue engineering constructs. The aim of this study was to develop and evaluate a simple and rapid cell seeding procedure on hydroxyapatite/ β-tricalcium phosphate (HA/βTCP), as well as define optimal cell density and control the biodistribution of grafted cells. To this end, human bone marrow-derived stromal cells (hBMSCs) were seeded on HA/βTCP scaffolds, and we have compared bone formation using an ectopic model. Our results demonstrated a significantly higher bone-forming capacity of hBMSCs directly loaded on HA/βTCP during surgery compared to hBMSCs preseeded for 7 days in vitro on HA/bTCP before ectopic implantation. The extent of new bone formation increases with increasing hBMSC densities quantitatively, qualitatively, and in frequency. Also, this study showed that grafted hBMSCs remained confined to the implantation site and did not spread toward other tissues, such as liver, spleen, lungs, heart, and kidneys. In conclusion, direct cell loading into a scaffold during surgery is more efficient for bone regeneration, as well as quick and safe. Therefore direct cell loading is suitable for clinical requirements and cell production control, making it a promising approach for orthopedic applications. Moreover, our results have provided evidence that the formation of a mature bone organ containing hematopoietic islets needs a sufficiently high local density of grafted hBMSCs, which should guide the optimal dose of cells for clinical use. © 2015 Cognizant Comm. Corp.


Poignard A.,University Paris Est Creteil | Lebouvier A.,University Paris Est Creteil | Cavet M.,Service de Radiologie Albert Chenevier | Rahmouni A.,Service de Radiologie Albert Chenevier | And 7 more authors.
International Orthopaedics | Year: 2014

Purpose: In order to evaluate new therapeutic approaches to human osteonecrosis of the femoral head (ONFH), this study proposed to improve the existing animal model by developing a new surgically induced pig model. Methods: First, ONFH was induced with an easy and minimally invasive technique: cryogenic insult with repeated freeze-thaw cycle. Then, to compare and improve the efficacy of this first method, we combined the cryogenic insult to vascular coagulation of the posterior circumflex vessels. Results: Cryoinjury with repeated freeze-thaw cycle alone is sufficient to induce, three weeks postsurgery, a subchondral necrosis as confirmed by magnetic resonance imaging (MRI) and histological analysis. However, a bone regeneration began at four weeks and was complete at eight weeks. To optimise this result, we combined cryoinjury with posterior circumflex vessel coagulation and observed the persistence of ONFH, with progression to collapse at 14 weeks postinduction. Conclusions: Cryoinjury associated with partial vascular coagulation is sufficient to obtain localised and sustainable necrosis in the subchondral area of the femoral head, reproducing all stages of the human disorder. The co-analysis by MRI and histology allowed us to confirm that the classic T1- and T2-weighted hyposignal regeneration front around a fatty high T1-weighted signal observed by MRI indicate signs of induced osteonecrosis. Our results indicate that our pig model induces all stages of human ONFH, which can be followed by MRI, making it relevant for clinical trials. © 2014 Springer-Verlag.


Lebouvier A.,Sanguine | Lebouvier A.,French Institute of Health and Medical Research | Poignard A.,Sanguine | Poignard A.,French Institute of Health and Medical Research | And 15 more authors.
Stem Cell Research and Therapy | Year: 2015

Introduction: Osteonecrosis of the femoral head (ONFH) is a degenerative disease progressing to a femoral head (FH) collapse. Injection of osteoprogenitor cells like bone marrow mesenchymal stromal cells (BMSCs) into the FH appears to be a good therapeutic treatment. However, safety and efficacy of BMSCs to treat bone defect are the main preclinical data required for clinical application. Efficacy and the lack of risk of cell transformation after amplification of BMSCs have been extensively described. The main objectives of this study were to develop a simple and usable procedure for clinicians and control its feasibility by evaluating the biodistribution of BMSCs after injection into the FH in a large animal model. The impact of this approach was evaluated on one natural pig ONFH. Methods: BMSCs were directly injected in the pig FH, and then the biodistribution of grafted cells was detected by quantitative real-time polymerase chain reaction, cytometry, or a combination of classic histology analysis and in situ hybridization (ISH). BMSC efficacy on bone regeneration was evaluated by magnetic resonance imaging (MRI) and histology. Results: After 30-minute and 24-hour follow-up, grafted cells were detected at the injection site and no BMSCs were detected in filter organs or body fluids. The combination of classic histology analysis and ISH showed a good homogeneity of cell distribution in FH. Local delivery of BMSCs onto a bone scaffold associated with bone formation in vivo confirmed the preferential tropism of BMSCs to the bone tissue as well as their efficacy to form bone. Treatment of a natural pig ONFH by autologous BMSCs indicated a beginning of bone healing as early as 2 weeks with a complete healing after 9 weeks. At this stage, MRI and histological analysis were similar to those of a normal FH. Conclusions: Intra-osseous injection of BMSCs in FH seems to be a good strategy for ONFH treatment as the safety concerning the biodistribution of BMSCs is ensured. Moreover, the efficacy of BMSCs in natural ONFH seems to indicate that this is a promising approach. Altogether, these results constitute the preclinical data necessary for the setup of a clinical application with expanded BMSCs in the context of advanced therapy medicinal products. © 2015 Lebouvier et al.; licensee BioMed Central.


Lebouvier A.,Sanguine | Poignard A.,Sanguine | Poignard A.,Service hospitalier | Coquelin-Salsac L.,Sanguine | And 12 more authors.
Stem Cell Research | Year: 2015

Osteonecrosis of the fwemoral head is a frequent complication in adult patients with sickle cell disease (SCD). To delay hip arthroplasty, core decompression combined with concentrated total bone marrow (BM) treatment is currently performed in the early stages of the osteonecrosis. Cell therapy efficacy depends on the quantity of implanted BMstromal cells. For this reason, expanded bone marrowstromal cells (BMSCs, also known as bone marrow derived mesenchymal stem cells) can be used to improve osteonecrosis treatment in SCD patients. In this study, we quantitatively and qualitatively evaluated the function of BMSCs isolated from a large number of SCD patients with osteonecrosis (SCD-ON) comparedwith control groups (patients with osteonecrosis not related to SCD (ON) and normal donors (N)). BM total nuclear cells and colony-forming efficiency values (CFE) were significantly higher in SCD-ON patients than in age and sex-matched controls. The BMSCs fromSCD-ON patients were similar to BMSCs from the control groups in terms of their phenotypic and functional properties. SCD-ON patients have a higher frequency of BMSCs that retain their bone regeneration potential. Our findings suggest that BMSCs isolated from SCD-ON patients can be used clinically in cell therapy approaches. This work provides important preclinical data that is necessary for the clinical application of expanded BMSCs in advanced therapies and medical products. © 2015.


PubMed | University of Nantes, Service de radiologie Albert Chenevier, Sanguine and Etablissement Francais du Sang dIle de France
Type: | Journal: Stem cell research & therapy | Year: 2015

Osteonecrosis of the femoral head (ONFH) is a degenerative disease progressing to a femoral head (FH) collapse. Injection of osteoprogenitor cells like bone marrow mesenchymal stromal cells (BMSCs) into the FH appears to be a good therapeutic treatment. However, safety and efficacy of BMSCs to treat bone defect are the main preclinical data required for clinical application. Efficacy and the lack of risk of cell transformation after amplification of BMSCs have been extensively described. The main objectives of this study were to develop a simple and usable procedure for clinicians and control its feasibility by evaluating the biodistribution of BMSCs after injection into the FH in a large animal model. The impact of this approach was evaluated on one natural pig ONFH.BMSCs were directly injected in the pig FH, and then the biodistribution of grafted cells was detected by quantitative real-time polymerase chain reaction, cytometry, or a combination of classic histology analysis and in situ hybridization (ISH). BMSC efficacy on bone regeneration was evaluated by magnetic resonance imaging (MRI) and histology.After 30-minute and 24-hour follow-up, grafted cells were detected at the injection site and no BMSCs were detected in filter organs or body fluids. The combination of classic histology analysis and ISH showed a good homogeneity of cell distribution in FH. Local delivery of BMSCs onto a bone scaffold associated with bone formation in vivo confirmed the preferential tropism of BMSCs to the bone tissue as well as their efficacy to form bone. Treatment of a natural pig ONFH by autologous BMSCs indicated a beginning of bone healing as early as 2weeks with a complete healing after 9weeks. At this stage, MRI and histological analysis were similar to those of a normal FH.Intra-osseous injection of BMSCs in FH seems to be a good strategy for ONFH treatment as the safety concerning the biodistribution of BMSCs is ensured. Moreover, the efficacy of BMSCs in natural ONFH seems to indicate that this is a promising approach. Altogether, these results constitute the preclinical data necessary for the setup of a clinical application with expanded BMSCs in the context of advanced therapy medicinal products.

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