Intramyocardial transplantation of mesenchymal stromal cells for chronic myocardial ischemia and impaired left ventricular function: Results of the MESAMI 1 pilot trial.

BACKGROUND: The MESAMI 1 trial was a bicentric pilot study designed to test the feasibility and safety of intramyocardially injected autologous bone marrow-derived mesenchymal stromal cells (MSCs) for the treatment of ischemic cardiomyopathy. METHODS AND RESULTS: The study included 10 patients with chronic myocardial ischemia, left ventricular (LV) ejection fractions (EFs) of ≤35%, and reversible perfusion defects who were on stable optimal medical therapy and were not candidates for revascularization. MSCs (mean: 61.5×10(6) cells per patient) were injected into 10-16 viable sites at the border of the LV scar via a NOGA-guided catheter. Both primary endpoints, feasibility (successful harvest, expansion, and injection of autologous MSCs) and safety (absence of severe adverse events [SAEs]) were met in all 10 patients at the 1-month follow-up time point, and none of the SAEs reported during the full 2-year follow-up period were attributable to the study intervention. The results of secondary efficacy endpoint analyses identified significant improvements from baseline to Month 12 in LVEF (29.4±2.0% versus 35.7±2.5%; p=0.003), LV end-systolic volume (167.8±18.8mL versus 156.1±28.6mL; p=0.04), 6-min walk test and NYHA functional class. CONCLUSIONS: Our results suggest that autologous MSCs can be safely administered to the hearts of patients with severe, chronic, reversible myocardial ischemia and impaired cardiac function and may be associated with improvements in cardiac performance, LV remodeling, and patient functional status. A randomized, double blind, multicenter, placebo-controlled clinical trial (MESAMI 2) will evaluate the efficacy of this treatment approach in a larger patient population. CLINICAL TRIAL REGISTRATION: Unique identifier: NCT01076920.

[Use of adipose tissue in regenerative medicine]. / Tissu adipeux et médecine régénératrice.

Adipose tissue is abundant and well known for its involvement in obesity and associated metabolic disorders. Its uses in regenerative medicine recently attracted many investigators, as large amounts of this tissue can be easily obtained using liposuction and it contains several populations of immature cells. The largest pool of such cells corresponds to immature stromal cells, called adipose-derived stromal cells (ADSCs). These cells are purified after proteolytic digestion of adipose tissue and selection by an adherent step. ADSCs display many common features with mesenchymal stem cells derived from bone marrow, including paracrine activity, but with some specific features, among which a greater angiogenic potential. This potential is now investigating at clinical level to treat critical ischemic hindlimb by autologous cells. Other potentials are also investigated and the treatment of fistula associated or not with Crohn's disease is reaching now phase III level.

Native human adipose stromal cells: localization, morphology and phenotype.

OBJECTIVES: Beside having roles in energy homeostasis and endocrine modulation, adipose tissue (AT) is now considered a promising source of mesenchymal stromal cells (adipose-derived stromal cells or ASCs) for regenerative medicine. Despite numerous studies on cultured ASCs, native human ASCs are rarely investigated. Indeed, the phenotype of ASCs in their native state, their localization within AT and comparison with bone marrow-derived mesenchymal stromal cells (BM-MSCs) has been poorly investigated. DESIGN: To address these issues, the stroma vascular fraction (SVF) of human AT was extracted and native cell subtypes were isolated by immunoselection to study their clonogenic potential in culture. Immunohistology on samples of human AT in combination with reconstruction of confocal sections were performed in order to localize ASCs. RESULTS: Compared with BM-MNCs, all native ASCs were found in the CD34(+) cell fraction of the AT-SVF. Native ASCs expressed classical mesenchymal markers described for BM-MSCs. Interestingly, CD34 expression decreased during ASC cell culture and was negatively correlated with cell proliferation rate. Immunohistological analysis revealed that native ASCs exhibited specific morphological features with protrusions. They were found scattered in AT stroma and did not express in vivo pericytic markers such as NG2, CD140b or alpha-smooth muscle actin, which appeared during the culture process. Finally, ASCs spontaneous commitment to adipocytic lineage was enhanced in AT from obese humans. CONCLUSIONS: The use of complementary methodological approaches to study native human ASCs revealed their immunophenotype, their specific morphology, their location within AT and their stemness. Furthermore, our data strongly suggest that human ASCs participate in adipogenesis during AT development.

Human adipose derived stroma/stem cells grow in serum-free medium as floating spheres.

With the goal of obtaining clinically safe human adipose-derived stroma/stem cells (ASC) and eliminating the use of serum, we have developed a new culture system that allows the expansion of ASC as spheres in a defined medium. These spheres can be passaged several times. They are not only aggregated cells but rather originate from single cells as clonal spheres can be obtained after seeding at very low density and reform clonal spheres after dissociation. These spheres can also revert to monolayer growth when plated in medium containing human plasma and even generate fibroblast-like colonies (CFU-f). Under several differentiation-specific media, spheres-derived ASC maintain their capacity to differentiate into osteoblasts, endothelial cells and adipocytes. These results indicate that human ASC can be maintained in a serum-free 3D culture system, which is of great interest for the expansion in bioreactors of autologous ASC and their use in clinical trials.

Mesenchymal stem cells for clinical application.

Mesenchymal Stem Cells/Multipotent Marrow Stromal Cells (MSC) are multipotent adult stem cells present in all tissues, as part of the perivascular population. As multipotent cells, MSCs can differentiate into different tissues originating from mesoderm ranging from bone and cartilage, to cardiac muscle. Conflicting data show that MSCs could be pluripotent and able to differentiate into tissues and cells of non-mesodermic origin as neurons or epithelial cells. Moreover, MSCs exhibit non-HLA restricted immunosuppressive properties. This wide range of properties leads to increasing uses of MSC for immunomodulation or tissue repair. Based on their immunosuppressive properties MSC are used particularly in the treatment of graft versus host disease, For tissue repair, MSCs can work by different ways from cell replacement to paracrine effects through the release of cytokines and to regulation of immune/inflammatory responses. In regenerative medicine, trials are in progress or planed for healing/repair of different tissue or organs as bone, cartilage, vessels, myocardium, or epithelia. Although it has been demonstrated that ex-vivo expansion processes using fetal bovine serum, recombinant growth factors (e.g. FGF2) or platelet lysate are feasible, definitive standards to produce clinical-grade MSC are still lacking. MSCs have to be produced according GMP and regulation constraints. For answering to the numerous challenges in this fast developing field of biology and medicine, integrative networks linking together research teams, cell therapy laboratories and clinical teams are needed.

[Early education for children with Prader-Willi syndrome]. / Préconisations d'education précoce pour les enfants porteurs d'un syndrome de Prader-Willi.

MATERIAL AND METHOD: A cohort of 21 children (under 42 months of age) with Prader-Willi syndrome were followed in a longitudinal way by the referral center team with evaluation of language and cognitive function (every six months). RESULTS: Disorders seen between birth and the second year are primarily related to hypotonia with oropharyngeal dysphagia and impact on the pre-lingual phase of communication. After 2 years the psychomotor delay and the disorders of language acquisition effect development. When compared to patients who had treatment after 4 years and with data published in the literature, those patients with an early diagnosis and early treatment using kinesitherapy and logopedy showed faster linguistic acquisition. However articulation disorders still remain. CONCLUSION AND RECOMMENDATIONS: This work highlights the importance of early readaptation associating kinesitherapy, logopedy and psychomotoricity which can improve the cognitive phenotype of these children.

[Validation of a self assessment for speech disorders (Phonation Handicap Index)]. / Validation d'un questionnaire d'auto-evaluation de la parole (Parole Handicap Index).

OBJECTIVE: The aim of this work is the psychometric validation of a self assessment questionnaire about speech in dysarthria. MATERIALS AND METHODS: A prospective study was performed on 91 persons to analyze reliability and validity of this new questionnaire. It is composed of 25 items subdivided in 3 domains: Physical, functional and emotional. We first compared 17 normal speakers to 21 dysarthric patients. Then we analysed the intra-individual variability with 53 patients who completed the questionnaire twice with two weeks interval between the completion. The statistical analysis verified internal consistency of each item, intra-subject reliability was analyzed through Pearson test and clinical validity was calculated through the non parametric Mann Whitney test. RESULTS: The internal consistency reliability was correct (Cronbach's alpha > 0.9). It appeared a statically significant difference between normal speakers and dysarthric patients (p < 0.05). The correlations to the handicap and severity felt were fair ensured us of the content's validity. However the absence of difference for 5 items drove us to eliminate them. Also, analysis the test / retest by the correlation matrix allowed to delete 5 other questions. The obtained total score was 0,861. As for the validity of contents, the correlations in the handicap and in the degree of severity felt by the dysarthric patients were satisfactory. CONCLUSION: The results of this study allows to resume the validation of this questionnaire, its short form of 15 items is particularly adapted to dysarthric patients. It now remains to test its reliability with the medical evolution of the patients. We propose to name it "Phonation Handicap Index".

[Study of the predictive value of detection tests for silent aspirations]. / Etude de la valeur prédictive des tests de détection des fausses routes silencieuses.

UNLABELLED: Screening for aspiration in patients with swallowing disorders is important in preventing complications. The tests used in this regard are insufficient due to silent aspiration relating to abnormal protective reflexes in many patients with swallowing problems. OBJECTIVE: The aim of this study is to determine the predictive values of simple tests in screening for silent aspiration. MATERIAL AND METHOD: The reference test used was videofluoroscopic examination on swallowing. In the presence of aspiration (FR+) the presence (ME+) or not (ME-) of a cough of throat clearing was noted. The tests being studied were a nasal test with isotonic saline and swallowing according to a set time. RESULTS: For screening for aspiration the presence of a "wet voice" was considered to be a sign of reduced protective reflexes. 1) During the nasal test, the results are 100% for the positive predictive value (VPp) and 83.3% for the negative predictive value (VPn); 2) These results are respectively 84.6% and 35.9% during the swallowing test. Regarding screening for silent aspiration, 1) during the nasal test, the results are 62.5% for the positive predictive value (VPp) and 36.3% for the negative predictive value (VPn); 2) These results are respectively 54.5% and 26.6% during the swallowing test. CONCLUSION: This preliminary study points out the lack of predictive value of the nasal test and the swallow test for the silent aspirations. However the results could be useful for other researchers developing other tests in this area.

[Positioning in the oropharyngeal dysphagia]. / Positionnement dans les dysphagies oropharyngées.

Sitting body posture and the movements for the feeding gesture are interdependent and can worsen an oropharyngeal dysphagia. Their management will have a direct impact on the patient's handicap and will be able to decrease the complications. Clinical process aiming at placing a person having postural disorders, positioning will ensure a base adapted to the person, comfortable, allowing mobility and independence. It requires a methodical accompaniment: evaluation, test, validation, control of adapted equipment for the patient and gesture readjustment.

[Erythrocyte removal from bone marrow by density gradient separation using the COBE 2991 cell processor with the triple-bag processing set]. / Désérythrocytation de moelle osseuse par gradient de densité sur COBE 2991 avec le kit triple "bérets".

ABO-incompatible bone marrow transplantation requires red blood cell depletion. Lots of laboratory adopted the technique of density gradient centrifugation (Ficoll-hypaque) using the COBE 2991 cell processor with simple-bag processing set. However, tubing of this set is not adapted to the currently available peristaltic pumps. Moreover, two other sets are required: one for the buffy-coat and one for postgradient cell washing. We developed a method using triple-bag processing set to conduct whole-step procedure (concentration, Ficoll and washing). Peristaltic PVC tubing is provided in one line of the set allowing a safe processing without several connections thus reducing risks of microbial contamination. First, we used buffy-coat of total blood for training, then, we carried out red cell depletion of healthy bone marrow donors. The red blood cell depletion was 97.9+/-1.1% and CD34+ recovery was 89.6+/-8.7%. These results are very close to those obtained with the simple-bag set (red cell depletion.=94.0+/-6.8% and CD34+ recovery=95.9+/-20.3%). We conclude that the triple-bag system, very little used in France, is practical, simplified the manipulation and is more safety than the simple-bag set.

[Management of swallowing disorders after brain injuries in adults]. / Prise en charge des troubles de déglutition après lésions cérébrales dans un centre de réveil pour adultes.

The management of swallowing disorders after brain injury must be soon as well. The physiopathological analysis and the organization of the therapeutic project of these patients require the intervention of an interdisciplinary team. Dysphagia falls under a complex clinical context associating impairments of cognition, communication and behavioural control. The management associates speech therapist, caregivers, otolaryngolologist, phoniatrician, physiotherapist and nutritional therapist without forgetting the family circle. The fluctuations of consciousness and concentration of our patients brings us to constantly readjusting and rehabilitating the strategies of feeding. Obstacles with their evolution towards a normal feeding are akinesia, limits of motor functions, impairements of cognition and behavioural control. In the located lesions swallow recovers can be fast, instead of in severe brain-injury the challenge is to ensure safe and adequate nutrition, using a variety of strategies depending on the presenting symptoms. The purpose of this article is to relate our experience beside patients with an acute or recent cerbrovascular event.

[The hopes of the mesenchymal stem cells in regenerative medicine]. / Les espoirs des cellules souches mésenchymateuses en médecine réparatrice.

The mesenchymal stem cells are a cell population of bone marrow, which have the capacity to differentiate towards all the cells from the locomotor apparatus. They also have immunomodulatory properties and can contribute to tissue repair, thanks to the secretion of many growth factors. Such cells are also found in the cord blood. In the same way, very close stem cells exist in great quantity in fat tissue. These cells are very good candidates in regenerative medicine. Besides, several clinical trials were carried out in order to highlight their effectiveness mainly in osseous repair and also during hematopoietic stem cells graft or cardiac repair after infarction. However, these trials will be able to develop fully only with the condition that culture techniques meeting the conditions of good manufacturing practice are set-up. This presentation gives a progress report on the whole of these subjects.

Bone marrow mesenchymal stem cells are abnormal in multiple myeloma.

Recent literature suggested that cells of the microenvironment of tumors could be abnormal as well. To address this hypothesis in multiple myeloma (MM), we studied bone marrow mesenchymal stem cells (BMMSCs), the only long-lived cells of the bone marrow microenvironment, by gene expression profiling and phenotypic and functional studies in three groups of individuals: patients with MM, patients with monoclonal gamopathy of undefined significance (MGUS) and healthy age-matched subjects. Gene expression profile independently classified the BMMSCs of these individuals in a normal and in an MM group. MGUS BMMSCs were interspersed between these two groups. Among the 145 distinct genes differentially expressed in MM and normal BMMSCs, 46% may account for a tumor-microenvironment cross-talk. Known soluble factors implicated in MM pathophysiologic features (i.e. IL (interleukin)-6, DKK1) were revealed and new ones were found which are involved in angiogenesis, osteogenic differentiation or tumor growth. In particular, GDF15 was found to induce dose-dependent growth of MOLP-6, a stromal cell-dependent myeloma cell line. Functionally, MM BMMSCs induced an overgrowth of MOLP-6, and their capacity to differentiate into an osteoblastic lineage was impaired. Thus, MM BMMSCs are abnormal and could create a very efficient niche to support the survival and proliferation of the myeloma cells.

[Cardiac cellular therapy: from cells to the first clinical uses]. / Thérapie cellulaire cardiaque: des cellules aux premières indications cliniques.

Despite the improvement in revascularisation techniques, coronary artery disease remains the principal aetiology of cardiac failure in developed countries. The therapeutic management of cardiac failure has been improved over recent years, yet cardiac failure is still associated with significant morbidity and mortality. As cardiac transplantation lacks donors, techniques that allow myocardial regeneration represent an attractive alternative. To date, several types of cells are under study and are suitable for implantation into infarcted myocardium (myoblasts, medullary stem cells...). Following good preclinical study results, the first human cell therapy trials, using the intramyocardial route, have begun, in the course of aorto-coronary bypass surgery in patients with chronic ischaemic cardiopathy and little altered left ventricular function, and then in those with ventricular dysfunction. Different modes of administration of these cell therapy products are under study and could be envisaged in clinical situations such as just after infarction in order to improve ventricular remodelling with an intracoronary injection technique. As for every new treatment, there are numerous problems to resolve, from understanding the relevant mechanisms of cellular transplantation, to the secondary effects that it could entail. Nevertheless, cardiac cellular transplantation is expanding rapidly and with the evolution of techniques it allows a glimpse of a new field of treatment for cardiac failure.

Proteomic study of Galectin-1 expression in human mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) are known to interact with hematopoietic stem cells (HSCs) and immune cells, and are of potential interest to be used as therapeutic agents for enhancing allogenic hematopoietic engraftment and preventing graft-versus-host disease (GVHD). Galectin 1 (Gal1) belongs to a family of structurally related molecules expressed in many vertebrate tissues that exert their functions both by binding to glycoconjugates, and by interaction with protein partners. In this work using a proteomic approach, we looked for the presence and the localization of Gal1 in short- and long-term culture of human (h) hMSC. We first determined, that Gal1 is one of the major proteins expressed in hMSC. We futher demonstrated that its expression is maintained when hMSC are expanded through a subculturing process up to five passages. Moreover, Gal1 is secreted and found at the cell surface of MSC, participating in extra cellular matrix (ECM)-cell interactions. Given the immunomodulatory properties of Gal1, its potential involvement in immunological functions of hMSC could be suggested.

Regulation of CD45-induced signaling by galectin-1 in Burkitt lymphoma B cells.

It has been well established that Galectin-1 (GAL1), a beta-galactoside-binding protein, regulates the viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood. As a first step towards the elucidation of GAL1-initiated signaling events leading to a reduced viability of Burkitt lymphoma B cells, we tried to characterize the initial events induced by the binding of GAL1 to its receptor. This characterization was performed in BL36 cells, a Burkitt lymphoma cell line sensitive to GAL1. The results were as follows: (1) when solubilized cell membrane lysates were affinity bound to immobilized GAL1 and eluted by competition, the tyrosine phosphatase glyco-protein CD45 was found in the eluate, highlighting the role of CD45 as a receptor of GAL1; (2) the phosphatase activity of cell membranes diminished after incubation with GAL1; (3) immunoprecipitation experiments demonstrated that the phosphotyrosine kinase Lyn was dysregulated in cells that have been cultured in medium containing 700 nM GAL1, and (4) that the ratio between two isoforms of Lyn was modified during the treatment with GAL1. The regulation of Lyn therefore seems to be a key event in the action of GAL1.

Immunolabeling of CD3-positive lymphocytes with a recombinant single-chain antibody/alkaline phosphatase conjugate.

G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigen-binding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.

Chemokine SDF-1 enhances circulating CD34(+) cell proliferation in synergy with cytokines: possible role in progenitor survival.

The chemokine stromal cell-derived factor-1 (SDF-1), and its receptor, CXCR-4, have been implicated in the homing and mobilization of human CD34(+) cells. We show here that SDF-1 may also be involved in hematopoiesis, promoting the proliferation of human CD34(+) cells purified from normal adult peripheral blood (PB). CXCR-4 was expressed on PB CD34(+) cells. The amount of CXCR-4 on PB CD34(+) cells was 10 times higher when CD34(+) cells were purified following overnight incubation. CXCR-4 overexpression was correlated with a primitive PB CD34(+) cell subset defined by a CD34(high) CD38(low)CD71(low)c-Kit(low)Thy-1(+) antigenic profile. The functional significance of CXCR-4 expression was ascertained by assessing the promoting effect of SDF-1alpha on cell cycle, proliferation, and colony formation. SDF-1 alone increased the percentage of CD34(+) cells in the S+G(2)/M phases and sustained their survival. In synergy with cytokines, SDF-1 increased PB CD34(+) and CD34(high)CD38(low) cell expansion and colony formation. SDF-1 also stimulated the growth of colonies derived from primitive progenitors released from quiescence by anti-TGF-beta treatment. Thus, our results shed new light on the potential role of this chemokine in the stem cell engraftment process, which involves migration, adhesion, and proliferation. Furthermore, both adhesion-induced CXCR-4 overexpression and SDF-1 stimulating activity may be of clinical relevance for improving cell therapy settings in stem cell transplantation.

Production and characterization of a monoclonal antibody able to discriminate galectin-1 from galectin-2 and galectin-3.

Antisera raised against galectin-1 exhibit crossreactivities with other galectins or related molecules. In order to overcome this problem, a monoclonal antibody to human brain galectin-1 was obtained by selecting clones without reactivity toward galectin-3. This mAb specifically bound galectin-1 of various animal origins but neither galectin-2 nor galectin-3. Western-blotting analysis of soluble human brain extracts after 2D gel electrophoresis revealed only the two most acidic isoforms of galectin-1. The ability of this mAb to bind galectin-1/asialofetuin complexes indicates that its epitope is not localized in the carbohydrate recognition domain of galectin-1. This particularity induces with efficiency its monospecificity.