Dendritic cells constitute a family of antigen presenting cells defined by their morphology and their capacity to initiate primary immune response. Langerhans cells are paradigmatic dendritic cells, described in 1868 by a young medical student, Paul Langerhans in Berlin. Langerhans cells are present with epithelial cells in the epidermis, bronchi and mucosae. After antigenic challenge, Langerhans cells migrate into the T cell areas of proximal lymph nodes where they act as professional antigen-presenting cells. Langerhans cells originate in the bone marrow and CD34+ hematopoïetic progenitors are present in cord blood or circulating blood. They are actively involved in skin lesions of allergic contact dermatitis or atopic dermatitis, in cancer immunosurveillance and are infected by HIV in AIDS. Since 1992, Langerhans cells may be generated in vitro from CD34+ cord blood or circulating blood progenitors by culture with GM-CSF and TNF alpha, as well as from peripheral blood monocytes by culture with GM-CSF, IL4 and TGF beta 1. The possibility to obtain from the blood, the circulating progenitors of dendritic cells and the subsequent possibility to harvest a large number of these cells through in vitro culture using growth factors, have given rise to several very interesting therapeutic perspectives, especially in the field of anti-cancer immunotherapy. In dermatology advanced studies have concerned malignant melanomas. Anti-melanoma immunization trials were performed in patients, through dendritic cells charged with melanoma antigens. Side effects appear to be limited. Injections of antigenically charged dendritic cells were performed subcutaneously, intravenously or in the lymph nodes. Positive clinical responses were obtained with, in some cases, complete remission of the metastasis. These results open a particularly interesting perspective in the field of cancer treatment.

The success of HSCT from HLA partially disparate donors depends on the development of new strategies able to efficiently prevent GVHD and to protect patients from infections and relapse. Using an immunotoxin (IT) directed against the alpha-chain (p55) of the human IL-2r (RFT5-SMPT-dgA), we have previously shown that it is possible to kill mature T cells activated towards a specific HLA complex by a one-way MLR. We designed a clinical trial assessing the effect of infusing increasing doses of T lymphocytes in the setting of children recipients of non HLA genetically identical HSCT. Thirteen patients have been enrolled from September 1998 to April 2000 and fourteen HSCT have been realized in 13 patients (pts). Donors were MUD in 3 cases and familial HLA partially disparate in the remaining cases. Allodepleted donor T cells were injected between day +14 and day +30 provided that ATG was undetectable in the serum and blood PMN counts was > 500/microliter. The mean age of these patients was 17 months (range 1 to 42). Diagnosis included immune deficient and malignant hemopathies. Three patients received 1 x 10(5) allodepleted T cell/kg, 7 patients received 4 x 10(5)/kg and 4 patients received 6 x 10(5)/kg allodepleted T cells. Full inhibition of MLR was achieved in 12 out of 14 cases. In two cases, a residual T cell reactivity to the recipient was observed (4 to 5%) and patients developed grade II aGVHD. aGVHD occurred in 4 out of 11 grafted patients (all grade II). No chronic GVHD has developed, so far. Three patients died from severe VOD or PHT at day +34, day 51 and day +166, while one infected patient by VZV, CMV and EBV before HSCT died 6 months after transplantation from meningoencephalitis and another patient died from relapse at day +291. The patient for which there was no engraftment died at day +48 from staphylococcus infection. Overall survival is 54%, with a median follow up of 8 months; the mean time to reach a blood lymphocyte count > 500 was 41 days, to reach a CD3 count > 300 microliters 63 days (20-111), CD4 > 200 microliters 97 days and positive mitogen-induced proliferation 90 days. In three patients, a tetanus-toxoid positive proliferation was detected before immunization. From this intermediate analysis, we conclude that 1) specific allodepletion is an effective approach to prevent aGVHD in a haploincompatible setting, 2) data on immunological reconstitution suggest that infused T cells do survive and expand. A higher number of patients must be enrolled to determine the optimal number of T cells to infuse.

Fetal neural allografts have already proven their therapeutic value in several hundreds of patients with Parkinson's disease, and have very recently provided promising results for patients with Huntington's disease in our center. Fetal neurons integrate readily into the neural parenchyma of the adult hosts, differentiate into mature neurons and substitute, anatomically and functionally for lost host neurons. Notable clinical improvements have been obtained using this procedure. Nevertheless, a major obstacle hampers the development of the technique, that provoked by the logistic difficulty in retrieving and preparing the tissue. Indeed, this requires, for each surgical session, the organization of a chain of expertise which cannot be taken up by an external provider (e.g. a biotech company). This is difficult to organize outside of specialized research centers. The future of the technique relies, therefore, upon the design of alternative sources of tissue. Two different ways are currently explored very actively, namely xenografting of neurons of porcine origin and human stem cells, in particular derived from ES cells. In both cases, but in different ways, the goal of both techniques is to allow the organisation of cell banking systems, relieving the constraints of obtaining the collaboration of specialized obstetricians and biologists. Obstacles foreseen for these two alternative ways of fetal neurons to be are identified and research laboratories are actively exploring ways to overcome them.

Regenerative medicine can be defined as the possibility to replace aged/damaged cells by genetically similar young and functional cells. This could be reached by using human embryonic stem cells, eventually from cloned human embryos, or pluripotent adult stem cells. The range of the possible differentiation fates of these latter cells has recently been shown to be strikingly large. Although considerable works remains necessary to develop this new type of medicine, to assure its efficacy and safety, it nevertheless represents one of the major medical breakthroughs expected for the future.

When proceeding normally, embryonic morphogenesis begins with germ layer formation through the process of gastrulation. Each primordial germ layer gives rise to a particular set of lineages. Until recently, it was considered that fate switches between germ layers were impossible. In the last two or three years however, a fair number of such switches have been described (Table I), the most spectacular of which entails the differentiation of neural stem cells into various derivatives. This unexpected plasticity opens important prospects for cell therapy. Stem cells, which are the cells that display this plasticity, are defined by the two properties of self renewal and pluripotency. They are set apart during ontogeny and are responsible for maintaining the homeostasis of a tissue. This notion, first established in the case of hematopoietic stem cells was later extended to other fast renewing cells, such as those in the intestinal epithelium or epidermis, and more recently to cells reputedly non-renewable, i.e. neurons. A new strategy has been described, which has the interesting feature that it can be applied to the isolation of stem cells from various lineages. It consists in sorting out cells on the basis of the efflux of Hoechst 33342 dye (Goodell et al., 1996). When a cell suspension stained with this dye is examined under two distinct wave lengths, a "side population" (SP), characterized by weak fluorescence, can be identified and sorted out. The dye efflux property of these cells is due to the activity of the mdr (multidrug resistance) gene, which encodes a protein responsible for the building of a canal which serves to extrude toxins from the cells. A means of distinguishing a truly multipotent stem cell from a progenitor committed to a specific lineage has been reported. This consists in the expression of the Pax7 gene. Pax7-/- mouse muscles have no satellite cells, i.e. they miss the cells normally responsible for the regeneration of muscle. In contrast they do have an SP population. These SP cells are incapable of differentiating into muscle, but give rise to 10 times more hematopoietic colonies, when cloned in vitro, than SP cells from wild type muscle do. Thus Pax7 appears to be a commitment gene, in the absence of which stem cells cannot become specified to the muscle lineage. As a conclusion, this review emphasizes various features of the recent findings: 1) the unexpected plasticity uncovered in recent years is restricted to the stem cells of each tissue; 2) the switch in phenotype has to be "forced" on these stem cells by drastic experimental conditions enforced in the host: often sublethal irradiation is superimposed on a genetic deficiency. Progress in this field, concerning both conceptual and applied aspects, will require the identification of the factors characterizing the niches which promote integration and fate switches of stem cells, probably a combination of growth factors and intercellular interactions. Finally a key issue, before any therapeutical applications can be considered, is how to control the proliferation of transplanted stem cells in their new environment.

The clinical trials of myoblast transplantation in Duchenne Muscular Dystrophy (DMD) patients produced disappointing results. The main problems responsible for these poor results have since then been identified and partially resolved. One of them was related to the use of an inadequate immunosuppression and, since then, immunosuppression with FK506 has permitted successful myoblast transplantation not only in mice but also in monkeys. The requirement for a sustained immunosuppression may be eventually avoided by developing a state of tolerance to the allogeneic cells or by autologous transplantation of genetically corrected myoblasts or stem cells. The rapid death of 75-80% of the injected myoblasts during the first five days has also contributed to the limited success of the early trials. This death was due to an inflammatory reaction and has been compensated in animal experiments by the injection of a larger number of cells (30 millions per cc). Finally, the myoblasts migrated only 0.5 mm away from their site of injection. This problem is currently compensated in animal experiments by injecting the myoblasts at every mm. The number of injections required may eventually be reduced by transfecting myoblasts with one or several metalloproteinase genes. The very good results obtained during the last two years in primates permit us to undertake a new phase I clinical trial to verify that myoblast transplantation can lead to the formation of muscle fibers expressing normal dystrophin in muscles of DMD patients.

Murine embryonic stem (ES) cells are cell lines established from blastocyst which can contribute to all adult tissues, including the germ-cell lineage, after reincorporation into the normal embryo. ES cell pluripotentiality is preserved in culture in the presence of LIF. LIF withdrawal induces ES cell differentiation to nervous, myocardial, endothelial and hematopoietic tissues. The model of murine ES cell hematopoietic differentiation is of major interest because ES cells are non transformed cell lines and the consequences of genomic manipulations of these cells are directly measurable on a hierarchy of synchronized in vitro ES cell-derived hematopoietic cell populations. These include the putative hemangioblast (which represents the emergence of both hematopoietic and endothelial tissues during development), myeloid progenitors and mature stages of myeloid lineages. Human ES cell lines have been recently derived from human blastocyst in the USA. Their manipulation in vitro should be authorized in France in a near future with the possibility of developing a model of human hematopoietic differentiation. This allows to envisage in the future the use of ES cells as a source of human hematopoietic cells.

Transfusion medicine has the logic of a therapeutic chain applied to labile blood components and cell therapy products, within a coherent structure, such as the recently created Etablissement français du sang. Faced to the threat of emerging--sometimes hypothetical--transfusion risks, such as the possible transmission of BSE by blood transfusion, the precaution principle requires developing strategies to reduce labile blood components consumption, by strictly defining the framework of blood transfusion prescription and encouraging the search for red blood cell and platelet substitutes. In the field of alternatives to labile blood components, research has however yielded few results. The future of transfusion medicine lies in biotechnology: cell (and gene) therapy will become part of novel therapeutic strategies for the treatment of numerous pathologies in man. Transfusion medicine will have to consider the significant advances achieved over the last few years in the field of multipotent stem cells. Transfusion medicine will thus find its place in the promising field of innovating therapies.

The presence in normal adult man of stem cells sharing the properties of embryonic stem cells opens new avenues for basic and therapeutic research. We describe a stem cell present in normal adult human blood, probably able to give rise to the "reserve" stem cells in charge of repair, present in different organs. These monocytoid circulating cells are able to transdifferentiate into several cell types. In normal man, they are almost quiescent and are strictly controlled by a special subpopulation of T lymphocytes. In diseases such as fibrosis and chondrosarcoma, these cells proliferate and the differentiated cells escape T lymphocyte control. As a consequence, these cells accumulate, giving rise in vitro to a tissue which evoke the lesions characterizing the disorder of the patient, showing spontaneously their pluripotentiality. Neural cell markers are present in this migrating cell, suggesting that pluripotent stem cells present in adult man may derive from the neural crest. These circulating cells could offer a source of stem cells for cellular and gene therapy provided the normal cells could be expanded, their transdifferentiation directed and the control by T lymphocytes maintained.

To evaluate the biological effects of guided bone regeneration (GBR) barrier materials on osteoblastic cell migration, migration of mouse osteoprogenitor cells (MC3T3-E1) was examined, in vitro, on various membranes. Eight commercially available GBR membranes - bovine type I collagen (BioMend; BM), porcine type I collagen (BioGide; BG), bovine type I atelocollagen (Tissue Guide; TG), polylactic acid (Epi-Guide; EG), co-polymer of polylactic acid and polyglycolic acid (Resolute; RL, Resolut XT; RL-XT), expanded polytetrafluoroethylene (e-PTFE; Gore Tex; GT) and co-polymer of cellulose acetate and nitrocellulose (Millipore filter; MP) - were tested. A 3x5 mm section of the membrane was fixed to the bottom of a culture dish with double-sided adhesive tape, and half of the membrane was closely covered by PARAFILM (American National Can) to leave an unexposed area for cell migration. The border between exposed and unexposed areas was marked as a baseline of cell migration. Membranes were then plated with 3 ml of cell suspension at an initial density of 1x105 cells/ml in alpha-MEM culture medium with 10% fetal bovine serum and ascorbic acid. After a 5-hour incubation, non-attached cells were completely washed out with phosphate buffered saline and the PARAFILM cover was removed. After 3 days cultivation, specimens were fixed with 10% buffered formalin and stained briefly with hematoxylin. The area of cell migration on a membrane was analyzed using a LA 500 Image Analysis System and migration area per unit length of the baseline (mm2/mm) was compared among membranes. Results demonstrated that cell migration was greater in the order: RL>RL-XT, BM, TG, MP>EG, BG. Membranes except for BG, EG and GT showed the migration rate equal to or higher than a plastic culture cover slip (Celldesk) (P<0.01) on which cells generally grow favorably. Only a small number of the cells attached to GT, and the net cell migration for the membrane could not be determined. These results indicate that GBR barrier materials per se may influence the process of bone regeneration in vivo through the effects of their presence on cell migration.

Hepatitis C virus (HCV) induces chronic persistent infection that can lead to the development of hepatocellular carcinoma. We have searched for the presence of HCV genomic RNA in cells from hematopoietic origin and have, among others, documented such sequences in B cells as well as dendritic cells (DC) derived from monocytes. The allostimulatory capacity of these latter cells was found altered in chronic patients while it appeared restored in long term responders to therapy.

Cell therapy offers today important perspectives for the treatment of type 1 diabetes. The current utilization of primary human islets of Langerhans nevertheless forbids all hope of developing this treatment on a large scale. The recent description of the persistence of stem cells capable of proliferating and differentiating in the adult pancreas offers an attractive alternative for the production in vitro of homologous insulin-secreting cells. We first reproduced in vitro from human islet preparations the proliferation of ductal epithelial structures and their progressive organization. Thereafter, we focused on the description of a reproducible source of human ductal cells by the transdifferentiation of exocrine preparations. More recently we described in these exocrine derived ductal cells the the expression the of insulin promoter factor-1 (IPF-1/otherwise known as PDX-1), a transcription factor essential for the differentiation of ductal cells into endocrine cells during both development and pancreatic regeneration. If the proliferation and differentiation of these cells is confirmed, this approach could lead to the description of an abundant source of human pancreatic stem cells for the production ex vivo of human insulin secreting cells and may even allow autologous cell therapy, in the absence of immunosuppression.

The transcription factor MyoD, member of the myogenic regulators family, induces differentiation in precursor cells by its ability to arrest cell proliferation and to activate muscle specific genes. MyoD plays a key role in the antagonism between proliferation and differentiation. The withdrawal from the cell cycle and the activation of muscle differentiation are related to the level of MyoD protein. The cyclin E-cdk2 complex, one of the key regulators of the G1/S transition is directly implicated in the degradation of MyoD by the ubiquitin-proteasome pathway, leading the myoblasts to proliferate. The display of this control in normal myoblasts suggests that its deficiency in the muscle stem cells could lead to the formation of rhabdomyosarcomas which have lost both the control of cell proliferation and the transcriptional activity of MyoD.

Myelodysplastic syndromes are clonal hematologic disorders, expanded from myeloid stem cells. A primitive immunologic disorder is discussed. This hypothesis could explain a non-casual association with systemic diseases. The aim of our study is to test this hypothesis.

We report two cases of atypical defibrination syndromes in patients with respectively acute monoblastic leukemia (chronic myeloid leukemia initially) and acute lymphoblastic leukemia. Hemostasis studies show low fibrinogen level, elevated D-dimers, decreased alpha 2 antiplasmin and factor V, normal antithrombin III values. Plasminogen is below the normal range in one patient. Soluble complexes, which are an important argument for diagnosis of intravascular coagulation disease, are not detected in both patients. Primary or secondary hyperfibrinolysis seems also excluded since euglobulin clot lysis time was normal. Enzymatic proteolysis of fibrinogen (or fibrin) by the blast cells has been reported by some authors; this mechanism could account for the hemostasis abnormalities observed in these two patients.

Comprehension of cell cycle regulation mechanisms has progressed very quickly these past few years and regulators of the cell cycle have gained widespread importance in cancer. This review first summarizes major advances in the understanding of the control of cell cycle mechanisms. Examples of how this control is altered in tumoral cells are then described.

Neuroblastoma is a very common solid tumor which arises in childhood and shows an extreme heterogeneity at the clinical, histological and genetic levels. Besides age and stage, N-myc amplification and 1p deletion are prognostic factors of the disease: in Europe, these genetic markers are used to conduct therapy. In France, N-myc amplification is a factor of bad prognosis which leads, in all forms of the disease including localised forms and metastatic forms of children aged of less than 1 year, to a myeloablative treatment with autologous hematopoietic stem cells transplantation. By contrast, N-myc amplification has no impact on the survival of children aged of more than 1 year with a poor prognosis (30% overall survival, 5 years) but this genetic abnormality is taken into account to treat primary tumor of these patients. In an attempt to find out prognostic factors of these aggressive forms of the disease, various pathways (apoptosis, differentiation angiogenesis, detoxication, immune response) have been recently surveyed, but studies have been carried out on a limited number of genes. Moreover, experimental models of human metastatic neuroblastoma have been obtained in which variations of genes transcript levels involved in these pathways, are observed. The current break-through of cDNA microarrays allows to develop a dynamic transcriptomic scanning of these models as well as of tumors and bone marrows from patients upon conventional chemotherapy. This technology will enable: i) to define molecular entities of the metastatic disease; ii) to apply adapted treatment; iii) to develop new therapeutic strategies.

The manipulation of embryonic stem (ES) cells allows to generate mice with specific alteration in any gene. This is therefore an invaluable tool for studying gene function. A number of genes involved in the regulation of hematopoiesis have been inactivated, including genes that encode transcription factors, cytokines and their receptors as well as those encoding for intracellular signalling proteins. Alternatively, ES cells are able to differentiate towards myeloid, lymphoid and endothelial lineages under specific culture conditions. The role of master genes controlling hematopoiesis can be investigated by substituting the in vitro hematopoietic differentiation model of ES cells to mice fabrication. This method can be applied for studying effects of gene inactivation or overexpression of normal or abnormal gene. Interestingly, in vitro differentiation of ES cells recapitulates some aspects of embryonic development, including the emergence of the hemangioblast, the common precursor of hematopoietic and endothelial lineages. Thus, hematopoietic differentiation of ES cells constitutes a model for studying effects of gene manipulation on both hematopoiesis and emergence and commitment of the more hematopoietic primitive cell, the hemangioblast, during embryogenesis. In our studies, we used ES cells inactivated for the c-mpl gene, the thrombopoietin receptor, for dissecting the functions of various intracytoplasmic domain of c-mpl in the response of ES cell-derived hematopoietic cells to TPO.

The possible use of embryonic stem cells for therapeutic purposes raises once more the ethical question concerning the position of the embryo in relation to medical needs. Since this technology treats the embryo as a raw material, the debate must incorporate a semantic clarification, in order to identify the conceptual ambiguities that exist between popular thought, science, anthropology and the religious and philosophical convictions of the individual. The problem is knowing whether the end always justifies the means and whether the future may be sacrificed to the present.

Embryo definition is without any ambiguity: it consists in a stage of development able to give rise to an autonomous organism. In this sense, it is obvious that human embryos could be produced by cloning. If the therapeutic prospects of therapeutic cloning are confirmed, there will be a tension between two ethical logics: to respect human embryos as possible persons ... and to improve the condition of severely affected patients. Whatever the definitive solution, its moral significance should be denied. In contrast, it seems possible to use spare embryons for selected research without considering them only as things.