Human T-lymphoid progenitors generated in a feeder-cell-free Delta-like-4 culture system promote T-cell reconstitution in NOD/SCID/γc(-/-) mice.

Slow T-cell reconstitution is a major clinical concern after transplantation of cord blood (CB)-derived hematopoietic stem cells. Adoptive transfer of in vitro-generated T-cell progenitors has emerged as a promising strategy for promoting de novo thymopoiesis and thus accelerating T-cell reconstitution. Here, we describe the development of a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Culture of human CD34(+) CB cells in this new DL-4 system enabled the in vitro generation of large amounts of T-cell progenitor cells that (a) displayed the phenotypic and molecular signatures of early thymic progenitors and (b) had high T lymphopoietic potential. When transferred into NOD/SCID/γc(-/-) (NSG) mice, DL-4 primed T-cell progenitors migrated to the thymus and developed into functional, mature, polyclonal αß T cells that subsequently left the thymus and accelerated T-cell reconstitution. T-cell reconstitution was even faster and more robust when ex vivo-manipulated and nonmanipulated CB samples were simultaneously injected into NSG mice (i.e., a situation reminiscent of the double CB transplant setting). This work provides further evidence of the ability of in vitro-generated human T-cell progenitors to accelerate T-cell reconstitution and also introduces a feeder-cell-free culture technique with the potential for rapid, safe transfer to a clinical setting.

CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors.

BACKGROUND: Tumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies. METHODS: We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas. RESULTS: A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide. CONCLUSIONS: Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.

[Stem cell therapy: an update]. / Cellules souches: état des lieux en thérapie cellulaire.

Medicine will be faced with a major challenge in coming years, namely how to treat for tissue dysfunction due to disease and aging There are two basic options: drug therapy and cell therapy. Stem cells have been the subject of intense speculation and controversy for several years, as they open up radically new therapeutic possibilities. Classical drugs can only smoothen consequences of tissue dysfunction, whereas cell therapy has the potential to restore tissue function by providing fresh cells. Cell therapy is totally different from organ transplantation, which can only benefit a limited number of patients. The use of the generic term "stem cells" to designate a whole variety of cell types that are present throughout life, is a source of confusion and ambiguity. It will take years of cognitive research to unravel the molecular mechanisms that govern a stem cell's multi- or totipotent status before we can fully exploit this therapeutic tool to the full. The younger a stem cell the greater its potential and, probably, the more durable its benefits, but the use of embryonic stem cells raises ethical issues. The redundancy or equivalence of diferent categories of cells is another source of controversy, yet researchers must be able to study stem cells in all their diversity, as complementary rather than competitive alternatives, in an acceptable ethical and regulatory environment. We briefly describe the three types of stem cells: pluripotent embryonic stem cells, fetal and adult stem cells, and pluripotent reprogrammed adult somatic cells. Only the former two categories have physiological functions: the first gives rise to tissues and organs while the second maintains tissue function during adulthood

CD4 regulation in human lymphoid non-T-cells: a role for the silencer element.

In humans, the CD4 molecule is expressed on a subset of T-cells and at various levels on myeloid and lymphoid cells. The mechanisms regulating human CD4 gene expression are yet poorly understood. We speculated that the CD4 silencer, which operates in CD8+ T-cells to repress CD4 expression, could be responsible for CD4 repression in human lymphoid non-T-cells. To test this possibility, we used lentiviral vectors carrying CD4 regulatory sequences, with or without the silencer element, to express an eGFP reporter gene. We observed that (i) in the absence of the silencer element, eGFP expression was detected in CD34+-derived B- and NK-cells that otherwise lacked endogenous CD4 mRNA, indicating active repression of the CD4 regulatory sequences and (ii) the addition of the CD4 silencer could repress eGFP expression in these same cells, as well as in human B-cells generated in vivo in NOD/SCID mice. Collectively, our results suggest that beyond its well-characterized function in T-cells, the CD4 silencer also regulates CD4 gene expression in human lymphoid non-T-cells.

[Human hematopoiesis: from CD34 cells to T lymphocytes]. / Hématopoïèse humaine: des cellules CD34 aux lymphocytes T.

Hematopoietic stem cell (HSC) has two key-properties : the self-renewal and the multipotentiality which guarantee the homeostasis of the hematopoietic system all along the lifespan. Inside this system, T lymphocytes are particular for several reasons. First and foremost, their differentiation takes place in a different organ from the one where the immature progenitors are generated and expanded. This implies the migration of an immature progenitor from the fetal liver and later on from the bone marrow to the thymus. Secondly, T cell differentiation is characterized by thymic selection and generation of T lymphocytes with a diverse repertoire able to answer to all foreign antigens one can meet. These complicated mechanisms underlying the T cell differentiation, completely different from those characterizing the myeloid system, at least partially explain our limited knowledge on human T cell lymphopoiesis. Finally, T cell differentiation pathway shows the particularity of profound ontogenic changes with the huge production of lymphoid progenitors during the fetal and the first years of life which declines during the ageing period. Recently, the discovery of new hematopoietic cytokines, the discovery of genes involved in primary immunodeficiencies and the detailed description of the role of Notch receptors have strongly developed our knowledge on T cell lymphopoiesis. In this review, we will attempt to describe where we stand in the description of this fundamental process and to underline the unresolved questions. The knowledge of this process is crucial, since it will lead us to set up new protocols with the aim to speed up immunological reconstitution after HLA partially compatible HSC and to treat the lymphocytopenia of patients affected by HIV.

Short exposure to Notch ligand Delta-4 is sufficient to induce T-cell differentiation program and to increase the T cell potential of primary human CD34+ cells.

OBJECTIVE: The Notch pathway plays a key role in cell fate choices and in T-cell development. The goal of our study was to evaluate whether a short in vitro stimulation of the Notch pathway may alter human progenitor cell behavior. METHODS: CD34+ cord blood progenitors were exposed for 4 days to either immobilized Notch ligand Delta-4 or in control conditions. Phenotypic and molecular changes induced by the short stimulation were assessed at day 4. Next, long-term alteration of the fate of these progenitors was assessed in culture conditions suitable for B (coculture with MS5 stromal cells) and T (FTOC and OP9 stromal cells expressing Delta-4 systems) cell differentiation. RESULTS: Notch activation was sufficient to trigger immunophenotypic and molecular changes consistent with early T-cell lineage differentiation. Delta-4 induced, in 4 days, CD7+cytCD3epsilon+ cells. This paralleled at the gene-transcription level with de novo expression of several T cell-related transcription factors and TCRgamma rearrangement, while B cell transcripts were simultaneous silenced. As compared to non-Delta-4 primed cells, these early changes translated to long-term alteration of the potential of cells. Delta-4 priming led to an acceleration of T-cell development, including a completion of the TCR rearrangement, when cells were cultured in systems suitable for T-cell development while B-cell development was inhibited. CONCLUSION: A transient Notch activation is sufficient to promote T-cell differentiation from cord blood CD34+ cells. This system may be a useful tool for the amplification and the quantification of the T potential of CD34+ cells in various disease conditions.

Identification of hematopoietic stem/progenitor cells: strength and drawbacks of functional assays.

A major challenge in hematopoiesis is to conceive assays that could bring useful insights into experimental and clinical hematology. This means identifying separately the various classes of hematopoietic progenitors that are produced sequentially during the progression from stem cells to differentiated functional cells. Standardized short-term colony assays easily quantify lineage-committed myeloid precursors, but identification of primitive cells, which have both the ability to repopulate durably myeloid and lymphoid lineages and perhaps to self-renew, still depends on in vivo assays. Whatever the assay, two important requisites have to be solved: one is the definition of appropriate read-outs that will depend solely on the function of these cells, and the second is to evaluate precisely their numbers and proliferative potential in quantitative assays. When evaluating hematopoiesis, three parameters have to be taken into account: (1) the lack of reliable correlation between the phenotype of a given cell and its function. This is especially problematic in post-transplantation situations where cells from transplanted animals are analysed; (2) functionally heterogeneous cells are identified in a single assay; and (3) ontogeny-related changes in hematopoietic cell proliferation and self-renewal that, in human beings, hampers the exploration of adult stem cells. Nevertheless, years of progress in the manipulation of hematopoietic stem cells have recently resulted in the purification of a cell subset that repopulates irradiated recipients with absolute efficiency.