Background PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na+-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.Methodology/Principal FindingsTo determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and null PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and null alleles in heterozygous compounds results in late embryonic lethality (E14.5–E16.5) with phenotypic features intermediate between null and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation.Conclusion/SignificanceThis work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.

Hematopoiesis is established from circulating blood stem cells that seed the embryonic rudiments of blood-forming tissues, a basic notion in developmental hematology. However, the assumption that these stem cells originate from the extraembryonic mesoderm, where primitive hematopoiesis is initiated by intrinsic precursors, has been reconsidered after analysis of blood cell development in avian embryo chimeras: yolk-sac-derived stem cells do not contribute significantly to the definitive blood system, whose first forerunners develop independently along the ventral aspect of the embryonic aorta. Recently, the homologous intraembryonic tissues of the mouse have been submitted to sensitive in vivo and in vitro assays, which showed that they also harbor multipotential hematopoietic stem cells. We have now identified a dense population of hematogenous cells, marked by the surface expression of the CD34 glycoprotein, associated with the ventral endothelium of the aorta in the 5-week human embryo. Therefore, we extend to the human species the growing evidence that intraembryonic hematopoietic cells developing independently of the yolk sac might be the real stem of the whole blood system.

The recent development of lentivirus-derived vectors is an important breakthrough in gene transfer technology because these vectors allow transduction of nondividing cells such as hematopoietic stem cells (HSC), due to an active nuclear import of reverse-transcribed vector DNA. We recently demonstrated that addition of the central DNA flap of HIV-1 to an HIV-derived lentiviral vector strikingly increases transduction of CD34(+) cells. We now describe improvements of the transduction protocol designed to preserve HSC properties and two modifications of the previously described TRIP-CMV vector. First, deletion of the enhancer/promoter of the 3' LTR in the TRIP-CMV vector resulted in a safer vector (TRIPDeltaU3-CMV) with conserved transduction efficiency and increased EGFP transgene expression. Second, the original internal CMV promoter was replaced with the promoter for the ubiquitously expressed elongation factor 1alpha (EF1alpha). This promoter substitution resulted in a significantly more homogeneous expression of the EGFP transgene in all hematopoietic cell types, including CD34(+)-derived T lymphocytes, in which the CMV promoter was inactive, and NOD/SCID mouse repopulating cells. We thus present here an HIV-derived lentiviral vector, TRIPDeltaU3-EF1alpha, which can very efficiently transduce human cord blood HSC and results in high long-term transgene expression in CD34(+)-derived T, B, NK, and myeloid hematopoietic cells.

BackgroundTumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies.MethodsWe 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.ResultsA 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.ConclusionsOur 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.

We found that when marrow cells from four patients with newly diagnosed Philadelphia chromosome-positive chronic myelogenous leukemia were maintained in culture for two to four weeks, a previously undetectable population of chromosomally normal hematopoietic cells (including erythroid, granulopoietic, and pluripotent progenitors) became readily demonstrable in three cases. Time-course studies showed that in such cultures the dominant Philadelphia chromosome-positive population rapidly disappeared, in contrast to coexisting chromosomally normal progenitors, which remained detectable for periods of two to three months. Long-term marrow cultures thus offer a new approach to the assessment of a suppressed but functionally intact population of chromosomally normal hematopoietic stem cells in patients with chronic myelogenous leukemia.

The aim of the present study was to determine if the human erythroid (E) and megakaryocytic (MK) lineages were closely linked to the existence of a bipotent burst-forming unit (BFU) E/MK progenitor. In methylcellulose cultures, BFU-E/MK colonies were observed at day 12 and closely resembled mature BFU-E with the exception that the erythroid component was surrounded by MK. These colonies were quite different from the colony forming unit (CFU)-GEMM-derived colonies, which were composed of a larger number of erythroblasts and which developed later in culture. The existence of these bilineage colonies composed of 100 to 1,000 erythroblasts intermingled with a few MK and without granulocytic cells was confirmed by the plasma clot technique and immunoalkaline phosphatase labeling of the MK. To investigate if this bipotent progenitor belonged to the compartment of primitive progenitors, CD34+ marrow cells were subfractionated according to expression of the CD38 antigen. The bipotent BFU-E/MK progenitor as well as a large fraction of MK progenitors were found in the CD34+ CD38+/- or in the CD34+ CD38- cell fractions. Growth of this bipotent BFU-E/MK progenitor required the combination of stem cell factor (SCF), Interleukin-3 (IL-3), and Epo in serum free conditions. Addition of IL- 6 had only a marginal effect, whereas megakaryocyte growth and development factor (MGDF) was not an absolute requirement, but slightly increased the plating efficiency of CFU-MK and of BFU-E/MK progenitors when combined with SCF, IL-3, and Epo. In contrast, when these cultures were performed in the presence of 30% fetal calf serum, no BFU-E/MK colonies were observed irrespective of the combination of growth factors used, including the presence of MGDF; however, inclusion of the MS-5 cell line restored the growth of this bipotent progenitor. In contrast, in cultures performed in the presence of human normal or aplastic plasma, MS-5 had only a slight effect on the cloning efficiency but improved MK cytoplasmic maturation and MK size, suggesting that the main effect of MS-5 is to diminish the inhibitory effect of the fetal calf serum on the MK differentiation. The clonal origin of bipotent BFU-E/MK colonies was demonstrated in liquid culture of single CD34+ CD38low cells by immunophenotyping individual clones. At day 12, 30% of the clones contained erythroblasts (glycophorin A+) and some MK (CD41+) without granulocytes (G) or macrophages (M) (CD14+ and CD15+). At day 20, clones containing erythroblasts and MK were rare (5%). In contrast multilineage clones could be frequently detected at this time without passage from BFU-E/MK clones at day 12 to GEMM at day 20. These results suggest that a bipotent BFU-E/MK progenitor may be a nonrandom step in the hierarchical development of stem cells.

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In the present study, we investigated the differentiation of human NK cells from bone marrow, cord blood and mobilized peripheral blood purified CD34+ stem cells using a potent culture system. Elutriated CD34+ stem cells were grown for several weeks in medium supplemented with stem cell factor (SCF) and IL-15 in the presence or absence of a murine stromal cell line (MS-5). Our data indicate that IL-15 induced the proliferation and maturation of highly positive CD56+ NK cells in both types of culture, although murine stromal cells slightly increased the proliferation of NK cells. NK cells differentiated in the presence of MS-5 were mostly CD56+ CD7 and a small subset expressed CD16. These in vitro differentiated CD56+ NK cells displayed cytolytic activity against the HLA class I- target K562. The CD56+ CD16+ subset also lysed NK-resistant Daudi cells. Neither of these NK subsets were shown to express Fas ligand. Total CD56+ cells expressed high amounts of transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor, but no IFN-gamma. Investigation of NK receptor expression showed that most CD56+ cells expressed membrane CD94 and NKG2-A mRNA. PCR analysis revealed that p58 was also expressed in these cells. The role of CD94 in NK cell-mediated cytotoxicity was assessed on human HLA-B7-transfected murine L cells. While a low cytotoxic activity towards HLA-B7 cells was observed, the HLA-DR4 control cells were killed with high efficiency. These studies demonstrate that cytolytic and cytokine-producing NK cells may be derived from adult and fetal precursors by IL-15 and that these cells express a CD94 receptor which may influence their lytic potential.