Ming Jiang Xu

In vitro expansion of murine multipotential hematopoietic progenitors from the embryonic aorta-gonad-mesonephros region

The origin of hematopoietic stem cells (HSCs) and their growth factor requirement are poorly understood. Here we describe a new in vitro culture system of the aorta-gonad-mesonephros (AGM) region, where long-term repopulating HSCs first arise. We demonstrate that oncostatin M (OSM) is expressed in the AGM and is absolutely required for the expansion of multipotential hematopoietic progenitors in vitro. In addition, OSM enhances the formation of endothelial cell clusters. Thus, OSM appears to be a key cytokine for the development of multipotential hematopoietic progenitors in the AGM, possibly acting on common precursor cells between HSCs and endothelial cells. By using the AGM culture derived from macrophage colony-stimulating factor (M-CSF)-deficient op/op mutant embryos, we also show a pivotal role for M-CSF in fetal myelopoiesis.

Extensive brain hemorrhage and embryonic lethality in a mouse null mutant of CREB-binding protein.

CREB-binding protein (CBP) is a transcriptional co-activator which is required by many transcription factors. Rubinstein-Taybi syndrome (RTS), which is an autosomal dominant syndrome characterized by abnormal pattern formation, is associated with mutations in the human CBP gene. Various abnormalities occur at high frequency in the skeletal system of heterozygous Cbp-deficient mice, but some features of RTS such as cardiac anomalies do not, suggesting that some symptoms of RTS are caused by a dominant-negative mechanism. Here we report the characterization of homozygous Cbp-deficient mice. Homozygous mutants died around E10.5-E12.5, apparently as a result of massive hemorrhage caused by defective blood vessel formation in the central nervous system, and exhibited apparent developmental retardation as well as delays in both primitive and definitive hematopoiesis. Cbp-deficient embryos exhibited defective neural tube closure which was similar to those observed in twist-deficient embryos. However, a decrease in the level of twist expression was not observed in Cbp-deficient embryos. Anomalous heart formation, a feature of RTS patients and mice mutated in the CBP-related molecule, p300, was not observed in Cbp-deficient embryos. Since both Cbp and p300 are ubiquitously expressed in embryonic tissues including the developing heart, these results suggest that cardiac anomalies observed in RTS patients may be caused by a dominant negative effect of mutant CBP.

Reconstitution of human haematopoiesis in non-obese diabetic/severe combined immunodeficient mice by clonal cells expanded from single CD34+CD38- cells expressing Flk2/Flt3.

Summary. In the present study, we examined the expression of Flk2/Flt3, a tyrosine kinase receptor, on human cord blood CD34+ haematopoietic progenitor/stem cells. In flow cytometric analysis, Flk2/Flt3 was expressed on 80% of CD34+ cells and their immature subpopulations, CD34+CD33– and CD34+CD38– cells. Methycellulose clonal culture of sorted CD34+Flk2/Flt3+ and CD34+Flk2/Flt3– cells showed that most of myelocytic progenitors expressed Flk2/Flt3, but erythroid and haematopoietic multipotential progenitors were shared by both fractions. When 1 × 104 lineage marker‐negative (Lin–)CD34+Flk2/Flt3– cells were transplanted into non‐obese diabetic/severe combined immunodeficient (NOD/SCID) mice, none of the recipients possessed human CD45+ cells in bone marrow 11–12 weeks after the transplantation. In contrast, all recipients transplanted with 1 × 104 Lin–CD34+Flk2/Flt3+ cells showed successful engraftment. Furthermore, clonal cells expanded from single Lin–CD34+CD38–Flk2/Flt3+ cells in the culture with Flk2/Flt3 ligand, stem cell factor, thrombopoietin, and a complex of interleukin 6/soluble interleukin 6 receptor were individually transplanted into NOD/SCID mice. At 20 to 21 weeks after the transplantation, three out of 10 clones harvested at d 7 of culture, and three out of six clones at d 14 could reconstitute human haematopoiesis in recipient marrow. These results demonstrated that Flk2/Flt3 was expressed on a wide variety of human haematopoietic cells including long‐term‐repopulating haematopoietic stem cells.

Exclusive expression of G-CSF receptor on myeloid progenitors in bone marrow CD34+ cells.

Although granulocyte colony‐stimulating factor (G‐CSF) has been reported to act on cells of neutrophilic lineage, the administration of G‐CSF to induce the mobilization of various haematopoietic progenitors into the circulation. We analysed the expression of receptors for G‐CSF (G‐CSFR) on human bone marrow and G‐CSF‐mobilized peripheral blood CD34+ cells, and examined the proliferation and differentiation capabilities of sorted CD34+G‐CSFR+ and CD34+G‐CSFR− cells using methylcellulose clonal culture. Flow cytometric analysis showed that G‐CSFR was expressed on 14.9 ± 4.9% of bone marrow CD34+ cells, most of which were included in CD34+CD33+ and CD34+CD38+ cell fractions. In clonal cultures, CD34+G‐CSFR+ cells produced only myeloid colonies, whereas CD34+G‐CSFR− cells produced erythroid bursts, megakaryocyte and multilineage colonies. When incubated with the cytokine cocktail for 5 d, CD34+G‐CSFR− cells generated CD34+G‐CSFR+ myeloid progenitors. In G‐CSF‐mobilized peripheral blood, CD34+ cells contained 10.8 ± 5.8% of G‐CSFR+ cells, most of which were also myeloid progenitors, although CD34+G‐CSFR− cells contained a substantial number of myeloid progenitors. These results indicated that the expression of G‐CSFR on CD34+ cells is restricted to myeloid progenitors, suggesting that the specific activity of G‐CSF on myelopoiesis depends on the exclusive expression of its receptor on myeloid progenitors, and that the mobilization of various haematopoietic progenitors is not a direct effect of G‐CSF in humans.

Analysis of signals and functions of the chimeric human granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells and transgenic mice.

Receptors for GM-CSF, IL-3, and IL-5 are composed of two subunits: α, which is specific for each cytokine, and βc, which is shared by all. Although the role of βc in signal transduction has been extensively studied, the role of the α subunit has remained to be clarified. To analyze the role of the human (h) GM-CSF receptor α subunit, we constructed a chimeric receptor subunit composed of extracellular and transmembrane regions of α fused with the cytoplasmic region of βc, designated α/β. In BA/F3 cells, chimeric receptor composed of α/β,β can transduce signals for mitogen-activated protein kinase cascade activation and proliferation in response to hGM-CSF. Although phosphorylation of Jak1 but not of Jak2 occurred with stimulation of hGM-CSF, the dominant-negative Jak2 but not the dominant-negative Jak1 suppresses c-fos promoter activation. To determine whether the chimeric receptor α/β,β is functional in vivo, we developed transgenic mice expressing the chimeric receptor α/β,β. Bone marrow cells from the transgenic mice expressing the α/β,β receptor form not only GM colonies but also various lineages of colonies in response to GM-CSF. In addition, mast cells were produced when bone marrow cells of the transgenic mouse were cultured with hGM-CSF. Thus, it appears that the cytoplasmic region of the α subunit is not required for hGM-CSF promoting activities, even in bone marrow cells.