The human poliovirus receptor related 2 protein is a new hematopoietic/endothelial homophilic adhesion molecule

We have recently described Poliovirus Receptor Related 2 (PRR2), a new cell surface molecule homologous to the poliovirus receptor (PVR/CD155). Both molecules are transmembrane glycoproteins belonging to the Ig superfamily (IgSF). They contain 3 Ig domains of V, C2, and C2 types in their extracellular regions that share 51% aa identity. The PRR2 gene encodes two mRNA isoforms of 3.0 kb (hPRR2 [short form]) and 4.4 kb (hPRR2delta [long form]), both widely expressed in human tissues, including hematopoietic cells. To further characterize PRR2 expression during hematopoiesis and to analyze its function, we have developed a monoclonal antibody (MoAb) directed against its extracellular region (R2.477). PRR2 was expressed in 96% of the CD34(+), 88% of the CD33(+), and 95% of the CD14(+) hematopoietic lineages and faintly in the CD41 compartment. Ectopic expression of both PRR2 cDNAs induced marked cell aggregation. A soluble chimeric receptor construct with the Fc fragment of human IgG1 (PRR2-Fc) as well as a fab fragment of the anti-PRR2 MoAb (R2.477) inhibit aggregation. PRR2-Fc binds specifically to PRR2-expressing cells. These results suggest that PRR2 is a homophilic adhesion receptor. PRR2 was also expressed at the surface of endothelial cells at the intercellular junctions of adjacent cells but not at the free cellular edges. Homophilic interactions are associated with dimerization of isoforms of PRR2 and lead to the tyrosine phosphorylation of PRR2delta. Altogether, these results suggest that homophilic properties of PRR2 could participate to the regulation of hematopoietic/endothelial cell functions.     

Expressions of PDGF receptor alpha, c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells

In gastrulating embryos, various types of cells are generated before differentiation into specific lineages. The mesoderm of the gastrulating mouse embryo represents a group of such intermediate cells. PDGF receptor alpha (PDGFRalpha), c-Kit and fetal liver kinase 1 (Flk1) are expressed in distinctive mesodermal derivatives of post-gastrulation embryos. Their expressions during gastrulation were examined by whole mount immunostaining with monoclonal antibodies against these three receptors. The antibodies stained different mesodermal subsets in gastrulating embryos. Flow cytometry of head fold stage embryos revealed that Flk1+ mesodermal cells could be further classified by the level of c-Kit expression. To examine the possibility that hematopoietic cell differentiation is initiated from the Flk1+ mesoderm, embryonic stem (ES) cells were cultured on the OP9 or PA6 stromal cell layer; the former but not the latter supported in vitro hematopoiesis from ES cells. Flk1+ cells were detected only on the OP9 cell layer from day 3 of differentiation before the appearance of hematopoietic cells. Thus, Flk1+ cells will be required for in vitro ES cell differentiation into hematopoietic cells. The results suggest that these three receptor tyrosine kinases will be useful for defining and sorting subsets of mesodermal cells from embryos or in vitro cultured ES cells.     

Molecular identity of hematopoietic precursor cells emerging in the human embryo

It is now accepted from studies in animal models that hematopoietic stem cells emerge in the para-aortic mesoderm-derived aorta-gonad-mesonephros region of the vertebrate embryo. We have previously identified the equivalent primitive hematogenous territory in the 4- to 6-week human embryo, under the form of CD34(+)CD45(+)Lin- high proliferative potential hematopoietic cells clustered on the ventral endothelium of the aorta. To characterize molecules involved in initial stem cell emergence, we first investigated the expression in that territory of known early hematopoietic regulators. We herein show that aorta-associated CD34(+) cells coexpress the tal-1/SCL, c-myb, GATA-2, GATA-3, c-kit, and flk-1/KDR genes, as do embryonic and fetal hematopoietic progenitors later present in the liver and bone marrow. Next, CD34(+)CD45(+) aorta-associated cells were sorted by flow cytometry from a 5-week embryo and a cDNA library was constructed therefrom. Differential screening of that library with total cDNA probes obtained from CD34(+) embryonic liver cells allowed the isolation of a kinase-related sequence previously identified in KG-1 cells. In addition to emerging blood stem cells, KG-1 kinase is also strikingly expressed in all developing endothelial cells in the yolk sac and embryo, which suggests its involvement in the genesis of both hematopoietic and vascular cell lineages in humans.     

Embryonic stem cells and hematopoietic stem cell biology

Two advances in murine embryonic stem (ES) cell technology and their applications for the study of hematopoietic stem cells (HSCs) are discussed in this article. First, ES cells induced to differentiate in vitro form hematopoietic lineages in a fashion that recapitulates the ontogeny of blood formation in the embryo. This system offers a unique opportunity to isolate, examine, and manipulate the most primitive hematopoietic progenitors. Second, targeted gene ablation (knockout) studies in ES cells have identified several genes that are required for normal hematopoiesis and may function in the formation, maintenance, and differentiation of HSCs. Insights into murine hematopoiesis gained through the study of ES cells generally should be applicable to other vertebrates, including humans.     

Hematopoietic induction and respecification of A-P identity by visceral endoderm signaling in the mouse embryo

The anteroposterior axis of the developing embryo becomes morphologically apparent at the onset of gastrulation with the formation of the primitive streak. This structure, where the first mesodermal cells arise, marks the posterior aspect of the embryo. To examine the potential role of non-mesodermal signals in specifying posterior (hematopoietic and endothelial) cell fates in the mouse embryo, we have devised a transgenic explant culture system. We show that interactions between primitive endoderm and adjacent embryonic ectoderm or nascent mesoderm are required early in gastrulation for initiation of hematopoiesis and vasculogenesis. Surprisingly, primitive endoderm signals can respecify anterior (prospective neural) ectoderm to a posterior mesodermal fate, resulting in formation of blood and activation of endothelial markers. Reprogramming of anterior ectoderm does not require cell contact and is effected by stage-dependent, short-range, diffusible signal(s). Therefore, primitive endoderm signaling is a critical early determinant of hematopoietic and vascular development and plays a decisive role in anterior-posterior patterning during mouse embryogenesis.     

Cellular expression of a GPI-linked T cell activation protein

A T cell activation protein was identified by generating a monoclonal antibody (anti-D2) against a gamma delta T cell receptor bearing gibbon ape T cell line (MLA144). Immunoprecipitation studies revealed three polypeptides of 180, 150, and 120 kDa. The antigen was also found to be expressed on endothelial cells in vivo and in vitro and on tumor cell lines from a variety of tissues. Studies performed using a variety of antibodies reveal this protein to be identical to an endothelial cell protein previously identified by several antibodies to T cell activation proteins (CDw109). We demonstrate that this protein is anchored in the membrane via a glycosylphosphatidylinositol (GPI) tail in T cells, tumor cells, and endothelial cells. An analysis of tissue sections reveals this protein to be normally highly expressed on vascular endothelial cells.     

Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice

Receptor tyrosine kinases Flt-1 and Flk-1/KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1/KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice (flt-1(TK-/-)) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in flt-1(TK-/-) mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.     

Activation of stress-activated protein kinases by hepatocyte isolation induces gene 33 expression

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis by acting as a potent inducer of vascular permeability as well as serving as a specific endothelial cell mitogen. The importance of angiogenic factors such as VEGF, although clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. We examined the expression of mRNA and protein for VEGF in 12 human hematopoietic tumor cell lines, representing multiple lineages and diseases, including leukemia, lymphoma, and multiple myeloma. Our results revealed that VEGF message was expressed in these cells and that the corresponding protein was secreted into the extracellular environment. Five of the 12 cell lines were also found to express the Flt-1 receptor for VEGF at a moderate to strong level, suggesting an autocrine pathway. When human vascular endothelial cells were exposed to recombinant human VEGF, there was an increase in the mRNA for several hematopoietic growth factors including macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 6. Plasma cells in the bone marrow from patients diagnosed with multiple myeloma were found to express VEGF, whereas both the Flt-1 and KDR high affinity VEGF receptors were observed to be markedly elevated in the normal bone marrow myeloid and monocytic cells surrounding the tumor. These data raise the possibility that VEGF may play a role in the growth of hematopoietic neoplasms such as multiple myeloma through either a paracrine or an autocrine mechanism.     

Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo

Hepatic hematopoiesis in the mouse embryo is preceded by two hematopoietic waves, one in the yolk sac, and the other in the paraaortic splanchnopleura, the presumptive aorta-gonad-mesonephros region that gives rise to prenatal and postnatal blood stem cells. An homologous intraembryonic site of stem cell emergence was previously identified at 5 weeks of human gestation, when hundreds of CD34(++ )Lin- high-proliferative potential hematopoietic cells border the aortic endothelium in the preumbilical region. In the present study, we have combined immunohistochemistry, semithin section histology, fluorescence-activated cell sorting and blood cell culture in an integrated study of incipient hematopoiesis in the human yolk sac, truncal arteries and embryonic liver from 21 to 58 days of development. The chronology of blood precursor cell emergence in these distinct tissues suggests a pivotal role in the settlement of liver hematopoiesis of endothelium-associated stem cell clusters, which emerge not only in the dorsal aorta but also in the vitelline artery. Anatomic features and in vitro functionality indicate that stem cells develop intrinsically to embryonic artery walls from a presumptive territory whose blood-forming potential exists from at least 24 days of gestation.     

Correlation of functional independence measure (FIM) and SPECT Iofetamine (I-123) as a predictor of functional return in stroke

Murine embryonic stem (ES) cells have been a useful model system for the study of various aspects of hematopoietic differentiation. Because we had observed a sharp peak of expression of the receptor tyrosine phosphatase gamma (Ptp gamma) gene between 14 and 18 days of ES-derived embryoid body differentiation, we investigated the effect of perturbation of expression of the Ptp gamma gene on ES cell differentiation, first by analyzing the effect of Ptp gamma overexpression. The murine full-length Ptp gamma cDNA in an expression vector was transfected into ES-D3 cells and stably transfected clones were isolated. Ptp gamma was expressed as an approximately 230-kD cell surface protein, and differentiating ES clones that overexpressed Ptp gamma gave rise to a normal number of hematopoietic colonies, approximately 1 CFU per 100 cells. There was, however, a significant increase of expression of early hematopoietic markers in colonies from Ptp gamma overexpressing ES cells. To confirm that the pertubation of hematopoietic differentiation was a result of Ptp gamma overexpression, we isolated ES stem cell clones expressing Ptp gamma antisense constructs and assayed embryoid bodies for the presence of hematopoietic precursors. We observed a complete absence of methylcellulose colonies, indicating absence of hematopoietic lineages. Results of these experiments point to an essential role for Ptp gamma in hematopoietic differentiation.     

Development of osteoclasts from embryonic stem cells through a pathway that is c-fms but not c-kit dependent

Osteoclasts are hematopoietic cells essential for bone resorption. To study the derivation of these interesting cells, we developed a stepwise culture system where stromal cells promote embryonic stem (ES) cells to differentiate into mature osteoclasts. Three phases to this differentiation process include (1) induction of hematopoiesis, along with the generation of osteoclast precursors, (2) expansion of these precursors, and (3) terminal differentiation into mature osteoclasts in the presence of 1alpha,25-dihydroxyvitamine D3 . Although the transition of ES cells to the hematopoietic lineage was not blocked by an antibody to c-fms, later phases were dependent on a signaling through this transmembrane receptor as indicated by the finding that anti-c-fms treatment of cells in the second and third phases reduced the number of osteoclasts produced by 75% and more than 99%, respectively. Blockade of signaling through another tyrosine kinase-type receptor, c-kit, did not affect any stages of osteoclastogenesis, although generation of other hemopoietic lineages was reduced to less than 10% of untreated. When small numbers of ES cells were directly cultured under conditions that promote osteoclast differentiation, tartrate-resistant acid phosphatase-positive multinucleated cells were observed at the edge but not inside of colonies. This suggests that some types of cell-cell interactions may inhibit development of mature osteoclasts. The culture system developed here provides an important tool for osteoclast biology.     

Expression of an L-selectin ligand on hematopoietic progenitor cells

The process of hematopoiesis is dependent on discrete cell-cell and cell-matrix interactions which are tightly regulated by expression of adhesion molecules. L-selectin, an adhesion protein best known for regulating leukocyte attachment to endothelium, is characteristically expressed on the earliest hematopoietic progenitor cells. Ligands for L-selectin have been extensively characterized on endothelial cells. We recently identified a ligand for L-selectin expressed on the human hematopoietic progenitor cell line KG1a. This molecule is an integral membrane glycoprotein which is structurally different from all ligands previously described. We hypothesize that this molecule may mediate L-selectin-specific adhesive interactions during hematopoiesis. This article discusses the biology of L-selectin and its ligands, and reviews our current understanding of the structure and distribution of the L-selectin ligand expressed on hematopoietic cells.     

Ligand-dependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2

The existence of a common precursor for endothelial and hemopoietic cells, termed the hemangioblast, has been postulated since the beginning of the century. Recently, deletion of the endothelial-specific vascular endothelial growth factor receptor 2 (VEGFR2) by gene targeting has shown that both endothelial and hemopoietic cells are absent in homozygous null mice. This observation suggested that VEGFR2 could be expressed by the hemangioblast and essential for its further differentiation along both lineages. However, it was not possible to exclude the hypothesis that hemopoietic failure was a secondary effect resulting from the absence of an endothelial cell microenvironment. To distinguish between these two hypotheses, we have produced a mAb directed against the extracellular domain of avian VEGFR2 and isolated VEGFR2+ cells from the mesoderm of chicken embryos at the gastrulation stage. We have found that in clonal cultures, a VEGFR2+ cell gives rise to either a hemopoietic or an endothelial cell colony. The developmental decision appears to be regulated by the binding of two different VEGFR2 ligands. Thus, endothelial differentiation requires VEGF, whereas hemopoietic differentiation occurs in the absence of VEGF and is significantly reduced by soluble VEGFR2, showing that this process could be mediated by a second, yet unidentified, VEGFR2 ligand. These observations thus suggest strongly that in the absence of the VEGFR2 gene product, the precursors of both hemopoietic and vascular endothelial lineages cannot survive. These cells therefore might be the initial targets of the VEGFR2 null mutation.