Identification of four human cDNAs that are differentially expressed by early hematopoietic progenitors

The molecular processes that maintain the stem cell pool are largely unknown. Using polymerase chain reaction-driven subtraction, we examined genes that are differentially expressed by early hematopoietic progenitors. We expected that identifying genes that are uniquely expressed by the earliest precursors would provide insight into the mechanism(s) through which stem cell number is maintained and differentiation is regulated.Using CD34(+)CD38(-) cells as starting material, we identified four mRNAs, expressed by these cells, that are either absent or present in reduced amounts in more mature CD34(+)CD38(+) cells. One of these cDNAs (C40) encodes a known member of the subfamily of protein phosphatases (CL100) that exhibits dual substrate specificity for phosphotyrosine- and phosphoserine/threonine-containing substrates and specifically inactivates MAP kinases. This phosphatase has been shown to play a role in regulating the differentiation of several cell types. The second cDNA (C23) is identical to LR11 (gp250), a member of the low-density lipoprotein receptor family. LR11 is unusual in that, in addition to 11 ligand-binding repeats, it contains a series of fibronectin type III repeats near its carboxyl terminal end that are similar to those found in cytokine receptors. It is highly expressed in developing brain, but hematopoietic expression has not been reported. The 178-bp fragment that we originally cloned is part of a 4,145-bp 3' untranslated region (UTR) that had not been previously sequenced and is among the largest human 3' UTRs ever reported. The other isolates (C21 and C12) do not correspond to known protein sequences. They are homologous to EST sequences from a fetal brain library. C21 encodes a previously unknown gene that is a member of the WD-40 family. An open reading frame encoding a 515 amino acid protein has been identified.Four mRNAs, differentially expressed by CD34(+)CD38(-) human bone marrow cells, have been identified. Although this population is highly enriched for early hematopoietic progenitors, none of these genes encodes a message whose expression is limited to the hematopoietic system. They all are expressed in a variety of tissues, suggesting that they are involved in processes that are fundamental to the development of many cell types. All of these cDNAs possess atypically long 3' UTRs, and one of them is among the longest ever described. Their differential expression by immature hematopoietic cells, in contrast to more mature cells, suggest that long 3' UTRs may be characteristic of genes that play a regulatory role during development.     

CD41 is developmentally regulated and differentially expressed on mouse hematopoietic stem cells

CD41 expression is associated with the earliest stages of mouse hematopoiesis. It is notably expressed on some cells of the intra-aortic hematopoietic clusters, an area where the first adult-repopulating hematopoietic stem cells (HSCs) are generated. Although it is generally accepted that CD41 expression marks the onset of primitive/definitive hematopoiesis, there are few published data concerning its expression on HSCs. It is as yet uncertain whether HSCs express CD41 throughout development, and if so, to what level. We performed a complete in vivo transplantation analysis with yolk sac, aorta, placenta, and fetal liver cells, sorted based on CD41 expression level. Our data show that the earliest emerging HSCs in the aorta express CD41 in a time-dependent manner. In contrast, placenta and liver HSCs are CD41?. Thus, differential and temporal expression of CD41 by HSCs in the distinct hematopoietic territories suggests a developmental/dynamic regulation of this marker throughout development.     

Ageing-exaggerated proliferation of vascular smooth muscle cells is related to attenuation of Jagged1 expression in endothelial cells

AIMS: Ageing has been shown to enhance neointima formation due to abnormal growth of vascular smooth muscular cells (VSMC), which is regulated by endothelial functions. The mechanism of how endothelium affects the growth of VSMC in the process remains unclear. We here examined the role of Jagged1, a regulator of cell growth. METHODS AND RESULTS: Male Sprague-Dawley rats at 3 (young) and 22 (old) months of age were subjected to a balloon catheter injury in the thoracic aorta. After 4 weeks, the neointima formation in the injured artery of old rats was more than that of young rats. Compared with the young rats, the increase in Jagged1 expression in the endothelium of old rats after the injury was delayed, weakened, and shortened, suggesting an impaired response of Jagged1 to the injury. In contrast, the increase in the expression of proliferating cell nuclear antigen in the neointima was more significant and maintained longer in old rats than in the young ones. Moreover, the expression of Jagged1 in the cultured arterial endothelial cells (EC) of old animals was less than those of the young ones, which promoted the Platelet-derived growth factor (PDGF)-induced growth and migration of the co-cultured VSMC. Furthermore, suppression of Jagged1 expression by a small interfering RNA in the EC of young rats reduced alpha-smooth muscle actin and calponin expression and also intensified the PDGF-increased growth and migration of the co-cultured VSMC. CONCLUSION: Ageing enhanced VSMC proliferation, at least in part, through impairing Jagged1 expression in the EC after vascular injury.     

Hydrocortisone promotes survival and proliferation of granulocyte-macrophage progenitors via monocytes/macrophages

We have determined the mechanism by which hydrocortisone (Hc) promotes the survival and proliferation of normal human granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM). Peripheral blood mononuclear cells were cultured in a liquid system with 0-10.0 microM Hc over 3 weeks. At 7-day intervals 50% of the culture media along with the cells (suspension cells) present in the media were removed and replaced with fresh media. No CFU-GM or very small numbers of CFU-GM were contained in the suspension cells of the 14- and 21-day-old liquid cultures without Hc; CFU-GM were present and increased with increasing concentrations of Hc. The CFU-GM content in suspension cells of 14- and 21-day-old liquid cultures with 1.0 microM Hc was at least threefold higher compared to liquid cultures without Hc. In a double-layer CFU-GM agar culture system, the suspension cells from liquid cultures with 1.0 microM Hc, but not from liquid cultures without Hc, supported CFU-GM proliferation from normal human bone marrow cells. The CFU-GM proliferation-inducing ability was confined to the monocytes/macrophages (Mo). CFU-GM colony inhibitory and stimulatory activities were detected in cell-free media recovered from liquid cultures without Hc, but only colony stimulatory activity was detected in the media from cultures with 1.0 microM Hc. These results indicate that greater than or equal to physiological concentrations of Hc (0.1-1.0 microM) are required for the persistence and proliferation of CFU-GM, and the effect of Hc is mediated through the Mo, probably by inhibiting the production of one or more of the CFU-GM colony inhibitory molecules.     

PECAM-1 is expressed on hematopoietic stem cells throughout ontogeny and identifies a population of erythroid progenitors

Platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31) is an adhesion molecule expressed on endothelial cells and subsets of leukocytes. Analysis of phenotypically defined hematopoietic stem cells (HSCs) from the yolk sac, fetal liver, and adult bone marrow demonstrates CD31 expression on these cells throughout development. CD31+ c-kit+ cells, but not CD31- c-kit+ cells, isolated from day-9.5 yolk sac give rise to multilineage hematopoiesis in vivo. Further evaluation of the CD31+ lineage marker-negative fraction of adult bone marrow reveals functionally distinct cell subsets. Transplantation of CD31+ Lin- c-kit- cells fails to protect lethally irradiated recipients, while CD31+ Lin- c-kit+ Sca-1- cells (CD31+ Sca-1-) provide radioprotection in the absence of long-term donor-derived hematopoiesis. Although donor-derived leukocytes were not detected in CD31+ Sca-1- recipients, donor-derived erythroid cells were transiently produced during the initial phases of bone marrow recovery. These results demonstrate CD31 expression on hematopoietic stem cells throughout ontogeny and identify a population of CD31+ short-term erythroid progenitors cells that confer protection from lethal doses of radiation.     

Hepatocyte growth factor is a survival factor for endothelial cells and is expressed in human atherosclerotic plaques

While the hypocholesterolemic effects of taurine have extensively been studied using experimental animals, the anti-atherosclerotic effects of taurine have been given less attention. We examined the effect of taurine on atherosclerotic lesions in Watanabe heritable hyperlipidemic (WHHL) rabbits. Treatment of WHHL rabbits with taurine (0.3% in drinking tap water) for 24 weeks decreased aortic lesions by 31%, estimated as intimal thickening. Taurine significantly decreased cholesteryl ester content of aortic arch, thoracic aorta, and abdominal aorta by 35, 43, and 54%, respectively. Concomitantly, activity of acyl-CoA:cholesterol acyltransferase (ACAT), an enzyme responsible for cholesterol esterification, was also significantly decreased. Immunohistochemical analysis revealed decreased macrophages in the intima of taurine-treated rabbits. Taurine had no apparent effect on blood pressure and serum cholesterol levels. Contents of thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, was reduced in serum and aorta by 29 and 50%, respectively, when taurine was ingested. In addition, LDL from taurine-treated rabbits was resistant to copper-induced oxidative modification. These results revealed that taurine prevents development of atherosclerosis and that the anti-atherosclerotic effects of taurine are independent of serum cholesterol levels. The anti-oxidant action of taurine may be involved in inhibiting atherosclerosis in these rabbits.     

Pleiotrophin is highly expressed by myeloma cells and promotes myeloma tumor growth

Pleiotrophin (PTN) is an important developmental cytokine that is highly expressed during embryogenesis but shows very limited expression in adult tissues, where it is largely restricted to the brain. High PTN serum levels are associated with a variety of solid tumors. We recently showed that patients with multiple myeloma (MM) also have elevated serum levels of this protein and the amount of PTN correlated with the patients' disease status and response to treatment. In this study, we demonstrate that MM cell lines and the malignant cells from MM patients' bone marrow produced PTN and secreted PTN protein into the supernatants during short-term culture. Moreover, Ptn gene expression correlated with the patients' disease status. Inhibition of PTN with a polyclonal anti-PTN antibody reduced growth and enhanced apoptosis of MM cell lines and freshly isolated bone marrow tumor cells from MM patients in vitro. Importantly, this antibody also markedly suppressed the growth of MM in vivo using a severe combined immunodeficiency (SCID)-hu murine model. This represents the first study showing the importance of PTN in the growth of any hematological disorder. Because the expression of this protein is very limited in normal adult tissues, PTN may represent a new target for the treatment of MM.     

Penumbra encodes a novel tetraspanin that is highly expressed in erythroid progenitors and promotes effective erythropoiesis

In a search for new genes involved in the regulation of erythropoiesis, we identified murine Penumbra cDNA from a multipotent hematopoietic cell line based on its predominant expression in erythroblasts. Subsequently, we identified the human PENUMBRA from a bone marrow cDNA library. Penumbra is a new member of the tetraspanin superfamily of membrane proteins, many of which are thought to function as organizers of supramolecular signaling complexes. Human and murine Penumbras contain 283 amino acids and are 97% identical. The human PENUMBRA gene is mapped to chromosome 7q32, a hot spot for deletions in myelodysplastic syndromes and acute myelogenous leukemias. Penumbra is targeted to the cell surface and forms disulfide-bonded homodimers. To study the effects of Penumbra deletions, we created a knockout mouse model by gene targeting. Penumbra(-/-) mice develop massive splenomegaly, basophilic macrocytic red blood cells, and anemia as they age. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra(-/-) mouse. EMX exhibits ineffective erythropoiesis in the presence of erythropoietin, a defect that is reversed by reexpression of Penumbra. These findings indicate that Penumbra has a positive function in erythropoiesis and its deletion or mutation may result in anemia.     

Contribution of endothelial progenitors and proangiogenic hematopoietic cells to vascularization of tumor and ischemic tissue

PURPOSE OF REVIEW: During the last several years, a substantial amount of evidence from animal as well as human studies has advanced our knowledge of how bone marrow derived cells contribute to neoangiogenesis. In the light of recent findings, we may have to redefine our thinking of endothelial cells as well as of perivascular mural cells. RECENT FINDINGS: Inflammatory hematopoietic cells, such as macrophages, have been shown to promote neoangiogenesis during tumor growth and wound healing. Dendritic cells, B lymphocytes, monocytes, and other immune cells have also been found to be recruited to neoangiogenic niches and to support neovessel formation. These findings have led to the concept that subsets of hematopoietic cells comprise proangiogenic cells that drive adult revascularization processes. While evidence of the importance of endothelial progenitor cells in adult vasculogenesis increased further, the role of these comobilized hematopoietic cells has been intensely studied in the last few years. SUMMARY: Angiogenic factors promote mobilization of vascular endothelial growth factor receptor 1-positive hematopoietic cells through matrix metalloproteinase-9 mediated release of soluble kit-ligand and recruit these proangiogenic cells to areas of hypoxia, where perivascular mural cells present stromal-derived factor 1 (CXCL-12) as an important retention signal. The same factors are possibly involved in mobilization of vascular endothelial growth factor receptor 2-positive endothelial precursors that may participate in neovessel formation. The complete characterization of mechanisms, mediators and signaling pathways involved in these processes will provide novel targets for both anti and proangiogenic therapeutic strategies.     

Transdifferentiation of mesenchymal stem cells into cardiomyocytes by direct cell-to-cell contact with neonatal cardiomyocyte but not adult cardiomyocytes

Recent studies have demonstrated that direct cell-to-cell interaction is one of the microenvironment factors for transdifferentiation of adult stem cells into cardiomyocytes. We investigated whether transdifferentiation of mesenchymal stem cells (MSCs) into cardiomyocytes was dependent on developmental stages of cocultured cardiomyocytes, and direct cell-to-cell interaction was essential for transdifferentiation. MSCs were isolated from adult rat and cocultured in four different ways: (1) with neonatal cardiomyocytes, (2) with adult cardiomyocytes, (3) with neonatal cardiomyocytes on the cell culture inserts, and (4) with the conditioned medium from neonatal cardiomyocytes. After 5 days of coculture with neonatal cardiomyocytes, 9.40±1.15% of 1,1-dioctadecyl-1-3,3,3,3-tetramethylindocarbocyanine perchlorate labeled MSCs expressed sarcomeric--actinin. Immunocytochemistry showed that only these MSCs expressed the cardiac markers and were not observed with other coculture condition as well as conditioned medium. Calcein-AM labeling of cardiomyocytes showed gap junctional communication between 56.1±2.0% of MSCs (24 h after labeling, n=5) and neonatal cardiomyocytes. These findings suggest that MSCs are capable of differentiating into cardiomyocytes when directly cocultured with neonatal cardiomyocytes by cell-to-cell interaction, but not with adult cardiomyocytes or conditioned medium.     

Cytokine signaling for proliferation, survival, and death in hematopoietic cells

The survival, proliferation, and differentiation of hematopoietic cells are regulated by cytokines. In the absence of cytokines, hematopoietic cells not only stop proliferation, but undergo apoptosis. This strict dependency of hematopoietic cells on cytokines is an important mechanism that maintains the homeostasis of blood cells. Cytokines induce various intracellular signaling pathways by activating the receptor-associated Janus kinases (Jaks), and distinct signals are responsible for cell cycle progression and cell survival. Induction of signals for cell cycle progression without suppressing apoptosis results in apoptotic cell death, indicating the essential role of anti-apoptotic signaling for cell growth. In hematopoietic cells, Ras, a cellular protooncogen product, and phosphatidylinositol 3 kinase are involved in the suppression of apoptosis. Cytokine depletion not only turns off anti-apoptotic signaling, but also actively induces cell death by activating caspases, a distinct family of cysteine proteases. Alterations in the mechanisms of cytokine signaling for cell cycle progression and anti-apoptotic function are implicated in hematological disorders.     

Effects of BCL-2 antisense oligodeoxynucleotides on in vitro proliferation and survival of normal marrow progenitors and leukemic cells

Previous studies have shown that the BCL-2 protooncogene encodes a mitochondrial protein that promotes cell survival by blocking programmed cell death. Bcl-2 protein has been detected in normal immature myeloid cells and in acute myeloid leukemia (AML) cells. To assess its functional role in normal and leukemic hematopoiesis, we performed serum-free cultures of CD34+ normal marrow cells, of bcl-2- positive myeloid lines, and of AML cells in the presence of bcl-2 sense, nonsense, and antisense phosphorothioate oligodeoxynucleotides. In all antisense-treated cultures, we observed (1) an inhibition of bcl- 2 protein expression by day 4 to 6 of culture; (2) a decrease in cell survival duration; and (3) a decrease in the number of clonogenic cells present in the culture. Moreover, exposure to chemotherapeutic drugs resulted in more effective killing of AML cells in the presence of antisense oligomers. We conclude that bcl-2 protein is necessary for the survival of myeloid cells in culture, and that it may be implicated in the resistance of AML cells to chemotherapy.