Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3 + cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24-or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.HSCs, defined by their ability to self-renew and to differentiate into all blood cell types, form the basis of bone marrow transplantation for treatment of cancers and hematopoietic disorders 1 , and are also a promising cell target for gene therapies that can potentially treat a broad variety of human diseases 2 . Development of these important clinical applications of HSCs is greatly hampered by the lack of understanding of the extracellular and intracellular signals that govern their fates as well as by the difficulty in carrying out ex vivo expansion of these cells.Numerous attempts have been made to increase the number of long-term HSCs in culture 3,4 . The use of stromal cell lines or combinations of cytokines have resulted in considerable selfrenewal of mouse HSCs assayed 4-6 weeks after transplant, and have led to as much as a sixfold increase in mouse long-term HSC activity in culture [5][6][7][8][9] . The introduction of exogenous transcription factors can expand HSCs more substantially [10][11][12] 13 . We subsequently developed a simple culture system using low but saturating levels of stem cell factor (SCF), thrombopoietin (TPO), IGF-2 and fibroblast growth factor 1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses, there was a greater than eightfold increase in numbers of long-term repopulating HSCs (LT-HSCs) after 10 d of culture of highly enriched bone marrow HSCs 14 .Here we further analyzed gene expression of E15 mouse liver CD3 + cells using Affymetrix U74Bv2 and U74Cv2 mouse microarrays, and identified the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3) as well as several other members of the family of angiopoietin-like proteins as potent stimulators of ex vivo expansion of HSCs. RESULTS Fetal liver CD3 + cells specifically express Angptl2 and Angptl3In Supplementary Fig. 1 online). These proteins are also expressed in adult bone marrow cells, including the side population (SP) CD45 + Sca-1 + highly enriched HSC population 17 ( Supplementary Fig. 1 online). We therefore hypothesized that these two proteins, not previously thought to be involved in HSC biology, might support expansion of HSCs. Angptl2 and Angptl3 stimulate ex ...
Adiponectin is a major insulin-sensitizing, multimeric hormone derived from adipose tissue that acts on muscle and liver to regulate whole-body glucose and lipid metabolism. Here, we describe a novel and highly conserved paralog of adiponectin designated as C1q/TNF-related protein (CTRP) 9. Of all the CTRP paralogs, CTRP9 shows the highest degree of amino acid identity to adiponectin in its globular C1q domain. CTRP9 is expressed predominantly in adipose tissue and females expresses higher levels of the transcript than males. Moreover, its expression levels in ob/ob mice changed in an age-dependent manner, with significant up-regulation in younger mice. CTRP9 is a secreted glycoprotein with multiple post-translational modifications in its collagen domain that include hydroxylated prolines and hydroxylated and glycosylated lysines. It is secreted as multimers (predominantly trimers) from transfected cells and circulates in the mouse serum with levels varying according to sex and metabolic state of mice. Furthermore, CTRP9 and adiponectin can be secreted as heterooligomers when cotransfected into mammalian cells, and in vivo, adiponectin/CTRP9 complexes can be reciprocally coimmunoprecipitated from the serum of adiponectin and CTRP9 transgenic mice. Biochemical analysis demonstrates that adiponectin and CTRP9 associate via their globular C1q domain, and this interaction does not require their conserved N-terminal cysteines or their collagen domains. Furthermore, we show that adiponectin and CTRP9 form heterotrimers. In cultured myotubes, CTRP9 specifically activates AMPK, Akt, and p44/42 MAPK signaling pathways. Adenovirus-mediated overexpression of CTRP9 in obese (ob/ob) mice significantly lowered serum glucose levels. Collectively, these results suggest that CTRP9 is a novel adipokine, and further study of CTRP9 will yield novel mechanistic insights into its physiological and metabolic function.
Background: Pancreatic cancer is the fourth leading cause of cancer death in the United States. Consequently, identification of clinically relevant biomarkers for the early detection of this cancer type is urgently needed. In recent years, proteomics profiling techniques combined with various data analysis methods have been successfully used to gain critical insights into processes and mechanisms underlying pathologic conditions, particularly as they relate to cancer. However, the high dimensionality of proteomics data combined with their relatively small sample sizes poses a significant challenge to current data mining methodology where many of the standard methods cannot be applied directly. Here, we propose a novel methodological framework using machine learning method, in which decision tree based classifier ensembles coupled with feature selection methods, is applied to proteomics data generated from premalignant pancreatic cancer.
The microRNA miR-150, a critical regulator of hematopoiesis, is downregulated in mixed-lineage leukemia (MLL). In this study, miR-150 acts as a potent leukemic tumor suppressor by blocking the oncogenic properties of leukemic cells. By using MLL-AF9-transformed cells, we demonstrate that ectopic expression of miR-150 inhibits blast colony formation, cell growth, and increases apoptosis in vitro. More importantly, ectopic expression of miR-150 in MLL-AF9-transformed cells completely blocked the development of myeloid leukemia in transplanted mice. Furthermore, gene expression profiling revealed that miR-150 altered the expression levels of more than 30 "stem cell signature" genes and many others that are involved in critical cancer pathways. In addition to the known miR-150 target Myb, we also identified Cbl and Egr2 as bona fide targets and shRNA-mediated suppression of these genes recapitulated the pro-apoptotic effects observed in leukemic cells with miR-150 ectopic expression.In conclusion, we demonstrate that miR-150 is a potent leukemic tumor suppressor that regulates multiple oncogenes.Implications: These data establish new, key players for the development of therapeutic strategies to treat MLL-AF9-related leukemia. Mol Cancer Res; 11(8); 912-22. Ó2013 AACR.
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