Bone marrow endothelial cells (ECs) are essential for reconstitution of hematopoiesis, but their role in self-renewal of long term-hematopoietic stem cells (LT-HSCs) is unknown. We have developed angiogenic models to demonstrate that EC-derived angiocrine growth factors support in vitro self-renewal and in vivo repopulation of authentic LT-HSCs. In serum/cytokine-free co-cultures, ECs through direct cellular contact, stimulated incremental expansion of repopulating CD34−Flt3−cKit+Lineage−Sca1+ LT-HSCs, which retained their self-renewal ability, as determined by single cell and serial transplantation assays. Angiocrine expression of Notch-ligands by ECs promoted proliferation and prevented exhaustion of LT-HSCs derived from wild-type, but not Notch1/Notch2 deficient mice. In transgenic notch-reporter (TNR.Gfp) mice, regenerating TNR.Gfp+ LT-HSCs were detected in cellular contact with sinusoidal ECs and interfering with angiocrine, but not perfusion function, of SECs impaired repopulation of TNR.Gfp+ LT-HSCs. ECs establish an instructive vascular niche for clinical scale expansion of LT-HSCs and a cellular platform to identify stem cell-active trophogens.

angiogenesis ͉ endothelium ͉ adenovirus ͉ tumor ͉ stem

Purpose In an effort to identify molecular markers of tumor aggressiveness and therapeutic targets in lung adenocarcinoma (ADC), we investigated the expression of mesothelin (MSLN) in lung ADC, as well as its biological and clinical relevance. Experimental Design In a training and validation set of patients with early-stage (I–III) lung ADC (n=1209), a tissue microarray consisting of tumors and normal lung tissue was used to examine the association between MSLN expression and recurrence-free survival (RFS) and overall survival (OS). The influence of MSLN overexpression on lung ADC was investigated in vitro and in vivo by use of clinically relevant orthotopic and metastatic xenogeneic and syngeneic mouse models. Results MSLN was expressed in 69% of lung ADC tumors, with one in five patients strongly expressing MSLN and no expression in normal lung tissue. Increased MSLN expression was associated with reduced OS (HR, 1.78 [95% CI, 1.26–2.50]; P<0.01) and RFS (HR, 1.67 [95% CI, 1.21–2.27]; P<0.01) in multivariate analyses, even after adjustment for currently known markers of tumor aggressiveness in lung ADC: male sex, smoking history, increasing stage, morphologic pattern, visceral pleural invasion, lymphatic or vascular invasion, and mutation status. In vitro, lung ADC cells overexpressing MSLN demonstrated increased cell proliferation, migration, and invasion; in vivo, mice with MSLN(+) tumors demonstrated decreased survival (P=0.001). Conclusions MSLN expression in patients with early-stage lung ADC is associated with increased risk of recurrence and reduced OS, indicating that MSLN expression is a molecular marker of tumor aggressiveness and a potential target for therapy.