No abstract

The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.

We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro-expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.

Background-Cardiomyocytes derived from human embryonic stem (hES) cells could be useful in restoring heart function after myocardial infarction or in heart failure.

Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel, a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here, we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin, fibronectin, and Matrigel but poorly to laminin ؉ entactin and collagen IV. Integrin-blocking antibodies demonstrated that ␣V␤5 integrins mediated adhesion to vitronectin, ␣5␤1 mediated adhesion to fibronectin, and ␣6␤1 mediated adhesion to laminin ؉ entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices, but in defined, nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel, supporting sustained self-renewal and pluripotency in three independent hESC lines. STEM

Hereditary hemorrhagic telangiectasia (HHT) is an inherited disorder characterized by vascular malformations. Many affected individuals develop recurrent nosebleeds, which can severely affect their quality of life and are clinically difficult to treat. We report here that treatment with thalidomide reduced the severity and frequency of nosebleeds (epistaxis) in the majority of a small group of subjects with HHT tested. The blood hemoglobin levels of the treated individuals rose as a result of reduced hemorrhage and enhanced blood vessel stabilization. In mice heterozygous for a null mutation in the Eng gene (encoding endoglin), an experimental model of HHT, thalidomide treatment stimulated mural cell coverage and thus rescued vessel wall defects. Thalidomide treatment increased platelet-derived growth factor-B (PDGF-B) expression in endothelial cells and stimulated mural cell activation. The effects of thalidomide treatment were partially reversed by pharmacological or genetic interference with PDGF signaling from endothelial cells to pericytes. Biopsies of nasal epithelium from individuals with HHT treated or not with thalidomide showed that similar mechanisms may explain the effects of thalidomide treatment in humans. Our findings demonstrate the ability of thalidomide to induce vessel maturation, which may be useful as a therapeutic strategy for the treatment of vascular malformations.

No abstract

Human embryonic stem cells (hESC) can proliferate indefinitely while retaining the capacity to form derivatives of all three germ layers. We have reported previously that hESC differentiate into cardiomyocytes when cocultured with a visceral endoderm-like cell line (END-2). Insulin/ insulin-like growth factors and their intracellular downstream target protein kinase Akt are known to protect many cell types from apoptosis and to promote proliferation, including hESC-derived cardiomyocytes. Here, we show that in the absence of insulin, a threefold increase in the number of beating areas was observed in hESC/ END-2 coculture. In agreement, the addition of insulin strongly inhibited cardiac differentiation, as evidenced by a significant reduction in beating areas, as well as in ␣-actinin and ␤-myosin heavy chain (␤-MHC)-expressing cells. Real-time reverse transcription-polymerase chain reaction and Western blot analysis showed that insulin inhibited cardiomyogenesis in the early phase of coculture by suppressing the expression of endoderm (Foxa2, GATA-6), mesoderm (brachyury T), and cardiac mesoderm (Nkx2.5, GATA-4). In contrast to previous reports, insulin was not sufficient to maintain hESC in an undifferentiated state, since expression of the pluripotency markers Oct3/4 and nanog declined independently of the presence of insulin during coculture. Instead, insulin promoted the expression of neuroectodermal markers. Since insulin triggered sustained phosphorylation of Akt in hESC, we analyzed the effect of an Akt inhibitor during coculture. Indeed, the inhibition of Akt or insulin-like growth factor-1 receptor reversed the insulin-dependent effects. We conclude that in hESC/END-2 cocultures, insulin does not prevent differentiation but favors the neuroectodermal lineage at the expense of mesendodermal lineages. STEM CELLS 2008;26:724 -733 Disclosure of potential conflicts of interest is found at the end of this article.