Maintenance of pluripotency and self-renewal ability of mouse embryonic stem cells in the absence of tetraspanin CD9.

We have previously demonstrated that the tetraspanin CD9 is necessary for membrane fusion between sperm and oocyte during fertilization. While knockout mice for CD9 are viable, CD9(-/-) females are sterile due to the inability of their oocytes to fuse with sperm. While CD9 is not essential for subsequent development, a role in embryonic stem (ES) cell self-renewal was hypothesised on the basis of two observations: CD9 is highly expressed in murine and human ES cells and the CD9-blocking antibody inhibits mouse ES cell colony formation and survival. To investigate whether CD9 has a direct effect on ES cells, we generated and characterised several CD9 knockout murine ES cell lines. These CD9(-/-) ES cell lines exhibited equivalent morphology and growth properties to wild-type ES cells. Furthermore, the CD9(-/-) ES cell lines also displayed similar expression of pluripotency factors Oct3/4, Sox2 and Nanog. CD9(-/-) ES cells were found to be pluripotent in vivo, as their cells injected into immunocompromised mice gave rise to teratomas consisting of tissues representative of all three germ layers. Additionally several high contribution mouse chimeras were generated by blastocyst injection with several CD9(-/-) ES cell lines. Taken together, our results reveal that CD9 is dispensable for mouse ES cell self-renewal and pluripotency. The generation of CD9(-/-) ES cells should prove to be a useful tool with which to study the function of this protein and a range of other associated cellular processes.

Shortening of human cell life span by induction of p16ink4a through the platelet-derived growth factor receptor beta.

Mesenchymal stem cells (MSCs) are attracting a great deal of attention because they represent a valuable source of cells for use in regenerative medicine. In human cell culture it is important to obtain large numbers of cells for use in therapy. In this study, we attempted to prolong life span of a marrow-derived mesenchymal stem cell using a combination of growth factors and hormones. Furthermore we tested whether chemically defined culture conditions are sufficient for maintenance of multipotent mesenchymal stem cells. Epidermal growth factor, platelet-derived growth factor-BB (PDGF-BB), acidic fibroblast growth factor (FGF), basic FGF, and leukemia inhibitory factor were found to be key factors for the mesenchymal stem cell proliferation. The combination of these growth factors showed extremely strong mitogenic activity, and simultaneously induced the expression of cyclin-dependent kinase inhibitor p16ink4a protein and premature senescence more rapidly than serum-supported culture conditions. The induction of p16ink4a by growth factors was mediated through the mitogen-activated protein kinase (MAPK) cascade. Excess growth stimulation by growth factors was thus one of the culture stress signals and a trigger of premature senescence at least in human cells.

Insulin hypersensitivity in mice lacking the V1b vasopressin receptor.

We have reported that [Arg(8)]-vasopressin-stimulated insulin release is blunted in islet cells isolated from V1b receptor-deficient (V1bR(-/-)) mice. In this study, we used V1bR(-/-) mice to examine the physiological role of the V1b receptor in regulating blood glucose levels in vivo, and we found that the fasting plasma glucose, insulin and glucagon levels were lower in V1bR(-/-) mice than in wild-type (V1bR(+/+)) mice. Next, we evaluated glucose tolerance by performing an intraperitoneal glucose tolerance test (GTT). The plasma glucose and insulin levels during the GTT were lower in V1bR(-/-) mice than in V1bR(+/+) mice. An insulin tolerance test (ITT) revealed that, after insulin administration, plasma glucose levels were lower in V1bR(-/-) mice than in V1bR(+/+) mice. In addition, a hyperinsulinaemic-euglycaemic clamp study showed that the glucose infusion rate was increased in V1bR(-/-) mice, indicating that insulin sensitivity was enhanced at the in vivo level in V1bR(-/-) mice. Furthermore, we found that the V1b receptor was expressed in white adipose tissue and that insulin-stimulated phosphorylation of Akt as an important signaling molecule was increased in adipocytes isolated from V1bR(-/-) mice. Thus, the blockade of the V1b receptor could result, at least in part, in enhanced insulin sensitivity by altering insulin signalling in adipocytes.

Impaired arginine-vasopressin-induced aldosterone release from adrenal gland cells in mice lacking the vasopressin V1A receptor.

We examined aldosterone release in response to stimulation with arginine-vasopressin (AVP) using adrenal gland cells. AVP caused a significant increase in aldosterone release from the dispersed adrenal gland cells of wild-type mice (V1AR+/+) at concentrations from 0.1 microM to 1 microM. In contrast, AVP-induced aldosterone release was impaired in adrenal gland cells from mice lacking the vasopressin V1A receptor (V1AR-/-), while adrenocorticotropic hormone (ACTH)-induced aldosterone release in V1AR-/- mice was not significantly different from that in V1AR+/+ mice. In addition, a vasopressin V1A receptor-selective antagonist 1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone (OPC-21268) potently inhibited AVP-induced aldosterone release. Thus, our study clearly demonstrates that AVP-induced aldosterone release from adrenal gland cells is mediated via the vasopressin V1A receptor in mice.

Blockade of both alpha1A- and alpha1B-adrenergic receptor subtype signaling is required to inhibit neointimal formation in the mouse femoral artery.

Attenuation of early restenosis after percutaneous coronary intervention (PCI) is important for the successful treatment of coronary artery disease. Some clinical studies have shown that hypertension is a risk factor for early restenosis after PCI. These findings suggest that alpha(1)-adrenergic receptors (alpha(1)-ARs) may facilitate restenosis after PCI because of alpha(1)-AR's remarkable contribution to the onset of hypertension. In this study, we examined the neointimal formation after vascular injury in the femoral artery of alpha(1A)-knockout (alpha(1A)-KO), alpha(1B)-KO, alpha(1D)-KO, alpha(1A)-/alpha(1B)-AR double-KO (alpha(1AB)-KO), and wild-type mice to investigate the functional role of each alpha(1)-AR subtype in neointimal formation, which is known to promote restenosis. Neointimal formation 4 wk after wire injury was significantly (P < 0.05) smaller in alpha(1AB)-KO mice than in any other group of mice, while blood pressures were not altered in any of the groups of mice after wire injury compared with those before it. These results suggest that lack of both alpha(1A)- and alpha(1B)-ARs could be necessary to inhibit neointimal formation in the mouse femoral artery.

Alteration of glucose homeostasis in V1a vasopressin receptor-deficient mice.

Arginine-vasopressin (AVP) is known to be involved in maintaining glucose homeostasis, and AVP-resistance is observed in poorly controlled non-insulin-dependent diabetes mellitus subjects, resulting in a lowered plasma volume. Recently we reported that V1a vasopressin receptor-deficient (V1aR(-/-)) mice exhibited a decreased circulating blood volume and hypermetabolism of fat accompanied with impaired insulin-signaling. Here we further investigated the roles of the AVP/V1a receptor in regulating glucose homeostasis and plasma volume using V1aR(-/-) mice. The plasma glucose levels at the baseline or during a glucose tolerance test were higher in V1aR(-/-) than wild-type (WT) mice. Moreover, a hyperinsulinemic-euglycemic clamp revealed that the glucose infusion rate was significantly lower in V1aR(-/-) mice than in WT mice and that hepatic glucose production was higher in V1aR(-/-) mice than WT mice. In contrast to the increased hepatic glucose production, the liver glycogen content was decreased in the mutant mice. These results indicated that the mutant mice had impaired glucose tolerance. Furthermore, feeding V1aR(-/-) mice a high-fat diet accompanied by increased calorie intake resulted in significantly overt obesity in comparison with WT mice. In addition, we found that the circulating plasma volume and aldosterone level were decreased in V1aR(-/-) mice, although the plasma AVP level was increased. These results suggested that the effect of AVP on water recruitment was disturbed in V1aR(-/-) mice. Thus, we demonstrated that one of the AVP-resistance conditions resulting from deficiency of the V1a receptor leads to decreased plasma volume as well as impaired glucose homeostasis, which can progress to obesity under conditions of increased calorie intake.

Hypermetabolism of fat in V1a vasopressin receptor knockout mice.

[Arg8]Vasopressin (AVP) has an antilipolytic action on adipocytes, but little is known about the mechanisms involved. Here, we examined the involvement of the V1a receptor in the antilipolytic effect of AVP using V1a receptor-deficient (V1aR-/-) mice. The levels of blood glycerol were increased in V1aR-/- mice. The levels of ketone bodies, such as acetoacetic acid and 3-hydroxybutyric acid, the products of the lipid metabolism, were increased in V1aR-/- mice under a fasting condition. Triacylglyceride and free fatty acid levels in blood were decreased in V1aR-/- mice. Furthermore, measurements with tandem mass spectrometry determined that carnitine and acylcarnitines in serum, the products of beta-oxidation, were increased in V1aR-/- mice. Most acylcarnitines were increased in V1aR-/- mice, especially in the case of 2-carbon (C2), C10:1, C10, C14:1, C16, C18:1, and hydroxy-18:1-carbon (OH-C18:1)-acylcarnitines under feeding rather than under fasting conditions. The analysis of tissue C2-acylcarnitine level showed that beta-oxidation was promoted in muscle under the feeding condition and in liver under the fasting condition. An in vitro assay using brown adipocytes showed that the cells of V1aR-/- mice were more sensitive to isoproterenol for lipolysis. These results suggest that the lipid metabolism is enhanced in V1aR-/- mice. The cAMP level was enhanced in V1aR-/- mice in response to isoproterenol. The phosphorylation of Akt by insulin stimulation was reduced in V1aR-/- mice. These results suggest that insulin signaling is suppressed in V1aR-/- mice. In addition, the total bile acid, taurine, and cholesterol levels in blood were increased, and an enlargement of the cholecyst was observed in V1aR-/- mice. These results indicated that the production of bile acid was enhanced by the increased level of cholesterol and taurine. Therefore, these results indicated that AVP could modulate the lipid metabolism by the antilipolytic action and the synthesis of bile acid via the V1a receptor.

In vitro differentiation of hepatocyte-like cells from embryonic stem cells promoted by gene transfer of hepatocyte nuclear factor 3 beta.

We investigated the ability of a genetically altered embryonic stem (ES) cell line to promote endodermal differentiation toward hepatocytes in vitro by transfecting the hepatocyte nuclear factor-3beta (HNF-3beta) gene. Parental and HNF-3beta-transfected ES cells were initiated toward differentiation in embryoid bodies (EBs) for 5 days and the resulting EBs were transferred to an attached culture system. Albumin production was observed using an immuno-cytochemical method 7 days after induction of differentiation in almost all differentiating HNF-3beta-transfected ES cells, whereas scant immuno-reactivity against albumin was found on the same day in the cultures of differentiating parental ES cells. An analysis using a reverse transcriptase polymerase chain reaction revealed the HNF-4alpha expression in the HNF-3beta-transfected ES cells and also demonstrated that the expression of endodermal and hepatocyte-related markers, such as transthyretin, alpha-fetoprotein, albumin, alpha-1 antitrypsin, tryptophan-2,3-dioxygenase and phosphoenol-pyruvate carboxykinase, could be observed at an early stage in the outgrowths of HNF-3beta-transfected ES cells compared to the parental ES cells. These results suggest that HNF-3beta-transfected ES cells may be useful for the efficient induction of hepatocytes in vivo.

Potential use of embryonic stem cells for the treatment of mouse parkinsonian models: improved behavior by transplantation of in vitro differentiated dopaminergic neurons from embryonic stem cells.

BACKGROUND: and Aims. The purpose of the present study was to examine the efficacy of transplantation of mouse embryonic-stem-(ES)-cell-derived tyrosine hydroxylase-positive (TH(+)) cells into Parkinsonian mice using behavioral tests and immunohistochemical evaluation. METHODS: Undifferentiated ES cells carrying the enhanced green fluorescent protein (EGFP) gene were differentiated into a cell population containing TH(+) neurons using a five-step in vitro differentiation method. These ES-cell-derived cells were used as allografts in Parkinsonian mice, made by administering injections of 6-hydroxydopamine (6-OHDA). Fifteen hemiparkinsonian mice were divided into three groups. Four weeks after 6-OHDA injection, mice in groups 1, 2, and 3 received phosphate-buffered saline, 1 x 10(4) graft cells, and 1 x 10(5) graft cells, respectively, into their dopamine-denervated striata. RESULTS: Improved rotational behavior was observed in the graft-transplanted groups (groups 2 and 3) 2 weeks after transplantation. Mice in group 2 displayed a continuous maintenance of reduced rotational behavior, while those in group 3 showed ipsilateral rotation toward the lesioned side at 4, 6, and 8 weeks after transplantation. Tumor formation was observed in one mouse in group 3. TH(+) cells were found at the grafted sites 8 weeks after transplantation in mice in groups 2 and 3, some of which were immunopositive to GFP, demonstrating the presence of dopaminergic neurons derived from the ES cells. CONCLUSION: Transplantation of in vitro differentiated ES cells changed rotational behavior in Parkinsonian mice. Our results suggest the potential availability of ES cells for Parkinson's disease.