Expression of the liver-specific gene Cyp7a1 reveals hepatic differentiation in embryoid bodies derived from mouse embryonic stem cells

Hepatic differentiation from mouse embryonic stem (ES) cells via the formation of embryoid bodies (EBs) has been revealed by the expression of hepatocyte-related genes such as alpha-fetoprotein and albumin. It is known, however, that the visceral endoderm differentiates in early EBs and expresses these hepatocyte-related genes. Thus, it remains unclear whether ES cells are capable of differentiating into hepatocytes derived from definitive endoderm in vitro. In the present study, yolk sac tissues isolated from the foetal mouse were found to express many hepatocyte-related genes. Among the hepatocyte-related genes examined, cytochrome P450 7A1 (Cyp7a1) was identified as a liver-specific gene that was not expressed in the yolk sac. Cyp7a1 was induced in developing EBs, and hepatic differentiation was preferentially observed in the developing EBs in attached culture as compared to those in suspension culture. Leukaemia inhibitory factor permitted the differentiation of visceral endoderm, but inhibited the expression of gastrulation-related genes and the hepatic differentiation in cultured EBs. ES cells expressing green fluorescent protein (GFP) under the control of the Cyp7a1 enhancer/promoter showed that cultured EBs contained GFP-positive epithelial-like cells. These results demonstrate that ES cells can differentiate in vitro into hepatocytes derived from definitive endoderm.     

Heterotopic Pancreas of the Stomach

Ordinary histological investigation has suggested that heterotopic pancreas of the stomach may have two types of histogenesis; one is development from immigrated fetal pancreas tissue, and the other is development from primitive gastric mucosal epithelium following penetration into the submucosa with subsequent erroneous differentiation into pancreas tissue. It is suspected that type I lesions include the majority of cases caused by immigration from fetal pancreas, and that some type II cases arise through erroneous differentiation of primitive gastric mucosal epithelium. With regard to immunohistochemical findings, cells positive for pancreatic polypeptide and amylase were much more numerous in the acini of type I cases compared with type II cases. Positive cells were found not infrequently in the acini of type II cases after staining for pancreatic polypeptide, insulin, glucagon, somatostatin, serotonin, and gastrin. On the other hand, a small number of cells in islets were not infrequently positive for α ₁–antitrypsin, α ₁,-antichimotrypsin, and amylase. It is considered that in the heterotopic pancreas, ductal cells have the potential to differentiate into acinar cells and islet cells, as is the cases in the orthotopic pancreas. Acta Pathol Jpn 42 : 249 254, 1992.     

Developmental platelet endothelial cell adhesion molecule expression suggests multiple roles for a vascular adhesion molecule

Platelet endothelial cell adhesion molecule (PECAM) is used extensively as a murine vascular marker. PECAM interactions have been implicated in both vasculogenesis and angiogenesis. To better understand the role of PECAM in mammalian development, PECAM expression was investigated during differentiation of murine embryonic stem (ES) cells and in early mouse embryos. Undifferentiated ES cells express PECAM, and as in vitro differentiation proceeds previously unidentified PECAM-positive cells that are distinct from vascular endothelial cells appear. PECAM expression is gradually restricted to endothelial cells and some hematopoietic cells of differentiated blood islands. In embryos, the preimplantation blastocyst contains PECAM-positive cells. PECAM expression is next documented in the postimplantation embryonic yolk sac, where clumps of mesodermal cells express PECAM before the development of mature blood islands. The patterns of PECAM expression suggest that undifferentiated cells, a prevascular cell type, and vascular endothelial cells express this marker during murine development. PECAM expression in blastocysts and by ES cells suggests that PECAM may function outside the vascular/hematopoietic lineage.     

Development, differentiation, and maturation of fetal mouse yolk sac macrophages in cultures

The development and differentiation of macrophages in the fetal mouse yolk sac were studied morphologically in four different culture experiments. In the culture of mouse embryos with yolk sac, the development of fetal macrophages was demonstrated to precede that of promonocytes and monocytes in the yolk sac. In vitro differentiation of the fetal macrophages was consistent with the results of our previous in vivo observation indicating that fetal macrophages were differentiated from primitive macrophages, but not from the monocytic cell series. Differentiation of primitive macrophages into fetal macrophages, before the development of promonocytes and monocytes, was reproduced in the culture of cell suspensions from the fetal mouse yolk sacs, with a mouse bone marrow stromal cell clone (ST2) particularly with those at 8 days of gestation. In the soft agar or liquid culture of yolk sac cells with LP3-conditioned medium, monocyte-macrophage colonies were effectively induced, but not fetal macrophage colonies. The results provide evidence for the existence, in yolk sac hematopoiesis, of two distinct macrophage populations: a fetal macrophage population and a monocyte-derived macrophage population. The data indicate an obvious difference in development and differentiation between the two populations and the temporal precedence of fetal macrophages appearing before monocyte-macrophages.     

Chromatin-remodeling factors allow differentiation of bone marrow cells into insulin-producing cells

Type 1 diabetes is caused by the destruction of pancreatic beta-cells by T cells of the immune system. Islet transplantation is a promising therapy for diabetes mellitus. Bone marrow stem cells (BMSC) have the capacity to differentiate into various cell lineages including endocrine cells of the pancreas. To investigate the conditions that allow BMSC to differentiate into insulin-producing cells, a novel in vitro method was developed by using the histone deacetylase inhibitor, trichostatin A (TSA). BMSC, cultured in presence of TSA, differentiated into islet-like clusters under appropriate culture conditions. These islet-like clusters were similar to the cells of the islets of the pancreas. The islet-like clusters showed endocrine gene expression typical for pancreatic beta-cell development and function, such as insulin (I and II), glucagon, somatostatin, GLUT-2, pancreatic duodenal homeobox-1 (PDX-1), and Pax 4. Immunocytochemistry confirmed islet-like clusters contained pancreatic hormones. The colocalization of insulin and C-peptide was also observed. Enzyme-linked immunosorbent assay analysis demonstrated that insulin secretion was regulated by glucose. Western blot analysis demonstrated the presence of stored insulin. Electron microscopy of the islet-like cells revealed an ultrastructure similar to that of pancreatic beta-cells, which contain insulin granules within secretory vesicles. These findings suggest that histone-deacetylating agents could allow the differentiation of BMSC into insulin-producing beta-cells.     

Histological study of heterotopic pancreas tissue of the stomach--histogenesis and immunohistochemical findings

Forty resected cases of heterotopic pancreas (15 of Heinrich's type I and 25 of type II) in the stomach were investigated. Acinic cells were more remarkably positive by pancreatic polypeptide and amylase in the cases of type I consisting of acinic cells, ducts and islet cells compared with those in type II consisting of acinic cells and ducts. Staining behavior by insulin, gastrin, glucagon, somatostatin and serotonin was similar to that of normal pancreas. However, the intestinal epithelium containing goblet cells and pyloric gland-like glands consisting of the cells with clear cytoplasm were not infrequently produced from the ducts in the lesions of type II. Muscle fibers with transition to muscularis mucosae were more frequently intermingled in the lesions of type II compared with type I. Acinic cell-differentiation in the mucosa apart from the main lesion was found in several cases of type II. It was suspected that there are two types of histogenesis for the heterotopic pancreas in the stomach; one arises from the fetal immigration of pancreas tissue in the stomach and the other from the immature gastric mucosal penetration into the submucosa with secondary differentiation to the pancreas tissue.     

Ontogeny of plasma cells in the early rat yolk sac

The present study investigated the development of plasma cells in the early rat yolk sac (days 10-16 of gestation) by light microscopy, transmission electron microscopy, immunoelectron microscopy, and indirect immunofluoresce techniques. Cells delineating the morphology of plasma cells in the yolk sac were observed as early as 12 days of embryonic life. As for positive immune staining for the intra-cytoplasmic immunoglobulin (Ig) production (IgA, IgM and IgG), the intensity of the immune staining was very weak on days 10 and 11 of gestation, while it turned very dense on day 12 of gestation. At 14 days of gestation, the number of positive cells was markedly reduced. Immunoelectron microscopy visualized products of the immune reaction in cisterns of the rough endoplasmic reticulum. Conventional electron microscopic examination of 12, 13, and 16-day yolk sacs confirmed the development and differentiation of plasma cells with their well-known ultrastructural features, making this the first study to demonstrate these in the early rat yolk sac. The development of plasma cells in the early yolk sac implies the ability of the yolk sac to effect a humoral immune response at this stage of fetal life. The probable role of plasma cells in the yolk sac is also discussed.     

Ultrastructural analysis of mouse embryonic stem cell-derived chondrocytes

Pluripotent embryonic stem (ES) cells cultivated as cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into different cell types of all three germ layers in vitro resembling processes of cellular differentiation during embryonic development. Regarding chondrogenic differentiation, murine ES cells differentiate into progenitor cells, which form pre-cartilaginous condensations in the EB-outgrowths and express marker molecules characteristic for mesenchymal cell types such as Sox5 and Sox6. Later, mature chondrocytes appear which express collagen type II, and the collagen fibers show a typical morphology as demonstrated by electron-microscopical analysis. These mature chondrogenic cells are organized in cartilage nodules and produce large amounts of extracellular proteoglycans as revealed by staining with cupromeronic blue. Finally, cells organized in nodules express collagen type X, indicating the hypertrophic stage. In conclusion, differentiation of murine ES cells into chondrocytes proceeds from the undifferentiated stem cell via progenitor cells up to mature chondrogenic cells, which then undergo hypertrophy. Furthermore, because the ES-cell-derived chondrocytes did not express elastin, a marker for elastic cartilage tissue, we suggest the cartilage nodules to resemble hyaline cartilage tissue.     

Expression of a novel antigen, EEM-1, in the mouse embryo and embryonic stem cell-derived embryoid bodies.

A novel monoclonal antibody designated EEM-1 is described. EEM-1 recognizes an intracellular protein with an apparent molecular weight of >250 kDa. Expression of the EEM-1 antigen is largely confined to extra-embryonic membranes, but some expression does occur in the embryo. In the embryonic day 6 (E6) and E7 embryo it is expressed in visceral and parietal endoderm; later derivatives of these structures in the yolk sac are negative. The outer layer of the amnion also stains. Within the embryo proper, antigen is expressed in limited regions of the gut, kidney, and pancreas. EEM-1 is also expressed in embryonic stem (ES) cells differentiating in vitro. Undifferentiated ES cells do not express the antigen. Embryoid bodies (EBs) derived from ES cells have patches of EEM-1-positive cells on their surface at 2 days in culture. Older EBs have increasing numbers of positive cells which are confined to the surface. A special class of EBs, termed "cystic EBs," are covered by a cell layer which strongly express EEM-1 antigen. The EEM-1 antibody will be useful for investigating the development of extra-embryonic membranes and their counterparts in the ES cell in vitro model.     

Heterotopic pancreas of the stomach. Histogenesis and immunohistochemistry.

Ordinary histological investigation has suggested that heterotopic pancreas of the stomach may have two types of histogenesis; one is development from immigrated fetal pancreas tissue, and the other is development from primitive gastric mucosal epithelium following penetration into the submucosa with subsequent erroneous differentiation into pancreas tissue. It is suspected that type-I lesions include the majority of cases caused by immigration from fetal pancreas, and that some type-II cases arise through erroneous differentiation of primitive gastric mucosal epithelium. With regard to immunohistochemical findings, cells positive for pancreatic polypeptide and amylase were much more numerous in the acini of type-I cases compared with type-II cases. Positive cells were found not infrequently in the acini of type-II cases after staining for pancreatic polypeptide, insulin, glucagon, somatostatin, serotonin, and gastrin. On the other hand, a small number of cells in islets were not infrequently positive for alpha 1-antitrypsin, alpha 1-antichymotrypsin, and amylase. It is considered that in the heterotopic pancreas, ductal cells have the potential to differentiate into acinar cells and islet cells, as is the cases in the orthotopic pancreas.     

胚胎干细胞R1诱导成胰岛素分泌细胞团的研究

目的 建立体外培养和扩增胚胎干细胞R1(ES-R1)的最佳条件;利用多种生长因子,优化培养条件,体外定向诱导ES-R1分化为胰岛素分泌细胞(IPCs).方法 以丝裂霉索-C处理的MEF为饲养层,培养液中加白血病抑制因子(LIF),ES-R1维持未分化状态并扩增,进行碱性磷酸酶染色.胚胎干细胞(ESCs)悬浮培养制成拟胚体(EBs),对培养至第4d的EBs开始定向诱导,依次加入无血清的ITS培养液,胰岛前体细胞增殖培养液和胰岛分化培养液,每种培养液内各培养6d,获得形态功能较成熟的IPCs.采用DTZ染色、免疫荧光染色、胰岛素刺激实验等方法对IPCs进行检测.结果 ESCs在饲养层细胞上呈克隆状生长,经碱性磷酸酶染色为阳性;EBs经3种诱导液诱导成三维立体的IPCs,IPCs被DTZ染成猩红色,胰岛素和胰高血糖素阳性表达,胰岛素刺激实验阳性.结论 ES-R1在体外培养时,用MEF做饲养层,培养液中添加一定浓度的LIF,可以最好地保持未分化状态并大量增殖.用分阶段添加不同生长因子的方法诱导ES-R1定向分化生成的IPCs在形态和功能上具有胰岛的特性.     

人卵黄囊间质干细胞的分离纯化及诱导成脂的实验研究

目的:分离纯化及体外定向诱导人卵黄囊间质干细胞(YS-MSC)分化为脂肪细胞。方法:显微分离人卵黄囊, 经酶消化得到卵黄囊细胞, 卵黄囊细胞经贴壁培养、传代纯化得到人YS-MSC, 流式细胞仪检测YS-MSC表面抗原表达, 钙钴法测定碱性磷酸酶(AKP)活性;地塞米松、消炎痛、胰岛素定向诱导YS-MSC分化为脂肪细胞。油红O检测中性脂肪。结果:人卵黄囊间质干细胞易于分离、纯化, 体外培养增殖潜能大。卵黄囊间质干细胞CD29、CD44、CD166及CD105表达阳性, CD34、CD45和CD86为阴性;AKP弱阳性。卵黄囊间质干细胞经成脂诱导转化为大小不等的园形或椭圆形细胞, 可见胞浆内有少量微小脂滴形成, 随时间延长, 胞浆中脂滴相互融合, 胞核被挤于细胞的一侧, 经油红O染色脂滴染橘红色, 符合脂肪细胞的生物学特征。结论:人卵黄囊间质干细胞与成体间质干细胞表型一致, 在体外可以分化为脂肪细胞。     

Insulin and C-peptide secretory responses to glucagon in man: studies on the dose-response relationships

The present study investigated the insulin and C-peptide secretory responses to glucagon in non-diabetic humans. Glucagon induced a transient increase in plasma insulin and C-peptide concentrations. At the dose level of 0.5 mg, glucagon elicited more efficient responses than at the dose level of 0.25 mg (p less than 0.05). However, the responses were not further potentiated by glucagon at 1.0 mg. Plasma glucose levels did not change during the first 2 min after glucagon injection, when already a marked increase in plasma insulin and C-peptide levels were observed. Thereafter, however, plasma glucose levels increased, to be maximal at 20 min after glucagon injection. Calculations of the minute-to-minute increase of plasma insulin and C-peptide levels revealed that plasma insulin levels increased by 32 +/- 7% of the increase in plasma C-peptide levels during the first 2 min, and by 36 +/- 6% of the increase in plasma C-peptide levels during the 3rd and 4th min after injection; the difference being the liver extraction of insulin. We conclude from this study in man that glucagon stimulates insulin secretion through both direct and indirect effects, that following glucagon injection, approximately 65% of the secreted insulin is extracted by the liver, and that the dose level of 0.5 mg glucagon is the optimal dose level for the stimulation of insulin secretion.     

Alpha-Fetoprotein Subtractions in Germ Cell Tumors Identified by Con A or LCH Crossed-Line Affinity Immunoelectrophoresis

ABSTRACT: Using concanavalin A (Con A) crossed-line affinity immunoelectrophoresis and lentil lectin (LCH) crossed-line affinity immunoelectrophoresis, alpha-fetoprotein (AFP) subfractions were studied in sera including three sera from nude mice heterotran-splanted with human yolk sac tumor of the ovary and three sera from patients with yolk sac tumor, mature solid teratoma, or immature solid teratoma of the ovary. In sera of nude mice bearing yolk sac tumor or from a patient with yolk sac tumor, subfractions from yolk sac and those from fetal liver were identified. Since AFP subfractions from yolk sac and fetal liver can be differentiated according to the carbohydrate moieties, our findings indicate that AFP produced by yolk sac tumor and fetal yolk sac are to some extent differently glycosylated. We also found that AFP in both mature and immature solid teratoma was composed of two subfractions ontogenetically originating from yolk sac or fetal liver. All these findings indicate that more than two different factors are responsible for the AFP synthesis in germ cell tumor of the ovary.