Generation of a functional mammary gland from a single stem cell

The existence of mammary stem cells (MaSCs) has been postulated from evidence that the mammary gland can be regenerated by transplantation of epithelial fragments in mice. Interest in MaSCs has been further stimulated by their potential role in breast tumorigenesis. However, the identity and purification of MaSCs has proved elusive owing to the lack of defined markers. We isolated discrete populations of mouse mammary cells on the basis of cell-surface markers and identified a subpopulation (Lin-CD29hiCD24+) that is highly enriched for MaSCs by transplantation. Here we show that a single cell, marked with a LacZ transgene, can reconstitute a complete mammary gland in vivo. The transplanted cell contributed to both the luminal and myoepithelial lineages and generated functional lobuloalveolar units during pregnancy. The self-renewing capacity of these cells was demonstrated by serial transplantation of clonal outgrowths. In support of a potential role for MaSCs in breast cancer, the stem-cell-enriched subpopulation was expanded in premalignant mammary tissue from MMTV-wnt-1 mice and contained a higher number of MaSCs. Our data establish that single cells within the Lin-CD29hiCD24+ population are multipotent and self-renewing, properties that define them as MaSCs.     

Purification and unique properties of mammary epithelial stem cells

Elucidation of the cellular and molecular mechanisms that maintain mammary epithelial tissue integrity is of broad interest and paramount to the design of more effective treatments for breast cancer. Evidence from both in vitro and in vivo experiments suggests that mammary cell differentiation is a hierarchical process originating in an uncommitted stem cell with self-renewal potential. However, analysis of the properties and regulation of mammary stem cells has been limited by a lack of methods for their prospective isolation. Here we report the use of multi-parameter cell sorting and limiting dilution transplant analysis to demonstrate the purification of a rare subset of adult mouse mammary cells that are able individually to regenerate an entire mammary gland within 6 weeks in vivo while simultaneously executing up to ten symmetrical self-renewal divisions. These mammary stem cells are phenotypically distinct from and give rise to mammary epithelial progenitor cells that produce adherent colonies in vitro. The mammary stem cells are also a rapidly cycling population in the normal adult and have molecular features indicative of a basal position in the mammary epithelium.     

Expression of Smad2 and Smad4 in rhesus monkey endometrium during the menstrual cycle and early pregnancy

Expression of Smad2 and Smad4 mRNAs in the endometrium of rhesus monkey on Days 8, 20 and 28 of the normal menstrual cycle and on Days 12, 18 and 26 of early pregnancy was detected using in situ hybridization. The results showed that Smad2 and Smad4 mRNAs were mainly localized in luminal epithelium and glandular epithelium. The expression of Smad2 mRNA in glandular epithelium was sustained at moderate level on Days 8, 20 and 28 of the menstrual cycle, while the expression of Smad4 gradually increased with the menstrual cycle. Both Smad2 and Smad4 mRNAs in functionalis glandular epithelium were expressed at the highest levels on Day 12 of early pregnancy, while in basalis glandular epithelium the most abundant expression of both Smads occurred on Days 12 and 18 of pregnancy. On Day 26, both Smads mRNAs were expressed at the lowest levels either in functionalis or in basalis. The data suggest that the epithelium is the major compartment where TGF-βs/activins exert their biological effects via Smads, and that Smad4 may play a role in the maintenance of endometrial gland function during secreting period of the menstrual cycle. During lacunar stage of early pregnancy, Smad2 and Smad4 are implicated in the tissue remodeling of endometrial functionalis and basalis, and during early villous stage both Smads are functional primarily in basalis.     

RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis

RANK ligand (RANKL), a TNF-related molecule, is essential for osteoclast formation, function and survival through interaction with its receptor RANK. Mammary glands of RANK- and RANKL-deficient mice develop normally during sexual maturation, but fail to form lobuloalveolar structures during pregnancy because of defective proliferation and increased apoptosis of mammary epithelium. It has been shown that RANKL is responsible for the major proliferative response of mouse mammary epithelium to progesterone during mammary lactational morphogenesis, and in mouse models, manipulated to induce activation of the RANK/RANKL pathway in the absence of strict hormonal control, inappropriate mammary proliferation is observed. However, there is no evidence so far of a functional contribution of RANKL to tumorigenesis. Here we show that RANK and RANKL are expressed within normal, pre-malignant and neoplastic mammary epithelium, and using complementary gain-of-function (mouse mammary tumour virus (MMTV)-RANK transgenic mice) and loss-of function (pharmacological inhibition of RANKL) approaches, define a direct contribution of this pathway in mammary tumorigenesis. Accelerated pre-neoplasias and increased mammary tumour formation were observed in MMTV-RANK transgenic mice after multiparity or treatment with carcinogen and hormone (progesterone). Reciprocally, selective pharmacological inhibition of RANKL attenuated mammary tumour development not only in hormone- and carcinogen-treated MMTV-RANK and wild-type mice, but also in the MMTV-neu transgenic spontaneous tumour model. The reduction in tumorigenesis upon RANKL inhibition was preceded by a reduction in pre-neoplasias as well as rapid and sustained reductions in hormone- and carcinogen-induced mammary epithelial proliferation and cyclin D1 levels. Collectively, our results indicate that RANKL inhibition is acting directly on hormone-induced mammary epithelium at early stages in tumorigenesis, and the permissive contribution of progesterone to increased mammary cancer incidence is due to RANKL-dependent proliferative changes in the mammary epithelium. The current study highlights a potential role for RANKL inhibition in the management of proliferative breast disease.     

Identification of stem cells in small intestine and colon by marker gene Lgr5

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. It is currently believed that four to six crypt stem cells reside at the +4 position immediately above the Paneth cells in the small intestine; colon stem cells remain undefined. Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5, also known as Gpr49) was selected from a panel of intestinal Wnt target genes for its restricted crypt expression. Here, using two knock-in alleles, we reveal exclusive expression of Lgr5 in cycling columnar cells at the crypt base. In addition, Lgr5 was expressed in rare cells in several other tissues. Using an inducible Cre knock-in allele and the Rosa26-lacZ reporter strain, lineage-tracing experiments were performed in adult mice. The Lgr5-positive crypt base columnar cell generated all epithelial lineages over a 60-day period, suggesting that it represents the stem cell of the small intestine and colon. The expression pattern of Lgr5 suggests that it marks stem cells in multiple adult tissues and cancers.     

粉红丝带引发的思考——Wnt信号通路在乳腺发育与乳腺癌变中的角色

 Wnt信号转导通路是参与乳腺发育和肿瘤形成的重要机制。本文综述了Wnt信号转导通路调控乳腺发育和干细胞稳态研究的进展,讨论了Wnt信号转导中成员分子在乳腺癌形成中的角色。     

Influence of local vascularity on hormone receptors in mammary gland

Procedures, such as teat removal (thelectomy) or teat duct ligation, which prevent removal of milk, lead to rapid involution of the lactating mammary gland; performed unilaterally they have been used previously to study the biochemistry of involution, enabling a comparison of normal and involuting glands in the same animal against the same systematic hormonal environment. Both the protein hormone prolactin and the steroid hormone oestrogen are of importance in the development and function of the mammary gland. In the present experiments, female Sprague-Dawley rats were unilaterally thelectomised and the binding to the mammary gland of prolactin and oestrogen was examined through pregnancy, lactation and weaning. There was an effect of thelectomy during lactation only, when levels of both receptors increased in the intact lactating gland but failed to rise in the thelectomised, involuting gland. Capillary closure is known to occur in the mammary glands of rats after 36-48 h of milk accumulation. The rate of delivery of hormones to the tissue will be drastically reduced and it is concluded that this, rather than systemic hormone levels, is of importance in controlling receptor levels.     

Alteration of the quality of milk by expression of sheep beta-lactoglobulin in transgenic mice

Milk contains a large amount of protein, most of which consists of a few major species synthesized in the mammary gland. The genes encoding these proteins are single-copy, and expressed during pregnancy and lactation. Although beta-lactoglobulin (BLG) is the major protein in the whey of ruminants, it is not present in rodent milk. We have generated transgenic mice carrying the sheep BLG gene, and show that in such mice, BLG is specifically and abundantly expressed in the mammary gland during lactation. This results in a remarkable alteration of milk composition. These findings suggest that the manipulation of milk composition by gene transfer has considerable potential for the improvement of dairy animals.     

体外培养人涎腺多形性腺瘤细胞及表面标记物的初步研究

体外培养15例人涎腺多形性腺瘤原发肿瘤组织,探讨体外培养的细胞与原发肿瘤组织的表面标志物表达情况。运用免疫组化法检测CK5/6、CK7、CK8/18、CK14、CK20,vimentin及肌上皮表面标记物α-SMA、calponin及p63在细胞和肿瘤组织中的表达。结果发现体外培养的多形性腺瘤细胞呈多样形,可同时表达角蛋白、肌上皮表面标记物及波形蛋白,与原发肿瘤表达基本一致。提示体外培养的多形性腺瘤细胞可能大部分来源于肌上皮细胞及导管上皮细胞等,这为研究涎腺多形性腺瘤肿瘤生物学多样性提供了实验依据。     

Viscous fingering of HCl through gastric mucin.

The HCl in the mammalian stomach is concentrated enough to digest the stomach itself, yet the gastric epithelium remains undamaged. One protective factor is gastric mucus, which forms a protective layer over the surface epithelium and acts as a diffusion barrier Bicarbonate ions secreted by the gastric epithelium are trapped in the mucus gel, establishing a gradient from pH 1-2 at the lumen to pH 6-7 at the cell surface. How does HCl, secreted at the base of gastric glands by parietal cells, traverse the mucus layer without acidifying it? Here we demonstrate that injection of HCl through solutions of pig gastric mucin produces viscous fingering patterns dependent on pH, mucin concentration and acid flow rate. Above pH 4, discrete fingers are observed, whereas below pH 4, HCl neither penetrates the mucin solution nor forms fingers. Our in vitro results suggest that HCl secreted by the gastric gland can penetrate the mucus gel layer (pH 5-7) through narrow fingers, whereas HCl in the lumen (pH 2) is prevented from diffusing back to the epithelium by the high viscosity of gastric mucus gel on the luminal side.     

人间充质干细胞成骨分化早期HOX家族基因转录的表观遗传调控

人间充质干细胞(human mesenchymal stem cells,hMSCs)是一类具有多分化潜能的成体干细胞,在体内外可以被人工定向诱导分化成多种不同的细胞.有报道表明,在干细胞分化的过程中,细胞核内染色质发生重塑.HOX家族基因作为一类转录因子,在胚胎发育以及细胞分化过程中发挥着十分重要作用.通过体外定向诱导骨髓间充质干细胞向成骨细胞分化,对比分化前后细胞中HOX家族基因的表达状况,发现HOX家族基因的表达水平在hMSCs早期成骨分化过程中显著下降.进一步的研究发现,HOX家族基因的这种表达变化是由其启动子区的组蛋白H3-Lys9乙酰化和二甲基化水平发生变化而导致的.一系列实验证据表明,在间充质干细胞的成骨分化过程中,HOX家族基因表达受到抑制,而这种抑制作用是与其分化过程中发生的染色质重塑事件密切相关的.     

Evidence that stem cells reside in the adult Drosophila midgut epithelium

Adult stem cells maintain organ systems throughout the course of life and facilitate repair after injury or disease. A fundamental property of stem and progenitor cell division is the capacity to retain a proliferative state or generate differentiated daughter cells; however, little is currently known about signals that regulate the balance between these processes. Here, we characterize a proliferating cellular compartment in the adult Drosophila midgut. Using genetic mosaic analysis we demonstrate that differentiated cells in the epithelium arise from a common lineage. Furthermore, we show that reduction of Notch signalling leads to an increase in the number of midgut progenitor cells, whereas activation of the Notch pathway leads to a decrease in proliferation. Thus, the midgut progenitor's default state is proliferation, which is inhibited through the Notch signalling pathway. The ability to identify, manipulate and genetically trace cell lineages in the midgut should lead to the discovery of additional genes that regulate stem and progenitor cell biology in the gastrointestinal tract.     

扁玉螺(Neverita didyma R  ding)消化系统的形态学研究

扁玉螺消化系统由消化道——口、食道、胃、肠和直肠以及消化腺——食道腺和肝脏组成,无唾液腺．消化道粘膜上皮有纤毛柱状细胞、杯状细胞和颗粒细胞三种细胞．食道腺仅有一种腺细胞．肝脏由消化细胞、隐窝细胞和贮藏细胞组成,其中贮藏细胞未在其他腹足类动物肝脏中发现     

Building of hFⅨ transgenic mice by spermatogenic cells

Human FⅨ expression vector pCMVⅨ was packaged by effectene^TM reagent and injected into mice seminiferous tubules with glass pipettes.The expressional frame of pCMVⅨ was examined by PCR and Southern blotting among 41 progenies.There were 2(4%) mice being integrated with hFⅨ gene into chromosomes.4.6ng/mL of hFⅨ protein was expressed in plasma of one mouse,which was tested by ELISA.We demonstrated that building of transgenic animals by spermatogonial stem cells is an efficient method.Meanwhile,it has also been proved to be an alternative choice for mammary gland bioreactor.     

Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population

The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1(+) (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDR(low)/C-KIT(CD117)(neg) population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDR(low)/C-KIT(neg) cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDR(low)/C-KIT(neg) fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.     

幼龄皱纹盘鲍消化系统的组织学研究

以组织学方法研究了45日龄皱纹盘鲍的消化系统,消化道粘膜上皮呈扁平状、立方状或柱状,食道可区分为3段,食道侧囊有3种细胞成分,嗦囊比胃小,肠有5种细胞成分,唾液腺由粘液细胞和纤毛细胞组成,消化腺由消化细胞和嗜碱性细胞组成。     

Oligopotent stem cells are distributed throughout the mammalian ocular surface

The integrity of the cornea, the most anterior part of the eye, is indispensable for vision. Forty-five million individuals worldwide are bilaterally blind and another 135 million have severely impaired vision in both eyes because of loss of corneal transparency; treatments range from local medications to corneal transplants, and more recently to stem cell therapy. The corneal epithelium is a squamous epithelium that is constantly renewing, with a vertical turnover of 7 to 14 days in many mammals. Identification of slow cycling cells (label-retaining cells) in the limbus of the mouse has led to the notion that the limbus is the niche for the stem cells responsible for the long-term renewal of the cornea; hence, the corneal epithelium is supposedly renewed by cells generated at and migrating from the limbus, in marked opposition to other squamous epithelia in which each resident stem cell has in charge a limited area of epithelium. Here we show that the corneal epithelium of the mouse can be serially transplanted, is self-maintained and contains oligopotent stem cells with the capacity to generate goblet cells if provided with a conjunctival environment. Furthermore, the entire ocular surface of the pig, including the cornea, contains oligopotent stem cells (holoclones) with the capacity to generate individual colonies of corneal and conjunctival cells. Therefore, the limbus is not the only niche for corneal stem cells and corneal renewal is not different from other squamous epithelia. We propose a model that unifies our observations with the literature and explains why the limbal region is enriched in stem cells.