Regulatory networks define phenotypic classes of human stem cell lines

Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal and adult sources have been called stem cells, even though they range from pluripotent cells-typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation-to adult stem cell lines, which can generate a far more limited repertoire of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine has highlighted the need for a general, reproducible method for classification of these cells. We report here the creation and analysis of a database of global gene expression profiles (which we call the 'stem cell matrix') that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent and differentiated cell types. Using an unsupervised clustering method to categorize a collection of approximately 150 cell samples, we discovered that pluripotent stem cell lines group together, whereas other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis we uncovered a protein-protein network (PluriNet) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas and induced pluripotent cells). Analysis of published data showed that the PluriNet seems to be a common characteristic of pluripotent cells, including mouse embryonic stem and induced pluripotent cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotency and self-renewal are under tight control by specific molecular networks.     

Self-renewal and expansion of single transplanted muscle stem cells

Adult muscle satellite cells have a principal role in postnatal skeletal muscle growth and regeneration. Satellite cells reside as quiescent cells underneath the basal lamina that surrounds muscle fibres and respond to damage by giving rise to transient amplifying cells (progenitors) and myoblasts that fuse with myofibres. Recent experiments showed that, in contrast to cultured myoblasts, satellite cells freshly isolated or satellite cells derived from the transplantation of one intact myofibre contribute robustly to muscle repair. However, because satellite cells are known to be heterogeneous, clonal analysis is required to demonstrate stem cell function. Here we show that when a single luciferase-expressing muscle stem cell is transplanted into the muscle of mice it is capable of extensive proliferation, contributes to muscle fibres, and Pax7(+)luciferase(+) mononucleated cells can be readily re-isolated, providing evidence of muscle stem cell self-renewal. In addition, we show using in vivo bioluminescence imaging that the dynamics of muscle stem cell behaviour during muscle repair can be followed in a manner not possible using traditional retrospective histological analyses. By imaging luciferase activity, real-time quantitative and kinetic analyses show that donor-derived muscle stem cells proliferate and engraft rapidly after injection until homeostasis is reached. On injury, donor-derived mononucleated cells generate massive waves of cell proliferation. Together, these results show that the progeny of a single luciferase-expressing muscle stem cell can both self-renew and differentiate after transplantation in mice, providing new evidence at the clonal level that self-renewal is an autonomous property of a single adult muscle stem cell.     

Continuous in vitro generation of multipotential stem cell clones from src-infected cultures

A molecular recombinant of Rous sarcoma virus and murine amphotropic leukaemia virus, src(MoMuLV), where the avian src oncogene has been placed under the influence of a murine virus promoter sequence, has been reported. Infection of long-term marrow cultures with this virus led to a dramatic change in the relative numbers of stem cells, granulocyte-macrophage progenitor cells and mature cells found in normal haematopoietic cell development. However, although the balance between self-renewal, differentiation and development was disturbed, injection of the cultured cells into irradiated syngeneic recipients did not lead to the development of leukaemia. Thus, although the control had been 'loosened', the host regulatory mechanisms were sufficient to impose a restraint on unlimited growth of the cells. We now show that the stem cells from the src-infected cultures show a remarkably increased capacity for self-renewal in vitro in situations which are inimical to the maintenance of self-renewal in normal uninfected stem cells and that self-renewal/differentiation can be modified by the culture conditions.     

Somatic support cells restrict germline stem cell self-renewal and promote differentiation

Stem cells maintain populations of highly differentiated, short-lived cell-types, including blood, skin and sperm, throughout adult life. Understanding the mechanisms that regulate stem cell behaviour is crucial for realizing their potential in regenerative medicine. A fundamental characteristic of stem cells is their capacity for asymmetric division: daughter cells either retain stem cell identity or initiate differentiation. However, stem cells are also capable of symmetric division where both daughters remain stem cells, indicating that mechanisms must exist to balance self-renewal capacity with differentiation. Here we present evidence that support cells surrounding the stem cells restrict self-renewal and control stem cell number by ensuring asymmetric division. Loss of function of the Drosophila Epidermal growth factor receptor in somatic cells disrupted the balance of self-renewal versus differentiation in the male germline, increasing the number of germline stem cells. We propose that activation of this receptor specifies normal behaviour of somatic support cells; in turn, the somatic cells play a guardian role, providing information that prevents self-renewal of stem cell identity by the germ cell they enclose.     

Generation of a prostate from a single adult stem cell

The existence of prostate stem cells (PSCs) was first postulated from the observation that normal prostate regeneration can occur after repeated cycles of androgen deprivation and replacement in rodents. Given the critical role of PSCs in maintaining prostate tissue integrity and their potential involvement in prostate tumorigenesis, it is important to define specific markers for normal PSCs. Several cell-surface markers have been reported to identify candidate PSCs, including stem cell antigen-1 (Sca-1, also known as Ly6a), CD133 (Prom1) and CD44 (refs 3-10). However, many non-PSCs in the mouse prostate also express these markers and thus identification of a more defined PSC population remains elusive. Here we identify CD117 (c-kit, stem cell factor receptor) as a new marker of a rare adult mouse PSC population, and demonstrate that a single stem cell defined by the phenotype Lin(-)Sca-1(+)CD133(+)CD44(+)CD117(+) can generate a prostate after transplantation in vivo. CD117 expression is predominantly localized to the region of the mouse prostate proximal to the urethra and is upregulated after castration-induced prostate involution-two characteristics consistent with that of a PSC marker. CD117(+) PSCs can generate functional, secretion-producing prostates when transplanted in vivo. Moreover, CD117(+) PSCs have long-term self-renewal capacity, as evidenced by serial isolation and transplantation in vivo. Our data establish that single cells in the adult mouse prostate with multipotent, self-renewal capacity are defined by a Lin(-)Sca-1(+)CD133(+)CD44(+)CD117(+) phenotype.     

Intrinsic and extrinsic control of haematopoietic stem-cell self-renewal

When stem cells divide, they can generate progeny with the same developmental potential as the original cell, a process referred to as self-renewal. Self-renewal is driven intrinsically by gene expression in a cell-type-specific manner and is modulated through interactions with extrinsic cues from the environment, such as growth factors. However, despite the prevalence of the term self-renewal in the scientific literature, this process has not been defined at the molecular level. Haematopoietic stem cells are an excellent model for the study of self-renewal because they can be isolated prospectively, manipulated relatively easily and assessed by using well-defined assays. Establishing the principles of self-renewal in haematopoietic stem cells will lead to insights into the mechanisms of self-renewal in other tissues.     

Wnt signalling regulates adult hippocampal neurogenesis

The generation of new neurons from neural stem cells is restricted to two regions of the adult mammalian central nervous system: the subventricular zone of the lateral ventricle, and the subgranular zone of the hippocampal dentate gyrus. In both regions, signals provided by the microenvironment regulate the maintenance, proliferation and neuronal fate commitment of the local stem cell population. The identity of these signals is largely unknown. Here we show that adult hippocampal stem/progenitor cells (AHPs) express receptors and signalling components for Wnt proteins, which are key regulators of neural stem cell behaviour in embryonic development. We also show that the Wnt/beta-catenin pathway is active and that Wnt3 is expressed in the hippocampal neurogenic niche. Overexpression of Wnt3 is sufficient to increase neurogenesis from AHPs in vitro and in vivo. By contrast, blockade of Wnt signalling reduces neurogenesis from AHPs in vitro and abolishes neurogenesis almost completely in vivo. Our data show that Wnt signalling is a principal regulator of adult hippocampal neurogenesis and provide evidence that Wnt proteins have a role in adult hippocampal function.     

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.     

Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision

Adult neurogenesis arises from neural stem cells within specialized niches. Neuronal activity and experience, presumably acting on this local niche, regulate multiple stages of adult neurogenesis, from neural progenitor proliferation to new neuron maturation, synaptic integration and survival. It is unknown whether local neuronal circuitry has a direct impact on adult neural stem cells. Here we show that, in the adult mouse hippocampus, nestin-expressing radial glia-like quiescent neural stem cells (RGLs) respond tonically to the neurotransmitter γ-aminobutyric acid (GABA) by means of γ2-subunit-containing GABAA receptors. Clonal analysis of individual RGLs revealed a rapid exit from quiescence and enhanced symmetrical self-renewal after conditional deletion of γ2. RGLs are in close proximity to terminals expressing 67-kDa glutamic acid decarboxylase (GAD67) of parvalbumin-expressing (PV+) interneurons and respond tonically to GABA released from these neurons. Functionally, optogenetic control of the activity of dentate PV+ interneurons, but not that of somatostatin-expressing or vasoactive intestinal polypeptide (VIP)-expressing interneurons, can dictate the RGL choice between quiescence and activation. Furthermore, PV+ interneuron activation restores RGL quiescence after social isolation, an experience that induces RGL activation and symmetrical division. Our study identifies a niche cell–signal–receptor trio and a local circuitry mechanism that control the activation and self-renewal mode of quiescent adult neural stem cells in response to neuronal activity and experience.     

肝脏干细胞研究进展

肝干细胞是一类具有自我更新能力和多项分化潜能的细胞 .在一定病理条件下可向肝细胞和胆管上皮细胞、胰腺外分泌上皮等有限的几种组织分化 ,具有多项分化增殖潜力 ,本文将就肝干细胞分类、表面标志、来源及人类肝干细胞的研究做一综述 .     

牦牛乳腺上皮细胞的体外培养的研究

采用胶原酶消化法和胰蛋白酶选择性消化法分离、培养和纯化牦牛乳腺上皮细胞．形态学观察表明,培养的细胞具有典型的上皮细胞形态特征．染色体数目分析表明,细胞核型稳定,在离体培养条件下不发生转化．牦牛乳腺上皮细胞染色体数目为60,染色体组型为2n=60．     

A role for Wnt signalling in self-renewal of haematopoietic stem cells

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.     

Distinct stem cells contribute to mammary gland development and maintenance

The mammary epithelium is composed of several cell lineages including luminal, alveolar and myoepithelial cells. Transplantation studies have suggested that the mammary epithelium is maintained by the presence of multipotent mammary stem cells. To define the cellular hierarchy of the mammary gland during physiological conditions, we performed genetic lineage-tracing experiments and clonal analysis of the mouse mammary gland during development, adulthood and pregnancy. We found that in postnatal unperturbed mammary gland, both luminal and myoepithelial lineages contain long-lived unipotent stem cells that display extensive renewing capacities, as demonstrated by their ability to clonally expand during morphogenesis and adult life as well as undergo massive expansion during several cycles of pregnancy. The demonstration that the mammary gland contains different types of long-lived stem cells has profound implications for our understanding of mammary gland physiology and will be instrumental in unravelling the cells at the origin of breast cancers.     

Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development

How environmental cues regulate adult stem cell and cancer cell activity through surface receptors is poorly understood. Angiopoietin-like proteins (ANGPTLs), a family of seven secreted glycoproteins, are known to support the activity of haematopoietic stem cells (HSCs) in vitro and in vivo. ANGPTLs also have important roles in lipid metabolism, angiogenesis and inflammation, but were considered 'orphan ligands' because no receptors were identified. Here we show that the immune-inhibitory receptor human leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse orthologue paired immunoglobulin-like receptor (PIRB) are receptors for several ANGPTLs. LILRB2 and PIRB are expressed on human and mouse HSCs, respectively, and the binding of ANGPTLs to these receptors supported ex vivo expansion of HSCs. In mouse transplantation acute myeloid leukaemia models, a deficiency in intracellular signalling of PIRB resulted in increased differentiation of leukaemia cells, revealing that PIRB supports leukaemia development. Our study indicates an unexpected functional significance of classical immune-inhibitory receptors in maintenance of stemness of normal adult stem cells and in support of cancer development.