Hippo信号对卵巢生殖干细胞增殖及卵巢功能的影响

已有研究表明,Hippo信号通路对干细胞的自我更新和分化至关重要,且Hippo信号通路在调控卵泡生长中起重要作用,然而,目前关于Hippo通路对卵巢生殖干细胞的增殖和分化以及卵巢功能重塑的影响相关的研究较少。为了明确Hippo信号通路效应因子YAP1与卵巢生殖干细胞体外增殖分化的关系,以及Hippo信号通路对卵巢癌的主要功能。我们采用两步法酶促分离和磁性分离技术分别鉴定卵巢生殖干细胞,通过测定MVH和OCT4标记物的表达,然后选择YAP1作为Hippo信号通路的主要效应分子,作为研究的靶基因。将含有过表达的YAP1或YAP1靶向的shRNA的慢病毒转导入卵巢生殖干细胞中。通过将过表达YAP1或YAP1 shRNA的慢病毒载体微量注射到不育小鼠模型中,观察调节Hippo信号通路对卵巢的增殖、分化和内分泌功能的影响。研究结果表明,在分离的卵巢生殖干细胞中观察到YAP1和MVH的共表达。与对照组相比,过表达YAP1的卵巢生殖干细胞中MVH和OCT4表达水平显著增加。而YAP1敲低后,MVH和OCT4水平显著降低;不育小鼠模型中YAP1过表达15 d后,E2和FSH含量显著升高,而YAP1 shRNA表达后,小鼠血清E2和FSH含量显著降低。YAP1可用于调控卵巢生殖干细胞的增殖和分化以及小鼠的卵巢功能。本研究表明,Hippo信号通路可能是调控卵巢功能重建的一个新的分子靶点。     

MiR-223 Regulates Human Embryonic Stem Cell Differentiation by Targeting the IGF-1R/Akt Signaling Pathway

Currently, there are difficulties associated with the culturing of pluripotent human embryonic stem cells (hESCs), and knowledge regarding their regulatory mechanisms is limited. MicroRNAs (miRNAs) regulate gene expression and have critical functions in stem cell self-renewal and differentiation. Moreover, fibroblast growth factor (FGF) and the insulin-like growth factor receptor (IGF-1R) are key activators of signaling in hESCs. Based on the identification of complementary binding sites in miR-223 and IGF-1R mRNA, it is proposed that miR-223 acts as a local regulator of IGF-1R. Therefore, levels of miR-223 were detected in differentiated versus undifferentiated hESCs. In addition, proliferation, apoptosis, and differentiation were assayed in these two hESC populations and were compared in the presence of exogenous miR-223 and miR-223 inhibitor. Inhibition of miR-223 was found to maintain the undifferentiated state of hESCs, while addition of miR-223 induced differentiation. Furthermore, these effects were found to be likely dependent on IGF-1R/Akt signaling.     

Scratching the niche that controls Caenorhabditis elegans germline stem cells

The Caenorhabditis elegans gonad provides a well-defined model for a stem cell niche and its control of self-renewal and differentiation. The distal tip cell (DTC) forms a mesenchymal niche that controls germline stem cells (GSCs), both to generate the germline tissue during development and to maintain it during adulthood. The DTC uses GLP-1/Notch signaling to regulate GSCs; germ cells respond to Notch signaling with a network of RNA regulators to control the decision between self-renewal and entry into the meiotic cell cycle.     

BMP signaling is required for controlling somatic stem cell self-renewal in the Drosophila ovary

BMP signaling is essential for promoting self-renewal of mouse embryonic stem cells and Drosophila germline stem cells and for repressing stem cell proliferation in the mouse intestine and skin. However, it remains unknown whether BMP signaling can promote self-renewal of adult somatic stem cells. In this study, we show that BMP signaling is necessary and sufficient for promoting self-renewal and proliferation of somatic stem cells (SSCs) in the Drosophila ovary. BMP signaling is required in SSCs to directly control their maintenance and division, but is dispensable for proliferation of their differentiated progeny. Furthermore, BMP signaling is required to control SSC self-renewal, but not survival. Moreover, constitutive BMP signaling prolongs the SSC lifespan. Therefore, our study clearly demonstrates that BMP signaling directly promotes SSC self-renewal and proliferation in the Drosophila ovary. Our work further suggests that BMP signaling could promote self-renewal of adult stem cells in other systems.     

BMP signaling and stem cell regulation

Stem cells play an essential role in cellular specialization and pattern formation during embryogenesis and in tissue regeneration in adults. This is mainly due to a stem cell's ability to replenish itself (self-renewal) and, at the same time, produce differentiated progeny. Realization of these special stem cell features has changed the prospective of the field. However, regulation of stem cell self-renewal and maintenance of its potentiality require a complicated regulatory network of both extracellular cues and intrinsic programs. Understanding how signaling regulates stem cell behavior will shed light on the molecular mechanisms underlying stem cell self-renewal. In this review, we focus on comparing the progress of recent research regarding the roles of the BMP signaling pathway in different stem cell systems, including embryonic stem cells, germline stem cells, hematopoietic stem cells, and intestinal stem cells. We hope this comparison, together with a brief look at other signaling pathways, will bring a more balanced view of BMP signaling in regulation of stem cell properties, and further point to a general principle that self-renewal of stem cells may require a combination of maintenance of proliferation potential, inhibition of apoptosis, and blocking of differentiation.     

Effect of GSK-3 inhibitor on the proliferation of multipotent male germ line stem cells (mGSCs) derived from goat testis

The glycogen synthase kinase 3 (GSK3) inhibitor, 6-bromoindirubin-3′-oxime (BIO), is a key regulator of many signaling pathways to maintain pluripotency of human and mouse embryonic stem cells (ESCs). However, the effect of BIO on derivation of dairy goat male germline stem cells (mGSCs) remains unclear. The objectives of this study were to investigate whether BIO influences derivation of dairy goat mGSCs. Dairy goat mGSCs were cultured in mTeSR containing BIO medium and its effects on the proliferation ability of goat mGSCs (derived from goats ≤2 mo of age) were evaluated by 5-Bromo-2-deoxyuridine (BrdU) incorporation and alkaline phosphatase (AP) staining. Furthermore, its effects on maintenance of the undifferentiated state of mGSCs in late passages of cultures, as well as the capacity of mGSCs to differentiate into embryoid bodies (EBs) were examined. The presence of BIO increased the mitosis index and the number of AP positive colonies, as well as expression of pluripotent markers, Oct4, Nanog, Sox2, C-myc, Klf4, E-cadherin, and the proliferative markers, Pcna and C-myc. In contrast, there was no significant change in expression of apoptosis markers, P53, P21 and cyclin-related genes (Cyclin A, CDK2, Cyclin D1), as determined by RT-PCR analysis. When mGSCs were cultured in mTeSR medium containing BIO, EBs were formed, which were capable of further differentiating into various cell types found in the three embryonic germ layers, as determined by immunofluorescence and/or histologic staining. In conclusion, adding BIO to cultures BIO significantly promoted establishment of goat mGSC colonies and maintained their undifferentiated state.     

Beta-catenin can induce hair follicle stem cell differentiation into transit-amplifying cells through c-myc activation

Hair follicle stem cells play important roles in maintaining homeostasis and skin tissue self-renewal. Transit-amplifying cells represent the transition of cells from hair follicle stem cells into differentiated epidermal cells. Thus far, the signaling pathway and the molecular biological mechanism that regulate the proliferation and differentiation of hair follicle stem cells remain unclear. In this paper, we studied the relationship between β-catenin and c-myc during the process of the differentiation of hair follicle stem cells into transit-amplifying cells. Based on our results, the expression of β-catenin can activate the nuclear gene c-myc and regulate the expression of transit-amplifying cell markers K15, K19, a6-integrin and β1-integrin, indicating that β-catenin is involved in the transformation process from hair follicle stem cells to transit-amplifying cells and suggesting that β-catenin plays an important biological role in the induction of this differentiation process.     

Cytosolic O-GlcNAcylation and PNG1 maintain Drosophila gut homeostasis by regulating proliferation and apoptosis

Tissue homeostasis requires a delicate balance between stem cell self-renewal, proliferation, and differentiation. Essential to this process is glycosylation, with both intra-and extra-cellular glycosylation being required for stem cell homeostasis. However, it remains unknown how intracellular glycosylation, O-GlcNAcylation, interfaces with cellular components of the extracellular glycosylation machinery, like the cytosolic N-glycanase NGLY1. In this study, we utilize the Drosophila gut and uncover a pathway in which O-GlcNAcylation cooperates with the NGLY1 homologue PNG1 to regulate proliferation in intestinal stem cells (ISCs) and apoptosis in differentiated enterocytes. Further, the CncC antioxidant signaling pathway and ENGase, an enzyme involved in the processing of free oligosaccharides in the cytosol, interact with O-GlcNAc and PNG1 through regulation of protein aggregates to contribute to gut maintenance. These findings reveal a complex coordinated regulation between O-GlcNAcylation and the cytosolic glycanase PNG1 critical to balancing proliferation and apoptosis to maintain gut homeostasis.     

The miRNA pathway intrinsically controls self-renewal of Drosophila germline stem cells

Stem cells uniquely self-renew and maintain tissue homoeostasis by differentiating into different cell types to replace aged or damaged cells [1]. During oogenesis of Drosophila melanogaster, self-renewal of germline stem cells (GSCs) requires both intrinsic signaling mechanisms and extrinsic signals from neighboring niche cells [2]. Emerging evidence suggests that microRNA (miRNA)-mediated translational regulation may also control Drosophila GSC self-renewal [3, 4]. It is unclear, however, whether the miRNA pathway functions within stem cells or niche cells to maintain GSCs. In Drosophila, Dicer-1 (Dcr-1) and the double-stranded RNA binding protein Loquacious (Loqs) catalyze miRNA biogenesis [3-5]. Here, we generate loqs knockout (loqs(KO)) flies by ends-out homologous recombination and show that loqs is essential for embryonic viability and ovarian GSC maintenance. Both developmental and miRNA processing defects are rescued by transgenic expression of Loqs-PB, but not Loqs-PA. Furthermore, mosaic germline analysis indicates that Loqs is required intrinsically for GSC maintenance. Consistently, GSCs are restored in loqs mutant ovaries by germline expression, but not somatic expression, of Loqs-PB. Together, these results demonstrate that Loqs-PB, but not Loqs-PA, is necessary and sufficient for Drosophila development and the miRNA pathway. Our study strongly suggests that miRNAs play an intrinsic, but not extrinsic, role in Drosophila female GSC self-renewal.     

Notch信号通路与生殖干细胞研究进展

Notch信号通路是一个在进化中高度保守的信号通道,具有调控细胞增殖、分化及凋亡的作用。近年来,随着研究的不断深入,发现Notch信号通路与生殖干细胞的增殖分化及干细胞微环境的作用机理密切关联,Notch信号通路在生殖系统发育及疾病治疗中的作用机制逐渐引起人们的广泛关注。该文综合论述了Notch信号通路的生理特性及功能,重点阐述Notch信号通路在精原干细胞、卵巢生殖干细胞及生殖干细胞微环境系统中的调控机制。     

Calcineurin-NFAT signaling critically regulates early lineage specification in mouse embryonic stem cells and embryos

Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, the signaling pathways that trigger their transition from self-renewal to differentiation remain elusive. Here, we report that calcineurin-NFAT signaling is both necessary and sufficient to switch ESCs from an undifferentiated state to lineage-specific cells and that the inhibition of this pathway can maintain long-term ESC self-renewal independent of leukemia inhibitory factor. Mechanistically, this pathway converges with the Erk1/2 pathway to regulate Src expression and promote the epithelial-mesenchymal transition (EMT), a process required for lineage specification in response to differentiation stimuli. Furthermore, calcineurin-NFAT signaling is activated when the earliest differentiation event occurs in mouse embryos, and its inhibition disrupts extraembryonic lineage development. Collectively, our results demonstrate that the NFAT and Erk1/2 cascades form a signaling switch for early lineage segregation in mouse ESCs and provide significant insights into the regulation of the balance between ESC self-renewal and early lineage specification.     

果蝇生殖腺干细胞和它们的微环境

干细胞微环境是由容纳一个或多个干细胞,并控制干细胞自我更新和子代细胞产生的组织细胞以及细胞外基质组成。干细胞必须在微环境内才能增殖,才能保持自我更新的特性。通过对果蝇生殖腺干细胞微环境的结构及其产生的信号路径（该路径可以调节干细胞自我更新）的研究,发现微环境中支持细胞和它们发出的信号路径在调节干细胞的增殖和分化中起重要的作用。     

FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b through MAP2K1 activation

Fibroblast growth factor 2 (FGF2) and glial cell line-derived neurotrophic factor (GDNF) are required to recapitulate spermatogonial stem cell (SSC) self-renewal in vitro. Although studies have revealed the role of the GDNF signaling pathway in SSCs, little is known about how FGF2 is involved. In the present study, we assessed the role of the FGF2 signaling pathway using a mouse germline stem (GS) cell culture system that allows in vitro expansion of SSCs. Adding GDNF or FGF2 induced phosphorylation of MAPK1/3, and adding the MAP2K1 inhibitor PD0325091 reduced GS cell proliferation and MAPK1/3 phosphorylation. Moreover, GS cells transfected with an activated form of Map2k1 not only upregulated Etv5 and Bcl6b gene expression, but also proliferated in an FGF2-independent manner, suggesting that they act downstream of MAP2K1 signaling to drive SSC self-renewal. Although GS cells transfected with Map2k1, Etv5 or Bcl6b showed normal spermatogonial markers, transplanting GS cells expressing Bcl6b into infertile mouse testes resulted in the formation of a germ cell tumor, suggesting that excessive self-renewal signals causes tumorigenic conversion. These results show that FGF2 depends on MAP2K1 signaling to drive SSC self-renewal via upregulation of the Etv5 and Bcl6b genes.     

LncRNA-cCSC1 modulates cancer stem cell properties in colorectal cancer via activation of the Hedgehog signaling pathway

Several long noncoding RNAs (lncRNAs) have been identified in various malignant tumors and determined to contribute to the process of tumorigenesis, including that of colorectal cancer (CRC). Cancer stem cells (CSCs) have been demonstrated to promote the expansion and maintain the invasion and metastasis of cancer cells, owing to their self-renewal capacity. However, the underlying modulation mechanism of CSC-associated lncRNAs in CRC remains largely unclear. Using integrated bioinformatic analysis, we identified a novel lncRNA (lncRNA-cCSC1) that is highly expressed in CRC and colorectal cancer stem cells (CRCSCs). The biological functions of lncRNA-cCSC1 were assessed in vitro and vivo through the silencing or upregulation of its expression. The depletion of lncRNA-cCSC1 markedly inhibited the self-renewal capacity of the CRCSCs and reduced their drug resistance to 5-fluorouracil. In contrast, lncRNA-cCSC1 overexpression increased the self-renewal effect. Furthermore, aberrant lncRNA-cCSC1 expression resulted in a concomitant alteration of smoothened (SMO) and GLI family zinc finger 1 (Gli1) expression in the Hedgehog (Hh) signaling pathway. Our study is the first to identify a novel lncRNA-cCSC1 in CRC and to indicate that it may regulate CSC-like properties via the Hh signaling pathway. Thus, lncRNA-cCSC1 could be a potential biomarker and promising therapeutic target for CRC.     

The Hippo pathway regulates stem cell proliferation,self-renewal,and differentiation

Stem cells and progenitor cells are the cells of origin for multi-cellular organisms and organs. They play key roles during development and their dysregulation gives rise to human diseases such as cancer. The recent development of induced pluripotent stem cell (iPSC) technology which converts somatic cells to stem-like cells holds great promise for regenerative medicine. Nevertheless, the understanding of proliferation, differentiation, and self-renewal of stem cells and organ-specific progenitor cells is far from clear. Recently, the Hippo pathway was demonstrated to play important roles in these processes. The Hippo pathway is a newly established signaling pathway with critical functions in limiting organ size and suppressing tumorigenesis. This pathway was first found to inhibit cell proliferation and promote apoptosis, therefore regulating cell number and organ size in both Drosophila and mammals. However, in several organs, disturbance of the pathway leads to specific expansion of the progenitor cell compartment and manipulation of the pathway in embryonic stem cells strongly affects their self-renewal and differentiation. In this review, we summarize current observations on roles of the Hippo pathway in different types of stem cells and discuss how these findings changed our view on the Hippo pathway in organ development and tumorigenesis.