The interaction of the Wnt and Notch pathways modulates natural killer versus T cell differentiation

The Wnt and Notch signaling pathways have been independently shown to play a critical role in regulating hematopoietic cell fate decisions. We previously reported that induction of Notch signaling in human CD34(+)CD38(-) cord blood cells by culture with the Notch ligand Delta 1 resulted in more cells with T or natural killer (NK) lymphoid precursor phenotype. Here, we show that addition of Wnt3a to Delta 1 further increased the percentage of CD34(-)CD7(+) and CD34(-)CD7(+)cyCD3(+) cells with increased expression of CD3 epsilon and preT alpha. In contrast, culture with Wnt3a alone did not increase generation of CD34(-)CD7(+) precursors or expression of CD3 epsilon or preT alpha gene. Furthermore, Wnt3a increased the amount of activated Notch1, suggesting that Wnt modulates Notch signaling by affecting Notch protein levels. In contrast, addition of a Wnt signaling inhibitor to Delta 1 increased the percentage of CD56(+) NK cells. Overall, these results demonstrate that regulation of Notch signaling by the Wnt pathway plays a critical role in differentiation of precursors along the early T or NK differentiation pathways. Disclosure of potential conflicts of interest is found at the end of this article.     

Isolation of multipotent neural crest-derived stem cells from the adult mouse cornea

We report the presence of neural crest-derived corneal precursors (COPs) that initiate spheres by clonal expansion from a single cell. COPs expressed the stem cell markers nestin, Notch1, Musashi-1, and ABCG2 and showed the side population cell phenotype. COPs were multipotent with the ability to differentiate into adipocytes, chondrocytes, as well as neural cells, as shown by the expression of beta-III-tubulin, glial fibrillary acidic protein, and neurofilament-M. COP spheres prepared from E/nestin-enhanced green fluorescent protein (EGFP) mice showed induction of EGFP expression that was not originally observed in the cornea, indicating activation of the neural-specific nestin second intronic enhancer in culture. COPs were Sca-1(+), CD34(+), CD45(-), and c-kit(-). Numerous GFP(+) cells were observed in the corneas of mice transplanted with whole bone marrow of transgenic mice ubiquitously expressing GFP; however, no GFP(+) COP spheres were initiated from these mice. On the other hand, COP spheres from transgenic mice encoding P0-Cre/Floxed-EGFP as well as Wnt1-Cre/Floxed-EGFP were GFP(+), indicating the neural crest origin of COPs, which was confirmed by the expression of the embryonic neural crest markers Twist, Snail, Slug, and Sox9. Taken together, these data indicate the existence of neural crest-derived, multipotent stem cells in the adult cornea.     

Wnt信号途径促进脂肪间充质干细胞向Ⅱ型肺泡上皮细胞分化

背景：脂肪间充质干细胞向Ⅱ型肺泡上皮细胞定向分化的能力以及调节机制尚未完全阐明。 目的：观察脂肪间充质干细胞在体外分化为Ⅱ型肺泡上皮的能力以及Wnt途径对分化的调节作用。 方法：取大鼠脂肪组织,体外分离培养脂肪间充质干细胞并通过流式细胞术进行鉴定。实验分为对照组、小气道生长液组和Wnt3a组,对照组用普通DMEM培养基培养,小气道生长液组和Wnt3a组均使用小气道生长液培养,且Wnt3a组加入Wnt信号通路激动剂Wnt3a培养。诱导10 d后分别通过qRT-PCR和免疫荧光检测Ⅱ型肺泡上皮标志物肺表面活性蛋白B,C,D的表达,并于诱导5 d和10 d时通过Western blot检测磷酸化β-catenin和GSK-3β。 结果与结论：大鼠脂肪组织中可成功分离出纯度较高的脂肪间充质干细胞,可表达CD44和CD29,不表达CD11b和CD45;经小气道生长液诱导后,脂肪间充质干细胞中肺表面活性蛋白B,C,D蛋白和mRNA表达均上调(P < 0.01),表明其可被诱导为Ⅱ型上皮细胞;加入Wnt3a后,经诱导的脂肪间充质干细胞中肺表面活性蛋白B,C,D蛋白和mRNA表达均高于小气道生长液组(P < 0.01),且在诱导过程中磷酸化β-catenin表达随时间逐渐上调而GSK-3β表达逐渐下调(P < 0.01)。结果证实,Wnt信号通路在脂肪间充质干细胞诱导分化为Ⅱ肺泡上皮细胞过程中激活并促进干细胞的定向分化。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程      

Wnt Signaling and the Control of Human Stem Cell Fate

Wnt signaling determines major developmental processes in the embryonic state and regulates maintenance, self-renewal and differentiation of adult mammalian tissue stem cells. Both β-catenin dependent and independent Wnt pathways exist, and both affect stem cell fate in developing and adult tissues. In this review, we debate the response to Wnt signal activation in embryonic stem cells and human, adult stem cells of mesenchymal, hematopoetic, intestinal, gastric, epidermal, mammary and neural lineages, and discuss the need for Wnt signaling in these cell types. Due to the vital actions of Wnt signaling in developmental and maintenance processes, deregulation of the pathway can culminate into a broad spectrum of developmental and genetic diseases, including cancer. The way in which Wnt signals can feed tumors and maintain cancer stem stells is discussed as well. Manipulation of Wnt signals both in vivo and in vitro thus carries potential for therapeutic approaches such as tissue engineering for regenerative medicine and anti-cancer treatment. Although many questions remain regarding the complete Wnt signal cell-type specific response and interplay of Wnt signaling with pathways such as BMP, Hedgehog and Notch, we hereby provide an overview of current knowledge on Wnt signaling and its control over human stem cell fate.     

Role for notch signaling in salivary acinar cell growth and differentiation

The Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. The Notch signaling is essential for Drosophila salivary gland development but its role in mammalian salivary gland remains unclear. The human salivary epithelial cell line, HSG, was studied to determine the role of Notch signaling in salivary epithelial cell differentiation. HSG expressed Notch 1 to 4, and the Notch ligands Jagged 1 and 2 and Delta 1. Treatment of HSG cells with inhibitors of γ‐secretase, which is required for Notch cleavage and activation, blocked vimentin and cystatin S expression, an indicator of HSG differentiation. HSG differentiation was also associated with Notch downstream signal Hes‐1 expression, and Hes‐1 expression was inhibited by γ‐secretase inhibitors. siRNA corresponding to Notch 1 to 4 was used to show that silencing of all four Notch receptors was required to inhibit HSG differentiation. Normal human submandibular gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1, with nuclear localization indicating Notch signaling in vivo. Hes‐1 was also expressed in the human tissue, with staining predominantly in the ductal cells. In salivary tissue from rats undergoing and recovering from ductal obstruction, we found that Notch receptors and ligands were expressed in the nucleus of the regenerating epithelial cells. Taken together, these data suggest that Notch signaling is critical for normal salivary gland cell growth and differentiation. Developmental Dynamics 238:724–731, 2009. © 2009 Wiley‐Liss, Inc.     

An evolving web of signaling networks regulated by Cripto-1

Over the past few decades, our understanding of the embryonic gene Cripto-1 has considerably advanced through biochemical, cell biology, and animal studies. Cripto-1 performs key functions during embryonic development, while it dramatically disappears in adult tissues, except possibly in adult tissue stem cells. Cripto-1 is re-expressed in human tumors promoting cell proliferation, migration, invasion, epithelial to mesenchymal transition, and tumor angiogenesis. This diversity of biological effects is dependent upon interaction of Cripto-1 with an extensive array of signaling molecules. In fact, Cripto-1 modulates signaling of transforming growth factor-β family members, including Nodal, GDF-1/-3, Activin, and TGF-β1, activates c-src/MAPK/Protein Kinase B (AKT) pathway in a Glypican-1 and GRP78-dependent manner, and cross-talks with erbB4, Wnt/β-catenin, Notch, Caveolin-1, and Apelin/putative receptor protein related to Angiotensin-type I receptor (APJ) pathways. This article provides an updated survey of the various signaling pathways modulated by Cripto-1 with a focus on mechanistic insights in our understanding of the biological function of Cripto-1 in eukaryotic cells.     

The H(+) vacuolar ATPase maintains neural stem cells in the developing mouse cortex

The vacuolar H(+) ATPase (v-ATPase) is crucial for endosome acidification, endocytosis, and trafficking in essentially all eukaryotic cells. Recent studies have shown that inhibition of the v-ATPase also leads to downregulation of important signaling pathways, including Notch and Wnt, which are key regulators of cell differentiation and tissue homeostasis across the animal kingdom. However, the requirement of endosome acidification and endocytosis in the transduction of Notch signaling is still highly debated. Moreover, no study has yet investigated the role of the v-ATPase during mammalian development. Here we show that expression of a dominant-negative subunit of the v-ATPase in neural precursors of the developing mouse cortex depleted neural stem cells by promoting their differentiation and the generation of neurons. Moreover, inhibition of the v-ATPase reduced endogenous Notch signaling and prevented the proliferative effect of a transmembrane, γ-secretase-dependent, active Notch without blocking the effects of its cytoplasmic intracellular domain (NICD). Our data are consistent with recent reports in Drosophila in which the v-ATPase has been suggested to be important for the transduction of Notch signaling. By extending these reports to mammalian embryos, our data may contribute to a better understanding of the role of the v-ATPase, endosome acidification, and endocytosis in signal transduction during neural stem cell differentiation and brain development.     

Hes1 regulates corneal development and the function of corneal epithelial stem/progenitor cells

Hes1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of Hes1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in Hes1-null mice. Hes1-null corneas manifested abnormal junctional specialization, cell differentiation, and less cell proliferation ability. Worthy of note, Hes1 is expressed mainly in the corneal epithelial stem/progenitor cells and is not detected in the differentiated corneal epithelial cells. Expression of Hes1 is closely linked with corneal epithelial stem/progenitor cell proliferation activity in vivo. Moreover, forced Hes1 expression inhibits the differentiation of corneal epithelial stem/progenitor cells and maintains these cells' undifferentiated state. Our data provide the first evidence that Hes1 regulates corneal development and the homeostatic function of corneal epithelial stem/progenitor cells.     

Effects of Delta1 and Jagged1 on early human hematopoiesis: correlation with expression of notch signaling-related genes in CD34+ cells

It has been shown that Notch signaling mediated by ligands of both Jagged and Delta families expands the hematopoietic stem cell compartment while blocking or delaying terminal myeloid differentiation. Here we show that Delta1- and Jagged1-expressing stromal cells have distinct effects on the clonogenic and differentiation capacities of human CD34(+) CD38(+) cells. Jagged1 increases the number of bipotent colony-forming unit-granulocyte macrophage (CFU-GM) and unipotent progenitors (CFU-granulocytes and CFU-macrophages), without quantitatively affecting terminal cell differentiation, whereas Delta1 reduces the number of CFU-GM and differentiated monocytic cells. Expression analysis of genes coding for Notch receptors, Notch targets, and Notch signaling modulators in supernatant CD34(+) cells arising upon contact with Jagged1 and Delta1 shows dynamic and differential gene expression profiles over time. At early time points, modest upregulation of Notch1, Notch3, and Hes1 was observed in Jagged1-CD34(+) cells, whereas those in contact with Delta1 strikingly upregulated Notch3 and Hes1. Later, myeloid progenitors with strong clonogenic potential emerging upon contact with Jagged1 upregulated Notch1 and Deltex and downregulated Notch signaling modulators, whereas T/NK progenitors originated by Delta1 strikingly upregulated Notch3 and Deltex and, to a lesser extent, Hes1, Lunatic Fringe, and Numb. Together, the data unravel previously unrecognized expression patterns of Notch signaling-related genes in CD34(+) CD38(+) cells as they develop in Jagged1- or Delta1-stromal cell environments, which appear to reflect sequential maturational stages of CD34(+) cells into distinct cell lineages.     

Mimicking cell-cell interactions at the biomaterial-cell interface for control of stem cell differentiation

The ability to regulate stem cell proliferation and differentiation has relevance in numerous medical applications, including medical devices, tissue engineering, and regenerative medicine. To control cellular behavior at the biomaterial or scaffold interface, many studies have employed surface modifications that mimic the extracellular matrix. Strikingly absent is the immobilization of cell-surface ligands to the biomaterial surface. One cell-to-cell signaling pathway that has been shown to regulate tissue development and stem cell fate is the Notch pathway. Recently, the Notch signaling pathway was identified as a key regulator of epithelial differentiation. Utilizing this knowledge, we applied an affinity immobilization scheme designed to attach and orient the Notch ligand, Jagged-1, in an active conformation on a biomaterial surface. When epithelial stem cells were plated on the bound ligand, the Notch/CBF-1 signaling pathway was stimulated and the cells upregulated both intermediate- and late-stage differentiation markers. In addition, the ligand promoted tight clustering and extensive stratification. Soluble Jagged-1 showed no Notch/CBF-1 signaling and very little, if any, cell differentiating activity. The high potency of bound Jagged-1 suggests that modification of a surface with a Notch ligand presents a powerful method to control stem cell differentiation at the cell-biomaterial interface.     

Notch ligand-bearing thymic epithelial cells initiate and sustain Notch signaling in thymocytes independently of T cell receptor signaling.

Thymic epithelial cells are specialized to play essential roles at multiple stages of T cell development in the thymus, yet the molecular basis of this specialization is largely unknown. Recently, the Notch family of transmembrane proteins has been implicated in thymocyte development. Such proteins interact with cell surface proteins of the Delta-like and Jagged families. It is known that Notch ligands are expressed intrathymically, and that Notch signaling is regulated by Notch ligands expressed on either the same or third-party cells. However, functional analysis of Notch ligand expression, and elucidation of the mechanism of Notch ligand signaling in thymocyte development, are unclear. Here, we find that Notch ligand expression in the thymus is compartmentalized, with MHC class II(+) thymic epithelium, but not thymocytes nor dendritic cells, expressing Jagged-1, Jagged-2 and Delta-like-1. We also provide evidence that contact with Notch ligands on thymic epithelium is necessary to activate and sustain Notch signaling in thymocytes, and that this can occur independently of positive selection induction. Our data suggest that Notch ligand expression by thymic epithelium may partly explain the specialization of these cells in supporting thymocyte development, by regulating Notch activation via an inductive signaling mechanism independently of signaling leading to positive selection.     

Notch和Wnt信号通路对神经干细胞增殖分化的影响

Notch和Wnt信号通路是调节神经干细胞(neural stem cells,NSCs)增殖、分化的重要通路,Notch信号通路的靶基因Hes1、Hes5及HES相关蛋白等分化抑制信号,通过旁侧抑制机制阻止NSCs的分化,并促进其自我更新;通过NICD与CSL DNA结合蛋白的直接结合,形成GFAP的转录激活复合物,上调GFAP的表达,从而促进NSCs向星形胶质细胞的分化。Wnt信号通过Wnt/β-catenin信号通路对细胞周期素D1和D2的转录调节,调控NSCs细胞周期的进程,使其量增殖;然而,过表达的Wnt3a和Wnt7a蛋白能够抑制NSCs的增殖,促进NSCs向神经元方向分化。     

Enrichment of prostate cancer stem cells from primary prostate cancer cultures of biopsy samples

This study was to enrich prostate cancer stem cells (PrCSC) from primary prostate cancer cultures (PPrCC). Primary prostate cancer cells were amplified in keratinocyte serum-free medium with epidermal growth factor (EGF) and bovine pituitary extract (BPE), supplemented with leukemia inhibitory factor (LIF), stem cell factor (SCF) and cholera toxin. After amplification, cells were transferred into ultra-low attachment dishes with serum-free DMEM/F12 medium, supplemented with EGF, basic fibroblast growth factor (bFGF), bovine serum albumin (BSA), insulin, and N2 nutrition. Expression of cell-type-specific markers was determined by RT-qPCR and immunostaining. Tumorigenicity of enriched PrCSC was determined by soft agar assay and xenograft assay in NOD/SCID mice. Biopsy samples from 19 confirmed prostate cancer patients were used for establishing PPrCC, and 18 cases (95%) succeeded. Both basal marker (CK5) and luminal markers (androgen receptor and CK8) strongly co-expressed in most of PPrCC, indicating their basal epithelial origin. After amplification under adherent culture condition in vitro, transient amplifying cells were the dominant cells. Sphere formation efficiency (SFE) of passaged PPrCC was about 0.5%, which was 27 times lower than SFE of LNCaP (13.67%) in the same condition. Compared with adherent cells from PPrCC, prostasphere from PPrCC showed up regulated stem cell markers and increased tumorigenic potential in soft-agar assay. However, spheroid cells from PPrCC prostasphere failed to initiate tumor in xenograft assay in 6 months. Thus, PPrCC can be established and amplified from prostate cancer biopsy samples. Our modified sphere culture system can enrich PrCSC from PPrCC.     

Bone morphogenetic protein signaling inhibits hair follicle anagen induction by restricting epithelial stem/progenitor cell activation and expansion

Epithelial stem cells (EP-SCs) located in the bulge region of a hair follicle (HF) have the potential to give rise to hair follicle stem/progenitor cells that migrate down to regenerate HFs. Bone morphogenetic protein (BMP) signaling has been shown to regulate the HF cycle by inhibiting anagen induction. Here we show that active BMP signaling functions to prevent EP-SC activation and expansion. Dynamic expression of Noggin, a BMP antagonist, releases EP-SCs from BMP-mediated restriction, leading to EP-SC activation and initiation of the anagen phase. Experimentally induced conditional inactivation of the BMP type IA receptor (Bmpr1a) in EP-SCs leads to overproduction of HF stem/progenitor cells and the eventual formation of matricomas. This genetic manipulation of the BMP signaling pathway also reveals unexpected activation of beta-catenin, a major mediator of Wnt signaling. We propose that BMP activity controls the HF cycle by antagonizing Wnt/beta-catenin activity. This is at least partially achieved by BMP-mediated enhancement of transforming growth factor-beta-regulated epithelial cell-specific phosphatase (PTEN) function. Subsequently, PTEN, through phosphatidyl inositol 3-kinase-Akt, inhibits the activity of beta-catenin, the convergence point of the BMP and Wnt signaling pathways.     

Wnt和Notch信号通路在肿瘤干细胞自我更新中的作用

肿瘤干细胞是一类能够导致肿瘤发生的具有自我更新能力的细胞，它与干细胞具有很多相似性，其中最重要的一点是自我更新能力。它们具有相似的自我更新调节通路，如：Wnt，Notch和Shh(Sonic hedgehog)。Wnt和Notch信号通路通过其受体和配体的相互作用在自我更新的增殖和分化中都起着重要的作用，两者均能促进干细胞增殖而抑制其分化，但各自侧重不同。此外，Wnt和Notch信号通路之间相互作用、协调共同完成干细胞的自我更新。对肿瘤干细胞的Wnt和Notch信号通路研究将为未来肿瘤的靶向治疗提供新的方向。