Control Stem Cell Maintenance Research Articles

The logistics of Wnt production and delivery.

Wnts are secreted proteins that control stem cell maintenance, cell fate decisions, and growth during development and adult homeostasis. Wnts carry a post-translational modification not seen in any other secreted protein: during biosynthesis, they are appended with a palmitoleoyl moiety that is required for signaling but also impairs solubility and hence diffusion in the extracellular space. In some contexts, Wnts act only in a juxtacrine manner but there are also instances of long range action. Several proteins and processes ensure that active Wnts reach the appropriate target cells. Some, like Porcupine, Wntless, and Notum are dedicated to Wnt function; we describe their activities in molecular detail. We also outline how the cell infrastructure (secretory, endocytic, and retromer pathways) contribute to the progression of Wnts from production to delivery. We then address how Wnts spread in the extracellular space and form a signaling gradient despite carrying a hydrophobic moiety. We highlight particularly the role of lipid-binding Wnt interactors and heparan sulfate proteoglycans. Finally, we briefly discuss how evolution might have led to the emergence of this unusual signaling pathway.

Molecular dynamics of estrogen-related receptors and their regulatory proteins: roles in transcriptional control for endocrine and metabolic signaling.

Estrogen-related receptor (ERR) is a member of the nuclear receptor (NR) superfamily and has three subtypes α, β, and γ. Despite their strong homology with estrogen receptor (ER) α, ERRs cannot accommodate endogenous hormones. However, they are able to regulate gene expression without ligand binding. ERRα and ERRγ orchestrate the expression of genes involved in bioenergetic pathways, while ERRβ controls placental development and stem cell maintenance. Evidence from recent studies, including clinical research, has also demonstrated close associations of ERRs with the pathophysiology of hormone-related cancers and metabolic disorders including type 2 diabetes mellitus. This review summarizes the basic knowledge and recent advances in ERRs and their associated proteins, focusing on the subcellular dynamics involved in transcriptional regulation. Fluorescent protein labeling enabled monitoring of ERRs in living cells and revealed previously unrecognized characteristics. Using this technique, we demonstrated a role of ERRβ in controlling estrogen signaling by regulating thesubnuclear dynamics of ligand-activated ERα. Visualization of ERRs and related proteins and subsequent analyses also revealed a function of ERRγ in promoting liver lactate metabolism in association with LRPGC1, a recently identified lactic acid-responsive protein. These findings suggest that ERRs activate unique transregulation mechanisms in response to extracellular stimuli such as hormones and metabolic signals, implying an adaptive system behind the cellular homeostatic regulation by orphan NRs. Control of subcellular ERR dynamics will contribute toward the development of therapeutic approaches to treat various diseases including hormone-related cancers and metabolic disorders associated with abnormal ERR signaling pathways.

Yap1-Driven Intestinal Repair Is Controlled by Group 3 Innate Lymphoid Cells

Tissue repair requires temporal control of progenitor cell proliferation and differentiation to replenish damaged cells. In response to acute insult, group 3 innate lymphoid cells (ILC3s) regulate intestinal stem cell maintenance and subsequent tissue repair. ILC3-derived IL-22 is important for stem cell protection, but the mechanisms of ILC3-driven tissue regeneration remain incompletely defined. Here we report that ILC3-driven epithelial proliferation and tissue regeneration are independent of IL-22. In contrast, ILC3s amplify the magnitude of Hippo-Yap1 signaling in intestinal crypt cells, ensuring adequate initiation of tissue repair and preventing excessive pathology. Mechanistically, ILC3-driven tissue repair is Stat3 independent, but it involves activation of Src family kinases. Our findings reveal that ILC3-driven intestinal repair entails distinct transcriptional networks to control stem cell maintenance and epithelial regeneration, which implies that tissue repair and crypt proliferation can be influenced by targeting innate immune cells independent of the well-established effects of IL-22.

P.105 - DMD Genetic registry in Russia.

The ability to sense and adapt to low oxygen levels (hypoxia) is central for most organisms and cell types. At the center of this process is a molecular mechanism, the cellular hypoxic response, in which the hypoxia inducible factors (HIFs) are stabilized by hypoxia, allowing the HIF proteins to act as master transcriptional regulators to adjust the cell to a low oxygen environment. In recent years, it has become increasingly appreciated that the cellular hypoxic response does not always operate in splendid isolation, but intersects with signaling mechanisms such as Notch signaling, a key regulatory signaling mechanism operating in most cell types controlling stem cell maintenance and differentiation. In this review, which is dedicated to the memory of Lorenz Poellinger,1 we discuss how the intersection between Notch and the cellular hypoxic response was discovered and our current understanding of the molecular basis for the cross-talk. We also provide examples of where Notch and hypoxia intersect in various physiological and disease contexts.

The interplay between the cellular hypoxic response and Notch signaling

The ability to sense and adapt to low oxygen levels (hypoxia) is central for most organisms and cell types. At the center of this process is a molecular mechanism, the cellular hypoxic response, in which the hypoxia inducible factors (HIFs) are stabilized by hypoxia, allowing the HIF proteins to act as master transcriptional regulators to adjust the cell to a low oxygen environment. In recent years, it has become increasingly appreciated that the cellular hypoxic response does not always operate in splendid isolation, but intersects with signaling mechanisms such as Notch signaling, a key regulatory signaling mechanism operating in most cell types controlling stem cell maintenance and differentiation. In this review, which is dedicated to the memory of Lorenz Poellinger,11This review article is dedicated to the memory of the late Lorenz Poellinger, who was one of the leaders in hypoxia signaling (see Refs. [2,3,11,13]) and was instrumental for the early phases of unraveling the Notch-hypoxia cross-talk (see Refs. [15,16,20,22,32,63,85]). He will be missed as a friend and scientist. we discuss how the intersection between Notch and the cellular hypoxic response was discovered and our current understanding of the molecular basis for the cross-talk. We also provide examples of where Notch and hypoxia intersect in various physiological and disease contexts.

Hedgehog signaling establishes precursors for germline stem cell niches by regulating cell adhesion.

Stem cells require different types of supporting cells, or niches, to control stem cell maintenance and differentiation. However, little is known about how those niches are formed. We report that in the development of the Drosophila melanogaster ovary, the Hedgehog (Hh) gradient sets differential cell affinity for somatic gonadal precursors to specify stromal intermingled cells, which contributes to both germline stem cell maintenance and differentiation niches in the adult. We also report that Traffic Jam (an orthologue of a large Maf transcription factor in mammals) is a novel transcriptional target of Hh signaling to control cell-cell adhesion by negative regulation of E-cadherin expression. Our results demonstrate the role of Hh signaling in niche establishment by segregating somatic cell lineages for differentiation.

HOXA5 Promotes Differentiation by Reducing WNT Signaling

Abstract HOXA5 and WNT negatively regulate each other to control stem cell maintenance and differentiation.

Wnt signaling in skin homeostasis and pathology.

The mammalian skin mediates the primary interphase between the body and the external environment and provides the first line of defense against pathogens, mechanical trauma, sunlight injuries, and chemical stress. Proper physical, biochemical, and immunological composition of the skin is necessary to maintain its barrier function. Therefore, the skin reflects a complex dynamic organ with high cellular turnover during normal tissue replacement and wound repair. Stem cell reservoirs ensure constant skin renewal. Wnt signaling controls stem cell maintenance and fate decisions in various tissues and also reflects a key pathway in controlling skin development and homeostasis. Disruption of Wnt signaling in the skin causes disorders such as alopecia, chronic inflammatory skin diseases or cancer. This review summarizes the role of Wnt signaling during skin development, homeostasis, and disease.

Neural Differentiation of Human Adipose Tissue-Derived Stem Cells Involves Activation of the Wnt5a/JNK Signalling.

Stem cells are a powerful resource for cell-based transplantation therapies, but understanding of stem cell differentiation at the molecular level is not clear yet. We hypothesized that the Wnt pathway controls stem cell maintenance and neural differentiation. We have characterized the transcriptional expression of Wnt during the neural differentiation of hADSCs. After neural induction, the expressions of Wnt2, Wnt4, and Wnt11 were decreased, but the expression of Wnt5a was increased compared with primary hADSCs in RT-PCR analysis. In addition, the expression levels of most Fzds and LRP5/6 ligand were decreased, but not Fzd3 and Fzd5. Furthermore, Dvl1 and RYK expression levels were downregulated in NI-hADSCs. There were no changes in the expression of ß-catenin and GSK3ß. Interestingly, Wnt5a expression was highly increased in NI-hADSCs by real time RT-PCR analysis and western blot. Wnt5a level was upregulated after neural differentiation and Wnt3, Dvl2, and Naked1 levels were downregulated. Finally, we found that the JNK expression was increased after neural induction and ERK level was decreased. Thus, this study shows for the first time how a single Wnt5a ligand can activate the neural differentiation pathway through the activation of Wnt5a/JNK pathway by binding Fzd3 and Fzd5 and directing Axin/GSK-3ß in hADSCs.

Class I KNOX transcription factors promote differentiation of cambial derivatives into xylem fibers in the Arabidopsis hypocotyl.

The class I KNOX transcription factors SHOOT MERISTEMLESS (STM) and KNAT1 are important regulators of meristem maintenance in shoot apices, with a dual role of promoting cell proliferation and inhibiting differentiation. We examined whether they control stem cell maintenance in the cambium of Arabidopsis hypocotyls, a wood-forming lateral meristem, in a similar fashion as in the shoot apical meristem. Weak loss-of-function alleles of KNAT1 and STM led to reduced formation of xylem fibers - highly differentiated cambial derivatives - whereas cell proliferation in the cambium was only mildly affected. In a knat1;stm double mutant, xylem fiber differentiation was completely abolished, but residual cambial activity was maintained. Expression of early and late markers of xylary cell differentiation was globally reduced in the knat1;stm double mutant. KNAT1 and STM were found to act through transcriptional repression of the meristem boundary genes BLADE-ON-PETIOLE 1 (BOP1) and BOP2 on xylem fiber differentiation. Together, these data indicate that, in the cambium, KNAT1 and STM, contrary to their function in the shoot apical meristem, promote cell differentiation through repression of BOP genes.

Paneth cells

Paneth cells

Angiomotins link F-actin architecture to Hippo pathway signaling

The Hippo pathway regulates the transcriptional coactivator YAP to control cell proliferation, organ size, and stem cell maintenance. Multiple factors, such as substrate stiffness, cell density, and G protein-coupled receptor signaling, regulate YAP through their effects on the F-actin cytoskeleton, although the mechanism is not known. Here we show that angiomotin proteins (AMOT130, AMOTL1, and AMOTL2) connect F-actin architecture to YAP regulation. First, we show that angiomotins are required to relocalize YAP to the cytoplasm in response to various manipulations that perturb the actin cytoskeleton. Second, angiomotins associate with F-actin through a conserved F-actin-binding domain, and mutants defective for F-actin binding show enhanced ability to retain YAP in the cytoplasm. Third, F-actin and YAP compete for binding to AMOT130, explaining how F-actin inhibits AMOT130-mediated cytoplasmic retention of YAP. Furthermore, we find that LATS can synergize with F-actin perturbations by phosphorylating free AMOT130 to keep it from associating with F-actin. Together these results uncover a mechanism for how F-actin levels modulate YAP localization, allowing cells to make developmental and proliferative decisions based on diverse inputs that regulate actin architecture.

The role of PML in hematopoietic and leukemic stem cell maintenance

The tumor suppressor promyelocytic leukemia (PML) was first identified as a component of PML-RARα fusion protein, one of the initiating cytogenetic abnormalities in acute promyelocytic leukemia. PML is now known to have diverse functions regulating the DNA-damage response, apoptosis, senescence, and angiogenesis. Recent investigations have identified PML as a regulator of metabolic pathways in stem cell compartments, including the hematopoietic system, and have provided researchers with new strategies for controlling stem cell maintenance and differentiation. Studies of PML in leukemia-initiating cells demonstrate that PML is also an essential component of their maintenance, which has drawn tremendous attention to PML from scientists in various stem cell fields. Here, we review research into PML and its associated pathways, including recent studies of PML as it relates to stem cell biology, as well as our finding that PML regulates fatty acid oxidation, which is essential to the maintenance of normal hematopoietic stem cells. We also discuss the therapeutic potential of controlling PML-associated pathways. In particular, we describe promising evidence for the use of arsenic trioxide in the treatment of chronic myeloid leukemia.

Stem cell maintenance in the adult mammalian hippocampus: A matter of signal integration?

The continuous generation of new neurons from stem cells in the hippocampal dentate gyrus is considered an important contributor to hippocampal plasticity. A prerequisite for the life-long generation of new dentate granule neurons is the maintenance of the neural stem cell pool. A number of essential molecular regulators and signals for hippocampal neural stem cell maintenance have been identified, but how these pathways interact to prevent precocious differentiation or exhaustion of the stem cell pool is currently unknown. Here, we summarize the current knowledge on the molecular regulation of the hippocampal stem cell pool and discuss the possibility that signal integration through Notch signaling controls stem cell maintenance in the adult hippocampus.

In Vitro Expansion and Transplantation of Intestinal Crypt Stem Cells

Adult tissue stem cells hold great promise for regenerative medicine strategies. Major hurdles in their clinical application persist and include the identification of such stem cells, their isolation and in vitro expansion, and—finally—their transplantation. These hurdles have recently been overcome for stem cells of the intestinal tract. Thus, exciting avenues are now opening up to apply these stem cells for a variety of therapeutic strategies. Herein, we highlight recent developments in intestinal stem cell biology and discuss this progress from the perspective of clinical application. Adult tissue maintenance and repair involves cell production by somatic stem cells and their transit-amplifying (TA) progenitor cells, followed by lineage commitment and terminal differentiation. The intestinal epithelium is the most vigorously regenerating tissue and is renewed every 3‐5 days. This rapid cell renewal, in combination with its modular architecture of crypts and villi, makes it an ideal model to study stem cell biology. The intestinal stem cells reside at the bottoms of crypts in the small intestine and colon. Their TA daughters occupy the remainder of the crypts. Differentiated cells reside on the villi in the small intestine and in crypt tops and in the flat surface epithelium of the colon. The differentiated cell types of the small intestine include the absorptive enterocytes and multiple secretory lineages (Paneth cells, goblet cells, enteroendocrine cells, and tuft cells), as well as the M cells of the Peyer’s patches. 1 Intestinal homeostasis is tightly controlled by wellcharacterized signaling pathways. In particular, the Wnt-, Bmp-, epidermal growth factor (EGF), and Notch pathways have been shown to be major players in this regulation. Wnt signaling constitutes the key pathway to maintain the stem cells and drive proliferation at crypt bottoms. Mice lacking Tcf7l2/Tcf4, a key transcription factor in this pathway, lose all proliferation in crypts. 2 Conversely, colon cancer is caused by loss of the Apc gene, which encodes a key negative regulator of the Wnt pathway. In addition to Wnt signals, Notch signaling is required to maintain crypt cells in the undifferentiated state. 3 EGF signals maintain the proliferative state of the crypt cells. Bmp and Tgf- signaling are activated in villus cells and are believed to regulate differentiation. Bmp ligands are expressed in villi, whereas Bmp antagonists such as noggin and gremlins are expressed at crypt mesenchyme to block Bmp signals from the crypts.4 All these pathways cross-talk with each other, forming complex molecular networks to govern tissue homeostasis.

Abstract SY28-02: Interactions between cancer stem cells and their niche govern metastatic colonization

Abstract Metastatic growth in distant organs is the major cause of cancer mortality. Formation of metastasis is a multi-step process with several rate-limiting steps. While dissemination of tumour cells appears to be an early and frequent event, successful initiation of metastatic growth, a process termed “metastatic colonization,” is rather inefficient for many cancer types and accomplished only by a minority of cancer cells that reach distant sites. Prevalent target sites are characteristic for many tumour entities. The inefficiency of the metastatic process suggests two possibilities: a differential potential of the disseminated cancer cells to grow at the secondary sites or an inadequate support by distant tissues. We aimed at exploring limiting factors that determine metastatic success using the MMTVPyMT mouse breast cancer model which spontaneously metastasizes to the lungs. Considering that the disseminated cancer cells might have a differential metastatic potential, we investigated whether the recently identified cancer stem cells (CSCs, also termed tumour initiating cells), might be also relevant for the metastatic process. We found that a small population of cancer stem cells (CSCs) is critical for metastatic colonization. These CSCs represent a stable population of tumour cells which selectively survive and proliferate upon metastatic seeding and thereby drive the initial expansion of cancer cells at the secondary site. In contrast, nonCSCs fail to grow, are rapidly lost and do not de-differentiate into CSCs. Yet, the numbers of CSCs appear to be too high to fully explain the inefficiency of metastatic nodule formation, indicating that additional factors restrict successful metastatic colonization. Stem cells are suspected to rely on signals from their stromal environment such as localized growth factors that can affect stem cell maintenance and proliferation. In this study, we find stromal niche signals to be crucial for this process. We identify the extracellular matrix (ECM) component POSTN to be expressed by fibroblasts in the normal tissue exclusively in response to tumour cell infiltration. Infiltrating tumour cells induce stromal POSTN expression in the secondary target organ via TGFb3 secretion. Importantly the induction of POSTN at the target site is essential for the metastatic process. Indeed, generation of a POSTN null mouse revealed that the metastatic process is heavily compromised in the absence of POSTN. Similarly, cancer cells unable to induce POSTN expression at the target site due to blocking of TGFb3 signaling show poor metastatic activity. We further show that this ECM protein is required to allow maintenance of cancer stem cells: the cells driving the metastatic process. To understand how POSTN connects to the mechanisms that control stem cell maintenance we characterize its binding partners. We found that POSTN is able to bind Wnt ligands and therefore increase Wnt signaling. In vivo we observed Wnt signaling activity selectively in the CSCs population and this activity is abrogated in absence of POSTN. Finally, the metastatic activity of tumour cells with constitutively active Wnt signaling is independent of POSTN presence. Thus, POSTN acts as a niche component that can promote stem cell maintenance and metastatic colonization by augmenting Wnt signaling. We suggest that the education of stromal cells by infiltrating tumour cells is an important step in metastatic colonization and that preventing de-novo niche formation may represent a novel treatment strategy against metastatic disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY28-02. doi:1538-7445.AM2012-SY28-02

Cytoneme-Mediated Delivery of Hedgehog Regulates the Expression of Bone Morphogenetic Proteins to Maintain Germline Stem Cells in Drosophila

Author SummaryThe Drosophila ovary contains a well-defined stem cell niche that hosts 2–3 germline stem cells (GSCs). The Hedgehog (Hh) family of signalling proteins mediates cellular homeostasis in several adult tissues, and here we decipher the detailed mechanism of action of Hh in the adult female GSC niche. We demonstrate that Hh acts in a juxtacrine manner (i.e., it requires physical contact between the cells involved) to maintain the normal pool of GSCs in the ovarian niche. Hh is produced in one type of niche support cell (the cap cells), and it is received, upon secretion, by a second, neighbouring population of niche cells (the escort cells). In the latter, we show that the Hh signalling pathway regulates the expression of the Drosophila Bone Morphogenetic Protein (BMP) homologues and essential stem cell factors decapentaplegic (dpp) and glass bottom boat (gbb). We also find that Hh distribution in the GSC niche is mediated by short cellular projections, reminiscent of wing disc cytonemes, although they grow from the (Hh) signal-producing cells towards the receiving cells. Under conditions of low levels of Hh protein and/or Hh signalling within the niche, cap cells emit up to 6-fold longer Hh-decorated cytonemes towards the signalling-deficient area of the niche. Our data reveal that stem cell niches are dynamic structures that can sense, and react to, changes in the activity of essential stem cell factors to prevent stem cell differentiation.

Histone H3K9 Trimethylase Eggless Controls Germline Stem Cell Maintenance and Differentiation

Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms.

Epigenetic regulation of cardiovascular differentiation

Epigenetic control mechanisms play a key role in the regulation of embryonic development and tissue homeostasis and modulate cardiovascular diseases. Increasing evidence suggests that lineage commitment of stem/progenitor cells is tightly regulated by epigenetic mechanisms. These epigenetic control mechanisms include DNA and histone modifications, which modulate the chromatin structure thereby regulating access of transcription factors. Particularly, the modification of histone acetylation and methylation, which is controlled by families of histone acetylases/deacetylases and methyltransferases/demethylases, respectively, controls stem cell maintenance, differentiation, and function. This review article summarizes our current understanding of epigenetic mechanisms regulating the differentiation of cardiovascular cells, specifically endothelial cells and cardiac muscle lineages. In particular, the article will focus on the enzymes which modify histones and are involved in chromatin remodelling.

TSC1/2 tumour suppressor complex maintainsDrosophilagermline stem cells by preventing differentiation

Tuberous sclerosis complex human disease gene products TSC1 and TSC2 form a functional complex that negatively regulates target of rapamycin (TOR), an evolutionarily conserved kinase that plays a central role in cell growth and metabolism. Here, we describe a novel role of TSC1/2 in controlling stem cell maintenance. We show that in the Drosophila ovary, disruption of either the Tsc1 or Tsc2 gene in germline stem cells (GSCs) leads to precocious GSC differentiation and loss. The GSC loss can be rescued by treatment with TORC1 inhibitor rapamycin, or by eliminating S6K, a TORC1 downstream effecter, suggesting that precocious differentiation of Tsc1/2 mutant GSC is due to hyperactivation of TORC1. One well-studied mechanism for GSC maintenance is that BMP signals from the niche directly repress the expression of a differentiation-promoting gene bag of marbles (bam) in GSCs. In Tsc1/2 mutant GSCs, BMP signalling activity is downregulated, but bam expression is still repressed. Moreover, Tsc1 bam double mutant GSCs could differentiate into early cystocytes, suggesting that TSC1/2 controls GSC differentiation via both BMP-Bam-dependent and -independent pathways. Taken together, these results suggest that TSC prevents precocious GSC differentiation by inhibiting TORC1 activity and subsequently differentiation-promoting programs. As TSC1/2-TORC1 signalling is highly conserved from Drosophila to mammals, it could have a similar role in controlling stem cell behaviour in mammals, including humans.