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1.
Elife ; 92020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33331818

RESUMO

WNT proteins are secreted symmetry breaking signals that interact with cell surface receptors of the FZD family to regulate a multitude of developmental processes. Studying selectivity between WNTs and FZDs has been hampered by the paucity of purified WNT proteins and by their apparent non-selective interactions with the FZD receptors. Here, we describe an engineered protein, called F7L6, comprised of antibody-derived single-chain variable fragments, that selectively binds to human FZD7 and the co-receptor LRP6. F7L6 potently activates WNT/ß-catenin signaling in a manner similar to Wnt3a. In contrast to Wnt3a, F7L6 engages only FZD7 and none of the other FZD proteins. Treatment of human pluripotent stem (hPS) cells with F7L6 initiates transcriptional programs similar to those observed during primitive streak formation and subsequent gastrulation in the mammalian embryo. This demonstrates that selective engagement and activation of FZD7 signaling is sufficient to promote mesendodermal differentiation of hPS cells.


Assuntos
Diferenciação Celular/fisiologia , Receptores Frizzled/fisiologia , Mesoderma/embriologia , Células-Tronco Pluripotentes/fisiologia , Western Blotting , Regulação da Expressão Gênica , Humanos , Mesoderma/citologia , Mesoderma/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes , Via de Sinalização Wnt/fisiologia
2.
Nat Cell Biol ; 21(6): 721-730, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110287

RESUMO

Wnt signalling drives many processes in development, homeostasis and disease; however, the role and mechanism of individual ligand-receptor (Wnt-Frizzled (Fzd)) interactions in specific biological processes remain poorly understood. Wnt9a is specifically required for the amplification of blood progenitor cells during development. Using genetic studies in zebrafish and human embryonic stem cells, paired with in vitro cell biology and biochemistry, we determined that Wnt9a signals specifically through Fzd9b to elicit ß-catenin-dependent Wnt signalling that regulates haematopoietic stem and progenitor cell emergence. We demonstrate that the epidermal growth factor receptor (EGFR) is required as a cofactor for Wnt9a-Fzd9b signalling. EGFR-mediated phosphorylation of one tyrosine residue on the Fzd9b intracellular tail in response to Wnt9a promotes internalization of the Wnt9a-Fzd9b-LRP signalosome and subsequent signal transduction. These findings provide mechanistic insights for specific Wnt-Fzd signals, which will be crucial for specific therapeutic targeting and regenerative medicine.


Assuntos
Células-Tronco Hematopoéticas/citologia , Receptores de Neurotransmissores/genética , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Receptores ErbB/genética , Humanos , Fosforilação , Via de Sinalização Wnt , Peixe-Zebra/crescimento & desenvolvimento , beta Catenina/genética
3.
Artigo em Inglês | MEDLINE | ID: mdl-29600540

RESUMO

The Wnt signaling pathway is a highly conserved system that regulates complex biological processes across all metazoan species. At the cellular level, secreted Wnt proteins serve to break symmetry and provide cells with positional information that is critical to the patterning of the entire body plan. At the organismal level, Wnt signals are employed to orchestrate fundamental developmental processes, including the specification of the anterior-posterior body axis, induction of the primitive streak and ensuing gastrulation movements, and the generation of cell and tissue diversity. Wnt functions extend into adulthood where they regulate stem cell behavior, tissue homeostasis, and damage repair. Disruption of Wnt signaling activity during embryonic development or in adults results in a spectrum of abnormalities and diseases, including cancer. The molecular mechanisms that underlie the myriad of Wnt-regulated biological effects have been the subject of intense research for over three decades. This review is intended to summarize our current understanding of how Wnt signals are generated and interpreted. This article is categorized under: Biological Mechanisms > Cell Signaling Developmental Biology > Stem Cell Biology and Regeneration.

4.
Prog Mol Biol Transl Sci ; 153: 321-341, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29389522

RESUMO

Leukemia and lymphoma are a wide encompassing term for a diverse set of blood malignancies that affect people of all ages and result in approximately 23,000 deaths in the United States per year (Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7-30.). Hematopoietic stem cells (HSCs) are tissue-specific stem cells at the apex of the hierarchy that gives rise to all of the terminally differentiated blood cells, through progressively restricted progenitor populations, a process that is known to be Wnt-responsive. In particular, the progenitor populations are subject to uncontrolled expansion during oncogenic processes, namely the common myeloid progenitor and common lymphoid progenitor, as well as the myeloblast and lymphoblast. Unregulated growth of these cell-types leads to mainly three types of blood cancers (i.e., leukemia, lymphoma, and myeloma), which frequently exhibit deregulation of the Wnt signaling pathway. Generally, leukemia is caused by the expansion of myeloid progenitors, leading to an overproduction of white blood cells; as such, patients are unable to make sufficient numbers of red blood cells and platelets. Likewise, an overproduction of lymphocytes leads to clogging of the lymph system and impairment of the immune system in lymphomas. Finally, cancer of the plasma cells in the blood is called myeloma, which also leads to immune system failure. Within each of these three types of blood cancers, there are multiple subtypes, usually characterized by their timeline of onset and their cell type of origin. Of these, 85% of leukemias are encompassed by the four most common diseases, that is, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL); AML accounts for the majority of leukemia-related deaths (Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7-30.). Through understanding how HSCs are normally developed and maintained, we can understand how the normal functions of these pathways are disrupted during blood cancer progression; the Wnt pathway is important in regulation of both normal and malignant hematopoiesis. In this chapter, we will discuss the role of Wnt signaling in normal and aberrant hematopoiesis. Our understanding the relationship between Wnt and HSCs will provide novel insights into therapeutic targets.


Assuntos
Neoplasias Hematológicas/patologia , Células-Tronco Hematopoéticas/citologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Neoplasias Hematológicas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos
5.
Genes (Basel) ; 9(2)2018 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-29382179

RESUMO

Hematopoietic stem cells (HSCs) differentiate into all cell types of the blood and can be used therapeutically to treat hematopoietic cancers and disorders. Despite decades of research, it is not yet possible to derive therapy-grade HSCs from pluripotent precursors. Analysis of HSC development in model organisms has identified some of the molecular cues that are necessary to instruct hematopoiesis in vivo, including Wnt9A, which is required during an early time window in zebrafish development. Although bona fide HSCs cannot be derived in vitro, it is possible to model human hematopoietic progenitor development by differentiating human pluripotent stem cells to hematopoietic cells. Herein, we modulate WNT9A expression during the in vitro differentiation of human embryonic stem cells to hematopoietic progenitor cells and demonstrate that WNT9A also regulates human hematopoietic progenitor cell development in vitro. Overexpression of WNT9A only impacts differentiation to CD34⁺/CD45⁺ cells during early time windows and does so in a dose-dependent manner. The cells that receive the Wnt signal-not the cells that secrete WNT9A-differentiate most efficiently to hematopoietic progenitors; this mimics the paracrine action of Wnt9a during in vivo hematopoiesis. Taken together, these data indicate that WNT9A is a conserved regulator of zebrafish and human hematopoietic development.

6.
Nat Commun ; 8(1): 1034, 2017 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-29044119

RESUMO

The WNT/ß-catenin signaling pathway is a prominent player in many developmental processes, including gastrulation, anterior-posterior axis specification, organ and tissue development, and homeostasis. Here, we use human pluripotent stem cells (hPSCs) to study the dynamics of the transcriptional response to exogenous activation of the WNT pathway. We describe a mechanism involving the WNT target gene SP5 that leads to termination of the transcriptional program initiated by WNT signaling. Integration of gene expression profiles of wild-type and SP5 mutant cells with genome-wide SP5 binding events reveals that SP5 acts to diminish expression of genes previously activated by the WNT pathway. Furthermore, we show that activation of SP5 by WNT signaling is most robust in cells with developmental potential, such as stem cells. These findings indicate a mechanism by which the developmental WNT signaling pathway reins in expression of transcriptional programs.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo , Proteína Wnt3A/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/genética , Via de Sinalização Wnt , Proteína Wnt3A/genética , beta Catenina/genética , beta Catenina/metabolismo
7.
Dev Neurobiol ; 77(11): 1239-1259, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28799266

RESUMO

The vertebrate central nervous system (CNS) is comprised of vast number of distinct cell types arranged in a highly organized manner. This high degree of complexity is achieved by cellular communication, including direct cell-cell contact, cell-matrix interactions, and cell-growth factor signaling. Among the several developmental signals controlling the development of the CNS, Wnt proteins have emerged as particularly critical and, hence, have captivated the attention of many researchers. With Wnts' evolutionarily conserved function as primordial symmetry breaking signals, these proteins and their downstream effects are responsible for simultaneously establishing cellular diversity and tissue organization. With their expansive repertoire of secreted agonists and antagonists, cell surface receptors, signaling cascades and downstream biological effects, Wnts are ideally suited to control the complex processes underlying vertebrate neural development. In this review, we will describe the mechanisms by which Wnts exert their potent effects on cells and tissues and highlight the many roles of Wnt signaling during neural development, starting from the initial induction of the neural plate, the subsequent patterning along the embryonic axes, to the intricately organized structure of the CNS. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1239-1259, 2017.


Assuntos
Sistema Nervoso Central , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Padronização Corporal/fisiologia , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Embrião não Mamífero/fisiologia , Vertebrados , Proteínas Wnt/genética , beta Catenina/genética
8.
Sci Signal ; 10(482)2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28588081

RESUMO

Increased protein translation in cells and various factors in the tumor microenvironment can induce endoplasmic reticulum (ER) stress, which initiates the unfolded protein response (UPR). We have previously reported that factors released from cancer cells mounting a UPR induce a de novo UPR in bone marrow-derived myeloid cells, macrophages, and dendritic cells that facilitates protumorigenic characteristics in culture and tumor growth in vivo. We investigated whether this intercellular signaling, which we have termed transmissible ER stress (TERS), also operates between cancer cells and what its functional consequences were within the tumor. We found that TERS signaling induced a UPR in recipient human prostate cancer cells that included the cell surface expression of the chaperone GRP78. TERS also activated Wnt signaling in recipient cancer cells and enhanced resistance to nutrient starvation and common chemotherapies such as the proteasome inhibitor bortezomib and the microtubule inhibitor paclitaxel. TERS-induced activation of Wnt signaling required the UPR kinase and endonuclease IRE1. However, TERS-induced enhancement of cell survival was predominantly mediated by the UPR kinase PERK and a reduction in the abundance of the transcription factor ATF4, which prevented the activation of the transcription factor CHOP and, consequently, the induction of apoptosis. When implanted in mice, TERS-primed cancer cells gave rise to faster growing tumors than did vehicle-primed cancer cells. Collectively, our data demonstrate that TERS is a mechanism of intercellular communication through which tumor cells can adapt to stressful environments.


Assuntos
Bortezomib/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Resistência a Medicamentos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Paclitaxel/farmacologia , Neoplasias da Próstata/patologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Antineoplásicos/farmacologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Moduladores de Tubulina/farmacologia , Células Tumorais Cultivadas , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
10.
Dev Cell ; 40(5): 439-452.e4, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28292423

RESUMO

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a-/-Wnt5b-/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry.


Assuntos
Padronização Corporal , Polaridade Celular , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteína Wnt-5a/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Comunicação Celular , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Domínio LIM/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Modelos Biológicos , Proteínas/metabolismo
11.
Zebrafish ; 14(4): 383-386, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-27829120

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been applied to edit genomes in a wide variety of model systems. Although this process can be quite efficient, editing at precise locations in the genome remains difficult without a suitable single guide RNA (sgRNA). We have developed a method for screening sgRNA function in vitro, using reagents that most zebrafish laboratories are already using. The results from our in vitro assay correlate with function in vivo in every sgRNA that we have examined so far. When combined with endonucleases with alternative protospacer adjacent motif site specificities and alternative sgRNAs, this method will streamline genome editing at almost any locus.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , RNA Guia/genética , Peixe-Zebra/genética , Animais , Endonucleases/metabolismo , Marcação de Genes , Técnicas In Vitro , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/genética
12.
Cell Rep ; 17(6): 1595-1606, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27806298

RESUMO

All mature blood cell types in the adult animal arise from hematopoietic stem and progenitor cells (HSPCs). However, the developmental cues regulating HSPC ontogeny are incompletely understood. In particular, the details surrounding a requirement for Wnt/ß-catenin signaling in the development of mature HSPCs are controversial and difficult to consolidate. Using zebrafish, we demonstrate that Wnt signaling is required to direct an amplification of HSPCs in the aorta. Wnt9a is specifically required for this process and cannot be replaced by Wnt9b or Wnt3a. This proliferative event occurs independently of initial HSPC fate specification, and the Wnt9a input is required prior to aorta formation. HSPC arterial amplification occurs prior to seeding of secondary hematopoietic tissues and proceeds, in part, through the cell cycle regulator myca (c-myc). Our results support a general paradigm, in which early signaling events, including Wnt, direct later HSPC developmental processes.


Assuntos
Aorta/citologia , Aorta/embriologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Contagem de Células , Ciclo Celular , Proliferação de Células , Hemangioblastos/metabolismo , Via de Sinalização Wnt
13.
Proc Natl Acad Sci U S A ; 113(46): 13150-13155, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27799526

RESUMO

Prodigiosin, a natural red pigment produced by numerous bacterial species, has exhibited promising anticancer activity; however, the molecular mechanisms of action of prodigiosin on malignant cells remain unclear. Aberrant activation of the Wnt/ß-catenin signaling cascade is associated with numerous human cancers. In this study, we identified prodigiosin as a potent inhibitor of the Wnt/ß-catenin pathway. Prodigiosin blocked Wnt/ß-catenin signaling by targeting multiple sites of this pathway, including the low-density lipoprotein-receptor-related protein (LRP) 6, Dishevelled (DVL), and glycogen synthase kinase-3ß (GSK3ß). In breast cancer MDA-MB-231 and MDA-MB-468 cells, nanomolar concentrations of prodigiosin decreased phosphorylation of LRP6, DVL2, and GSK3ß and suppressed ß-catenin-stimulated Wnt target gene expression, including expression of cyclin D1. In MDA-MB-231 breast cancer xenografts and MMTV-Wnt1 transgenic mice, administration of prodigiosin slowed tumor progression and reduced the expression of phosphorylated LRP6, phosphorylated and unphosphorylated DVL2, Ser9 phosphorylated GSK3ß, active ß-catenin, and cyclin D1. Through its ability to inhibit Wnt/ß-catenin signaling and reduce cyclin D1 levels, prodigiosin could have therapeutic activity in advanced breast cancers.


Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Prodigiosina/farmacologia , Prodigiosina/uso terapêutico , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclina D1/genética , Proteínas Desgrenhadas/genética , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Carga Tumoral/efeitos dos fármacos , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética
14.
Methods Mol Biol ; 1481: 161-81, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27590161

RESUMO

Human pluripotent stem cells (hPSCs) may revolutionize medical practice by providing: (a) a renewable source of cells for tissue replacement therapies, (b) a powerful system to model human diseases in a dish, and (c) a platform for examining efficacy and safety of novel drugs. Furthermore, these cells offer a unique opportunity to study early human development in vitro, in particular, the process by which a seemingly uniform cell population interacts to give rise to the three main embryonic lineages: ectoderm, endoderm. and mesoderm. This process of lineage allocation is regulated by a number of inductive signals that are mediated by growth factors, including FGF, TGFß, and Wnt. In this book chapter, we introduce a set of tools, methods, and protocols to specifically manipulate the Wnt signaling pathway with the intention of altering the cell fate outcome of hPSCs.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Células-Tronco Embrionárias Humanas/citologia , Via de Sinalização Wnt/genética , Ectoderma/crescimento & desenvolvimento , Endoderma/crescimento & desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/crescimento & desenvolvimento , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
15.
Elife ; 42015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26554899

RESUMO

The field of tissue engineering entered a new era with the development of human pluripotent stem cells (hPSCs), which are capable of unlimited expansion whilst retaining the potential to differentiate into all mature cell populations. However, these cells harbor significant risks, including tumor formation upon transplantation. One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential. Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties. This cell population, referred to as intermediate mesodermal progenitor (IMP) cells, is capable of unlimited expansion, lacks tumor formation potential, and, upon appropriate stimulation, readily acquires properties of a sub-population of kidney cells. Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate.


Assuntos
Diferenciação Celular , Mesoderma , Células-Tronco Pluripotentes/fisiologia , Células-Tronco/fisiologia , Técnicas de Cultura de Células , Humanos , Engenharia Tecidual
16.
Elife ; 4: e07091, 2015 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-26126266

RESUMO

Wnt signaling is essential for tissue homeostasis and its dysregulation causes cancer. Wnt ligands trigger signaling by activating Frizzled receptors (FZDRs), which belong to the G-protein coupled receptor superfamily. However, the mechanisms of G protein activation in Wnt signaling remain controversial. In this study, we demonstrate that FZDRs activate G proteins and trigger non-canonical Wnt signaling via the Dishevelled-binding protein, Daple. Daple contains a Gα-binding and activating (GBA) motif, which activates Gαi proteins and an adjacent domain that directly binds FZDRs, thereby linking Wnt stimulation to G protein activation. This triggers non-canonical Wnt responses, that is, suppresses the ß-catenin/TCF/LEF pathway and tumorigenesis, but enhances PI3K-Akt and Rac1 signals and tumor cell invasiveness. In colorectal cancers, Daple is suppressed during adenoma-to-carcinoma transformation and expressed later in metastasized tumor cells. Thus, Daple activates Gαi and enhances non-canonical Wnt signaling by FZDRs, and its dysregulation can impact both tumor initiation and progression to metastasis.


Assuntos
Receptores Frizzled/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Via de Sinalização Wnt , Humanos
17.
Stem Cell Reports ; 3(6): 1015-28, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25458891

RESUMO

Neural progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) are a multipotent cell population that is capable of nearly indefinite expansion and subsequent differentiation into the various neuronal and supporting cell types that comprise the CNS. However, current protocols for differentiating NPCs toward neuronal lineages result in a mixture of neurons from various regions of the CNS. In this study, we determined that endogenous WNT signaling is a primary contributor to the heterogeneity observed in NPC cultures and neuronal differentiation. Furthermore, exogenous manipulation of WNT signaling during neural differentiation, through either activation or inhibition, reduces this heterogeneity in NPC cultures, thereby promoting the formation of regionally homogeneous NPC and neuronal cultures. The ability to manipulate WNT signaling to generate regionally specific NPCs and neurons will be useful for studying human neural development and will greatly enhance the translational potential of hPSCs for neural-related therapies.


Assuntos
Diferenciação Celular , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Via de Sinalização Wnt , Biomarcadores , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Fenótipo
18.
Cell Rep ; 9(5): 1770-1780, 2014 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-25464842

RESUMO

WNT signaling promotes the reprogramming of somatic cells to an induced pluripotent state. We provide genetic evidence that WNT signaling is a requisite step during the induction of pluripotency. Fibroblasts from individuals with focal dermal hypoplasia (FDH), a rare genetic syndrome caused by mutations in the essential WNT processing enzyme PORCN, fail to reprogram with standard methods. This blockade in reprogramming is overcome by ectopic WNT signaling and PORCN overexpression, thus demonstrating that WNT signaling is essential for reprogramming. The rescue of reprogramming is critically dependent on the level of WNT signaling: steady baseline activation of the WNT pathway yields karyotypically normal iPSCs, whereas daily stimulation with Wnt3a produces FDH-iPSCs with severely abnormal karyotypes. Therefore, although WNT signaling is required for cellular reprogramming, inappropriate activation of WNT signaling induces chromosomal instability, highlighting the precarious nature of ectopic WNT activation and its tight relationship with oncogenic transformation.


Assuntos
Reprogramação Celular , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Via de Sinalização Wnt , Biomarcadores/metabolismo , Forma Celular , Transdiferenciação Celular , Células Cultivadas , Hipoplasia Dérmica Focal/patologia , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução Genética
19.
Proc Natl Acad Sci U S A ; 111(4): 1409-14, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24474766

RESUMO

WNT signaling is involved in maintaining stem cells in an undifferentiated state; however, it is often unclear which WNTs and WNT receptors are mediating these activities. Here we examined the role of the WNT receptor FZD7 in maintaining human embryonic stem cells (hESCs) in an undifferentiated and pluripotent state. FZD7 expression is significantly elevated in undifferentiated cells relative to differentiated cell populations, and interfering with its expression or function, either by short hairpin RNA-mediated knockdown or with a fragment antigen binding (Fab) molecule directed against FZD7, disrupts the pluripotent state of hESCs. The FZD7-specific Fab blocks signaling by Wnt3a protein by down-regulating FZD7 protein levels, suggesting that FZD7 transduces Wnt signals to activate Wnt/ß-catenin signaling. These results demonstrate that FZD7 encodes a regulator of the pluripotent state and that hESCs require endogenous WNT/ß-catenin signaling through FZD7 to maintain an undifferentiated phenotype.


Assuntos
Células-Tronco Embrionárias/citologia , Receptores Frizzled/fisiologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Linhagem Celular , Receptores Frizzled/metabolismo , Humanos , Camundongos , Transdução de Sinais , Proteína Wnt3A/metabolismo
20.
Stem Cell Reports ; 1(5): 464-78, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24286033

RESUMO

SOX2 is involved in several cell and developmental processes, including maintenance of embryonic stem cells, differentiation of neural progenitor cells, and patterning of gut endoderm. To study its role in a human system, we generated a human embryonic stem cell (hESC) line harboring a reporter gene encoding GFP in the SOX2 locus. This SOX2 reporter line faithfully recapitulates expression of the SOX2 gene in undifferentiated human pluripotent stem cells (hPSCs), neural progenitor cells (NPCs), and anterior foregut endoderm (AFE). In undifferentiated hESCs, GFP expression corresponds to those cells with highest levels of expression of genes associated with the pluripotent state. In NPCs, expression of GFP can be employed to isolate cells expressing markers associated with NPC multipotency. In AFE, we used transcriptome-wide expression analysis to identify cell surface markers with elevated expression in this population, thereby facilitating isolation and purification of this hPSC-derived cell population.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXB1/metabolismo , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/citologia , Humanos , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/genética , Transcriptoma
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