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1.
Development ; 142(7): 1375-86, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25804741

RESUMO

The efficient generation of striatal neurons from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) is fundamental for realising their promise in disease modelling, pharmaceutical drug screening and cell therapy for Huntington's disease. GABAergic medium-sized spiny neurons (MSNs) are the principal projection neurons of the striatum and specifically degenerate in the early phase of Huntington's disease. Here we report that activin A induces lateral ganglionic eminence (LGE) characteristics in nascent neural progenitors derived from hESCs and hiPSCs in a sonic hedgehog-independent manner. Correct specification of striatal phenotype was further demonstrated by the induction of the striatal transcription factors CTIP2, GSX2 and FOXP2. Crucially, these human LGE progenitors readily differentiate into postmitotic neurons expressing the striatal projection neuron signature marker DARPP32, both in culture and following transplantation in the adult striatum in a rat model of Huntington's disease. Activin-induced neurons also exhibit appropriate striatal-like electrophysiology in vitro. Together, our findings demonstrate a novel route for efficient differentiation of GABAergic striatal MSNs from human pluripotent stem cells.


Assuntos
Ativinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Neostriado/citologia , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Gânglios/efeitos dos fármacos , Gânglios/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Doença de Huntington/patologia , Doença de Huntington/terapia , Neurônios/metabolismo , Neurônios/transplante , Células-Tronco Pluripotentes/metabolismo , Ratos , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo
2.
Mol Syst Biol ; 13(10): 946, 2017 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-29038337

RESUMO

Polycomb repression in mouse embryonic stem cells (ESCs) is tightly associated with promoter co-occupancy of RNA polymerase II (RNAPII) which is thought to prime genes for activation during early development. However, it is unknown whether RNAPII poising is a general feature of Polycomb repression, or is lost during differentiation. Here, we map the genome-wide occupancy of RNAPII and Polycomb from pluripotent ESCs to non-dividing functional dopaminergic neurons. We find that poised RNAPII complexes are ubiquitously present at Polycomb-repressed genes at all stages of neuronal differentiation. We observe both loss and acquisition of RNAPII and Polycomb at specific groups of genes reflecting their silencing or activation. Strikingly, RNAPII remains poised at transcription factor genes which are silenced in neurons through Polycomb repression, and have major roles in specifying other, non-neuronal lineages. We conclude that RNAPII poising is intrinsically associated with Polycomb repression throughout differentiation. Our work suggests that the tight interplay between RNAPII poising and Polycomb repression not only instructs promoter state transitions, but also may enable promoter plasticity in differentiated cells.


Assuntos
Neurônios Dopaminérgicos/citologia , Genes Controladores do Desenvolvimento , Células-Tronco Embrionárias Murinas/citologia , Proteínas do Grupo Polycomb/metabolismo , RNA Polimerase II/metabolismo , Animais , Diferenciação Celular , Neurônios Dopaminérgicos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de RNA , Fatores de Transcrição/genética
3.
Development ; 138(20): 4363-74, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21880784

RESUMO

Effective induction of midbrain-specific dopamine (mDA) neurons from stem cells is fundamental for realizing their potential in biomedical applications relevant to Parkinson's disease. During early development, the Otx2-positive neural tissues are patterned anterior-posteriorly to form the forebrain and midbrain under the influence of extracellular signaling such as FGF and Wnt. In the mesencephalon, sonic hedgehog (Shh) specifies a ventral progenitor fate in the floor plate region that later gives rise to mDA neurons. In this study, we systematically investigated the temporal actions of FGF signaling in mDA neuron fate specification of mouse and human pluripotent stem cells and mouse induced pluripotent stem cells. We show that a brief blockade of FGF signaling on exit of the lineage-primed epiblast pluripotent state initiates an early induction of Lmx1a and Foxa2 in nascent neural progenitors. In addition to inducing ventral midbrain characteristics, the FGF signaling blockade during neural induction also directs a midbrain fate in the anterior-posterior axis by suppressing caudalization as well as forebrain induction, leading to the maintenance of midbrain Otx2. Following a period of endogenous FGF signaling, subsequent enhancement of FGF signaling by Fgf8, in combination with Shh, promotes mDA neurogenesis and restricts alternative fates. Thus, a stepwise control of FGF signaling during distinct stages of stem cell neural fate conversion is crucial for reliable and highly efficient production of functional, authentic midbrain-specific dopaminergic neurons. Importantly, we provide evidence that this novel, small-molecule-based strategy applies to both mouse and human pluripotent stem cells.


Assuntos
Dopamina/metabolismo , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Modelos Neurológicos , Neurogênese/fisiologia , Fatores de Transcrição Otx/metabolismo , Transdução de Sinais , Proteína Wnt1/metabolismo
4.
J Neurosci ; 31(46): 16814-25, 2011 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-22090507

RESUMO

HCN1-4 subunits form Na+/K+-permeable ion channels that are activated by hyperpolarization and carry the current known as I(h). I(h) has been characterized in vestibular hair cells of the inner ear, but its molecular correlates and functional contributions have not been elucidated. We examined Hcn mRNA expression and immunolocalization of HCN protein in the mouse utricle, a mechanosensitive organ that contributes to the sense of balance. We found that HCN1 is the most highly expressed subunit, localized to the basolateral membranes of type I and type II hair cells. We characterized I(h) using the whole-cell, voltage-clamp technique and found the current expressed in 84% of the cells with a mean maximum conductance of 4.4 nS. I(h) was inhibited by ZD7288, cilobradine, and by adenoviral expression of a dominant-negative form of HCN2. To determine which HCN subunits carried I(h), we examined hair cells from mice deficient in Hcn1, 2, or both. I(h) was completely abolished in hair cells of Hcn1⁻/⁻ mice and Hcn1/2⁻/⁻ mice but was similar to wild-type in Hcn2⁻/⁻ mice. To examine the functional contributions of I(h), we recorded hair cell membrane responses to small hyperpolarizing current steps and found that activation of I(h) evoked a 5-10 mV sag depolarization and a subsequent 15-20 mV rebound upon termination. The sag and rebound were nearly abolished in Hcn1-deficient hair cells. We also found that Hcn1-deficient mice had deficits in vestibular-evoked potentials and balance assays. We conclude that HCN1 contributes to vestibular hair cell function and the sense of balance.


Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Orelha Interna/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Equilíbrio Postural/genética , Canais de Potássio/metabolismo , Sáculo e Utrículo/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Benzazepinas/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/deficiência , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Orelha Interna/efeitos dos fármacos , Estimulação Elétrica , Feminino , Fatores de Transcrição Forkhead/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Movimento (Física) , Proteínas do Tecido Nervoso/genética , Proteínas de Neurofilamentos/metabolismo , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Canais de Potássio/deficiência , Canais de Potássio/genética , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Teste de Desempenho do Rota-Rod , Sáculo e Utrículo/citologia , Potenciais Evocados Miogênicos Vestibulares/genética , Potenciais Evocados Miogênicos Vestibulares/fisiologia
5.
J Gen Physiol ; 143(4): 481-97, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24638995

RESUMO

The hyperpolarization-activated, cyclic nucleotide-sensitive current, Ih, is present in vestibular hair cells and vestibular ganglion neurons, and is required for normal balance function. We sought to identify the molecular correlates and functional relevance of Ih in vestibular ganglion neurons. Ih is carried by channels consisting of homo- or heteromeric assemblies of four protein subunits from the Hcn gene family. The relative expression of Hcn1-4 mRNA was examined using a quantitative reverse transcription PCR (RT-PCR) screen. Hcn2 was the most highly expressed subunit in vestibular neuron cell bodies. Immunolocalization of HCN2 revealed robust expression in cell bodies of all vestibular ganglion neurons. To characterize Ih in vestibular neuron cell bodies and at hair cell-afferent synapses, we developed an intact, ex vivo preparation. We found robust physiological expression of Ih in 89% of cell bodies and 100% of calyx terminals. Ih was significantly larger in calyx terminals than in cell bodies; however, other biophysical characteristics were similar. Ih was absent in calyces lacking Hcn1 and Hcn2, but small Ih was still present in cell bodies, which suggests expression of an additional subunit, perhaps Hcn4. To determine the contributions of hair cell mechanotransduction and Ih to the firing patterns of calyx terminals, we recorded action potentials in current-clamp mode. Mechanotransduction currents were modulated by hair bundle defection and application of calcium chelators to disrupt tip links. Ih activity was modulated using ZD7288 and cAMP. We found that both hair cell transduction and Ih contribute to the rate and regularity of spontaneous action potentials in the vestibular afferent neurons. We propose that modulation of Ih in vestibular ganglion neurons may provide a mechanism for modulation of spontaneous activity in the vestibular periphery.


Assuntos
Potenciais de Ação/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Mecanotransdução Celular/fisiologia , Gânglio Espiral da Cóclea/fisiologia , Vestíbulo do Labirinto/fisiologia , Animais , Animais Recém-Nascidos , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout
6.
Stem Cell Rev Rep ; 8(2): 459-71, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21761283

RESUMO

A better understanding of the control of stem cell maintenance and differentiation fate choice is fundamental to effectively realising the potential of human pluripotent stem cells in disease modelling, drug screening and cell therapy. Dlk1 is a Notch related transmembrane protein that has been reportedly expressed in several neurogenic regions in the developing brain. In this study, we investigated the ability of Dlk1 in modulating the maintenance and differentiation of human and mouse ESC-derived neural progenitors. We found that DLK1, either employed as an extrinsic factor, or via transgene expression, consistently promoted the generation of neurons in both the mouse and human ESC-derived neural progenitors. DLK1 exerts this function by inducing cell cycle exit of the progenitors, as evidenced by an increase in the number of young neurons retaining BrdU labelling and cells expressing the cycling inhibitor P57Kip2. DLK1 antagonised the cell proliferation activity of Notch ligands Delta 1 and Jagged and inhibited Hes1-mediated Notch signaling as demonstrated by a luciferase reporter assay. Interestingly, we found that DLK1 promotes the neurogenic potential of human neural progenitor cells via suppression of Smad activation when they are challenged with BMP. Together, our data demonstrate for the first time a regulatory role for DLK1 in human and mouse neural progenitor differentiation and establish an interaction between DLK1 and Hes1-mediated Notch signaling in these cells. Furthermore, this study identifies DLK1 as a novel modulator of BMP/Smad signalling.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Neurais/citologia , Neurogênese , Células-Tronco Pluripotentes/citologia , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Proteínas de Ligação ao Cálcio , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Humanos , Ligantes , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fatores de Transcrição HES-1
7.
PLoS One ; 6(8): e24169, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21887381

RESUMO

In vitro generation of functional neurons from embryonic stem (ES) cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP) and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP(+) neurons in culture display functional neuronal properties even at early stages of differentiation.


Assuntos
Fenômenos Eletrofisiológicos , Células-Tronco Embrionárias/fisiologia , Neurônios/citologia , Potenciais de Ação , Animais , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Proteínas de Fluorescência Verde , Camundongos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Sódio/metabolismo
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