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1.
J Huntingtons Dis ; 13(1): 41-53, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38427495

RESUMO

Background: Mutations in the Huntingtin (HTT) gene cause Huntington's disease (HD), a neurodegenerative disorder. As a scaffold protein, HTT is involved in numerous cellular functions, but its normal and pathogenic functions during human forebrain development are poorly understood. Objective: To investigate the developmental component of HD, with a specific emphasis on understanding the functions of wild-type and mutant HTT alleles during forebrain neuron development in individuals carrying HD mutations. Methods: We used CRISPR/Cas9 gene-editing technology to disrupt the ATG region of the HTT gene via non-homologous end joining to produce mono- or biallelic HTT knock-out human induced pluripotent stem cell (iPSC) clones. Results: We showed that the loss of wild-type, mutant, or both HTT isoforms does not affect the pluripotency of iPSCs or their transition into neural cells. However, we observed that HTT loss causes division impairments in forebrain neuro-epithelial cells and alters maturation of striatal projection neurons (SPNs) particularly in the acquisition of DARPP32 expression, a key functional marker of SPNs. Finally, young post-mitotic neurons derived from HTT-/- human iPSCs display cellular dysfunctions observed in adult HD neurons. Conclusions: We described a novel collection of isogenic clones with mono- and biallelic HTT inactivation that complement existing HD-hiPSC isogenic series to explore HTT functions and test therapeutic strategies in particular HTT-lowering drugs. Characterizing neural and neuronal derivatives from human iPSCs of this collection, we show evidence that HTT loss or mutation has impacts on neuro-epithelial and striatal neurons maturation, and on basal DNA damage and BDNF axonal transport in post-mitotic neurons.


Assuntos
Doença de Huntington , Células-Tronco Pluripotentes Induzidas , Adulto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Huntington/metabolismo , Neurônios/metabolismo , Corpo Estriado/metabolismo , Alelos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo
2.
Hum Gene Ther ; 34(17-18): 958-974, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37658843

RESUMO

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a toxic gain-of-function CAG expansion in the first exon of the huntingtin (HTT) gene. The monogenic nature of HD makes mutant HTT (mHTT) inactivation a promising therapeutic strategy. Single nucleotide polymorphisms frequently associated with CAG expansion have been explored to selectively inactivate mHTT allele using the CRISPR/Cas9 system. One of such allele-selective approaches consists of excising a region flanking the first exon of mHTT by inducing simultaneous double-strand breaks at upstream and downstream positions of the mHTT exon 1. The removal of the first exon of mHTT deletes the CAG expansion and important transcription regulatory sites, leading to mHTT inactivation. However, the frequency of deletion events is yet to be quantified either in vitro or in vivo. Here, we developed accurate quantitative digital polymerase chain reaction-based assays to assess HTT exon 1 deletion in vitro and in fully humanized HU97/18 mice. Our results demonstrate that dual-single guide RNA (sgRNA) strategies are efficient and that 67% of HTT editing events are leading to exon 1 deletion in HEK293T cells. In contrast, these sgRNA actively cleaved HTT in HU97/18 mice, but most editing events do not lead to exon 1 deletion (10% exon 1 deletion). We also showed that the in vivo editing pattern is not affected by CAG expansion but may potentially be due to the presence of multiple copies of wildtype (wt)/mHTT genes HU97/18 mice as well as the slow kinetics of AAV-mediated CRISPR/Cas9 delivery.


Assuntos
Doenças do Sistema Nervoso Central , Doença de Huntington , Humanos , Animais , Camundongos , RNA Guia de Sistemas CRISPR-Cas , Células HEK293 , Éxons/genética , Alelos , Doença de Huntington/genética , Doença de Huntington/terapia , Sistema Nervoso Central
3.
Methods Mol Biol ; 2551: 357-378, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36310215

RESUMO

Aggregated alpha-synuclein (α-Syn) in neurons is a hallmark of Parkinson's disease (PD) and other synucleinopathies. Recent advances (1) in the production and purification of synthetic assemblies of α-Syn, (2) in the design and production of microfluidic devices allowing the construction of oriented and compartmentalized neuronal network on a chip, and (3) in the differentiation of human pluripotent stem cells (hPSCs) into specific neuronal subtypes now allow the study of cellular and molecular determinants of the prion-like properties of α-Syn in vitro. Here, we described the methods we used to reconstruct a cortico-cortical human neuronal network in microfluidic devices and how to take advantage of this cellular model to characterize (1) the prion-like properties of different α-Syn strains and (2) the neuronal dysfunctions and the alterations associated with the exposure to α-Syn strains or the nucleation of endogenous α-Syn protein in vitro.


Assuntos
Doença de Parkinson , Príons , Sinucleinopatias , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Príons/metabolismo
4.
Brain ; 145(5): 1584-1597, 2022 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-35262656

RESUMO

There has been substantial progress in the development of regenerative medicine strategies for CNS disorders over the last decade, with progression to early clinical studies for some conditions. However, there are multiple challenges along the translational pipeline, many of which are common across diseases and pertinent to multiple donor cell types. These include defining the point at which the preclinical data are sufficiently compelling to permit progression to the first clinical studies; scaling-up, characterization, quality control and validation of the cell product; design, validation and approval of the surgical device; and operative procedures for safe and effective delivery of cell product to the brain. Furthermore, clinical trials that incorporate principles of efficient design and disease-specific outcomes are urgently needed (particularly for those undertaken in rare diseases, where relatively small cohorts are an additional limiting factor), and all processes must be adaptable in a dynamic regulatory environment. Here we set out the challenges associated with the clinical translation of cell therapy, using Huntington's disease as a specific example, and suggest potential strategies to address these challenges. Huntington's disease presents a clear unmet need, but, importantly, it is an autosomal dominant condition with a readily available gene test, full genetic penetrance and a wide range of associated animal models, which together mean that it is a powerful condition in which to develop principles and test experimental therapeutics. We propose that solving these challenges in Huntington's disease would provide a road map for many other neurological conditions. This white paper represents a consensus opinion emerging from a series of meetings of the international translational platforms Stem Cells for Huntington's Disease and the European Huntington's Disease Network Advanced Therapies Working Group, established to identify the challenges of cell therapy, share experience, develop guidance and highlight future directions, with the aim to expedite progress towards therapies for clinical benefit in Huntington's disease.


Assuntos
Doença de Huntington , Doenças Neurodegenerativas , Animais , Encéfalo/metabolismo , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/terapia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/terapia
5.
Int J Mol Sci ; 23(3)2022 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-35163606

RESUMO

Generation of relevant and robust models for neurological disorders is of main importance for both target identification and drug discovery. The non-cell autonomous effects of glial cells on neurons have been described in a broad range of neurodegenerative and neurodevelopmental disorders, pointing to neuroglial interactions as novel alternative targets for therapeutics development. Interestingly, the recent breakthrough discovery of human induced pluripotent stem cells (hiPSCs) has opened a new road for studying neurological and neurodevelopmental disorders "in a dish". Here, we provide an overview of the generation and modeling of both neuronal and glial cells from human iPSCs and a brief synthesis of recent work investigating neuroglial interactions using hiPSCs in a pathophysiological context.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/transplante , Doenças Neurodegenerativas , Transtornos do Neurodesenvolvimento , Neuroglia/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/terapia , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/terapia
6.
Sci Adv ; 7(14)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33789888

RESUMO

Huntington disease (HD) damages the corticostriatal circuitry in large part by impairing transport of brain-derived neurotrophic factor (BDNF). We hypothesized that improving vesicular transport of BDNF could slow or prevent disease progression. We therefore performed selective proteomic analysis of vesicles transported within corticostriatal projecting neurons followed by in silico screening and identified palmitoylation as a pathway that could restore defective huntingtin-dependent trafficking. Using a synchronized trafficking assay and an HD network-on-a-chip, we found that increasing brain palmitoylation via ML348, which inhibits the palmitate-removing enzyme acyl-protein thioesterase 1 (APT1), restores axonal transport, synapse homeostasis, and survival signaling to wild-type levels without toxicity. In human HD induced pluripotent stem cell-derived cortical neurons, ML348 increased BDNF trafficking. In HD knock-in mice, it efficiently crossed the blood-brain barrier to restore palmitoylation levels and reverse neuropathology, locomotor deficits, and anxio-depressive behaviors. APT1 and its inhibitor ML348 thus hold therapeutic interest for HD.


Assuntos
Doença de Huntington , Animais , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Lipoilação , Camundongos , Proteômica
8.
Nat Commun ; 10(1): 4357, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554807

RESUMO

Cell therapy products (CTP) derived from pluripotent stem cells (iPSCs) may constitute a renewable, specifically differentiated source of cells to potentially cure patients with neurodegenerative disorders. However, the immunogenicity of CTP remains a major issue for therapeutic approaches based on transplantation of non-autologous stem cell-derived neural grafts. Despite its considerable side-effects, long-term immunosuppression, appears indispensable to mitigate neuro-inflammation and prevent rejection of allogeneic CTP. Matching iPSC donors' and patients' HLA haplotypes has been proposed as a way to access CTP with enhanced immunological compatibility, ultimately reducing the need for immunosuppression. In the present work, we challenge this paradigm by grafting autologous, MHC-matched and mis-matched neuronal grafts in a primate model of Huntington's disease. Unlike previous reports in unlesioned hosts, we show that in the absence of immunosuppression MHC matching alone is insufficient to grant long-term survival of neuronal grafts in the lesioned brain.


Assuntos
Rejeição de Enxerto/imunologia , Doença de Huntington/terapia , Células-Tronco Pluripotentes Induzidas/transplante , Complexo Principal de Histocompatibilidade/imunologia , Neurônios/transplante , Animais , Diferenciação Celular/imunologia , Citotoxicidade Imunológica/imunologia , Modelos Animais de Doenças , Teste de Histocompatibilidade , Humanos , Doença de Huntington/imunologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/imunologia , Neurônios/citologia , Neurônios/imunologia , Primatas , Ratos Nus , Transplante Autólogo
9.
Stem Cell Reports ; 13(3): 448-457, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31447328

RESUMO

The mechanisms underlying the selective degeneration of medium spiny neurons (MSNs) in Huntington disease (HD) remain largely unknown. CTIP2, a transcription factor expressed by all MSNs, is implicated in HD pathogenesis because of its interactions with mutant huntingtin. Here, we report a key role for CTIP2 in protein phosphorylation via governing protein kinase A (PKA) signaling in human striatal neurons. Transcriptomic analysis of CTIP2-deficient MSNs implicates CTIP2 target genes at the heart of cAMP-Ca2+ signal integration in the PKA pathway. These findings are further supported by experimental evidence of a substantial reduction in phosphorylation of DARPP32 and GLUR1, two PKA targets in CTIP2-deficient MSNs. Moreover, we show that CTIP2-dependent dysregulation of protein phosphorylation is shared by HD hPSC-derived MSNs and striatal tissues of two HD mouse models. This study therefore establishes an essential role for CTIP2 in human MSN homeostasis and provides mechanistic and potential therapeutic insight into striatal neurodegeneration.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Neurônios/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Corpo Estriado/metabolismo , Edição de Genes , Células-Tronco Embrionárias Humanas/citologia , Humanos , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Neurônios/citologia , Estresse Oxidativo , Fosforilação , Receptores de AMPA/metabolismo , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Transdução de Sinais , Transcriptoma , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética
10.
Neurobiol Dis ; 130: 104484, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31132407

RESUMO

As research progresses in the understanding of the molecular and cellular mechanisms underlying neurodegenerative diseases like Huntington's disease (HD) and expands towards preclinical work for the development of new therapies, highly relevant animal models are increasingly needed to test new hypotheses and to validate new therapeutic approaches. In this light, we characterized an excitotoxic lesion model of striatal dysfunction in non-human primates (NHPs) using cognitive and motor behaviour assessment as well as functional imaging and post-mortem anatomical analyses. NHPs received intra-striatal stereotaxic injections of quinolinic acid bilaterally in the caudate nucleus and unilaterally in the left sensorimotor putamen. Post-operative MRI scans showed atrophy of the caudate nucleus and a large ventricular enlargement in all 6 NHPs that correlated with post-mortem measurements. Behavioral analysis showed deficits in 2 analogues of the Wisconsin card sorting test (perseverative behavior) and in an executive task, while no deficits were observed in a visual recognition or an episodic memory task at 6 months following surgery. Spontaneous locomotor activity was decreased after lesion and the incidence of apomorphine-induced dyskinesias was significantly increased at 3 and 6 months following lesion. Positron emission tomography scans obtained at end-point showed a major deficit in glucose metabolism and D2 receptor density limited to the lesioned striatum of all NHPs compared to controls. Post-mortem analyses revealed a significant loss of medium-sized spiny neurons in the striatum, a loss of neurons and fibers in the globus pallidus, a unilateral decrease in dopaminergic neurons of the substantia nigra and a loss of neurons in the motor and dorsolateral prefrontal cortex. Overall, we show that this robust NHP model presents specific behavioral (learning, execution and retention of cognitive tests) and metabolic functional deficits that, to the best of our knowledge, are currently not mimicked in any available large animal model of striatal dysfunction. Moreover, we used non-invasive, translational techniques like behavior and imaging to quantify such deficits and found that they correlate to a significant cell loss in the striatum and its main input and output structures. This model can thus significantly contribute to the pre-clinical longitudinal evaluation of the ability of new therapeutic cell, gene or pharmacotherapy approaches in restoring the functionality of the striatal circuitry.


Assuntos
Disfunção Cognitiva , Modelos Animais de Doenças , Doença de Huntington , Transtornos Motores , Animais , Disfunção Cognitiva/induzido quimicamente , Corpo Estriado/patologia , Corpo Estriado/fisiopatologia , Doença de Huntington/induzido quimicamente , Doença de Huntington/patologia , Doença de Huntington/fisiopatologia , Estudos Longitudinais , Macaca fascicularis , Masculino , Transtornos Motores/induzido quimicamente , Ácido Quinolínico/toxicidade
11.
Stem Cell Reports ; 12(2): 230-244, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30639210

RESUMO

Reappraisal of neuropathological studies suggests that pathological hallmarks of Alzheimer's disease and Parkinson's disease (PD) spread progressively along predictable neuronal pathways in the human brain through unknown mechanisms. Although there is much evidence supporting the prion-like propagation and amplification of α-synuclein (α-Syn) in vitro and in rodent models, whether this scenario occurs in the human brain remains to be substantiated. Here we reconstructed in microfluidic devices corticocortical neuronal networks using human induced pluripotent stem cells derived from a healthy donor. We provide unique experimental evidence that different strains of human α-Syn disseminate in "wild-type" human neuronal networks in a prion-like manner. We show that two distinct α-Syn strains we named fibrils and ribbons are transported, traffic between neurons, and trigger to different extents, in a dose- and structure-dependent manner, the progressive accumulation of PD-like pathological hallmarks. We further demonstrate that seeded aggregation of endogenous soluble α-Syn affects synaptic integrity and mitochondria morphology.


Assuntos
Neurônios/metabolismo , alfa-Sinucleína/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/metabolismo
12.
Stem Cell Reports ; 11(5): 1199-1210, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30409508

RESUMO

Recent studies highlighted the importance of astrocytes in neuroinflammatory diseases, interacting closely with other CNS cells but also with the immune system. However, due to the difficulty in obtaining human astrocytes, their role in these pathologies is still poorly characterized. Here, we develop a serum-free protocol to differentiate human induced pluripotent stem cells (hiPSCs) into astrocytes. Gene expression and functional assays show that our protocol consistently yields a highly enriched population of resting mature astrocytes across the 13 hiPSC lines differentiated. Using this model, we first highlight the importance of serum-free media for astrocyte culture to generate resting astrocytes. Second, we assess the astrocytic response to IL-1ß, TNF-α, and IL-6, all cytokines important in neuroinflammation, such as multiple sclerosis. Our study reveals very specific profiles of reactive astrocytes depending on the triggering stimulus. This model provides ideal conditions for in-depth and unbiased characterization of astrocyte reactivity in neuroinflammatory conditions.


Assuntos
Astrócitos/patologia , Citocinas/farmacologia , Células-Tronco Pluripotentes Induzidas/patologia , Esclerose Múltipla/patologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Estudos de Casos e Controles , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura Livres de Soro , Humanos , Mediadores da Inflamação/metabolismo , Esclerose Múltipla/genética , Fenótipo , Remielinização/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética
13.
eNeuro ; 5(4)2018.
Artigo em Inglês | MEDLINE | ID: mdl-30310861

RESUMO

The neurodegenerative Huntington's disease (HD) is caused by a polyglutamine (polyQ) amplification in the huntingtin protein (HTT). Currently there is no effective therapy available for HD; however, several efforts are directed to develop and optimize HTT-lowering methods to improve HD phenotypes. To validate these approaches, there is an immediate need for reliable, sensitive, and easily accessible methods to quantify HTT expression. Using the AlphaLISA platform, we developed two novel sensitive and robust assays for quantification of HTT in biological samples using commercially available antibodies. The first, a polyQ-independent assay, measures the total pool of HTT, while the second, a polyQ-dependent assay, preferentially detects the mutant form of HTT. Using purified HTT protein standards and brain homogenates from an HD mouse model, we determine a lower limit of quantification of 1 and 3 pm and optimal reproducibility with CV values lower than 7% for intra- and 20% for interassay. In addition, we used the assays to quantify HTT in neural stem cells generated from patient-derived induced pluripotent stem cells in vitro and in human brain tissue lysates. Finally, we could detect changes in HTT levels in a mouse model where mutant HTT was conditionally deleted in neural tissue, verifying the potential to monitor the outcome of HTT-lowering strategies. This analytical platform is ideal for high-throughput screens and thus has an added value for the HD community as a tool to optimize novel therapeutic approaches aimed at modulating HTT protein levels.


Assuntos
Proteína Huntingtina/análise , Doença de Huntington/diagnóstico , Imunoensaio/normas , Animais , Modelos Animais de Doenças , Células HEK293 , Humanos , Imunoensaio/métodos , Camundongos , Mutação , Células-Tronco Neurais , Reprodutibilidade dos Testes
14.
Brain ; 141(5): 1434-1454, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29534157

RESUMO

The neurobiological functions of a number of kinases expressed in the brain are unknown. Here, we report new findings on DCLK3 (doublecortin like kinase 3), which is preferentially expressed in neurons in the striatum and dentate gyrus. Its function has never been investigated. DCLK3 expression is markedly reduced in Huntington's disease. Recent data obtained in studies related to cancer suggest DCLK3 could have an anti-apoptotic effect. Thus, we hypothesized that early loss of DCLK3 in Huntington's disease may render striatal neurons more susceptible to mutant huntingtin (mHtt). We discovered that DCLK3 silencing in the striatum of mice exacerbated the toxicity of an N-terminal fragment of mHtt. Conversely, overexpression of DCLK3 reduced neurodegeneration produced by mHtt. DCLK3 also produced beneficial effects on motor symptoms in a knock-in mouse model of Huntington's disease. Using different mutants of DCLK3, we found that the kinase activity of the protein plays a key role in neuroprotection. To investigate the potential mechanisms underlying DCLK3 effects, we studied the transcriptional changes produced by the kinase domain in human striatal neurons in culture. Results show that DCLK3 regulates in a kinase-dependent manner the expression of many genes involved in transcription regulation and nucleosome/chromatin remodelling. Consistent with this, histological evaluation showed DCLK3 is present in the nucleus of striatal neurons and, protein-protein interaction experiments suggested that the kinase domain interacts with zinc finger proteins, including the transcriptional activator adaptor TADA3, a core component of the Spt-ada-Gcn5 acetyltransferase (SAGA) complex which links histone acetylation to the transcription machinery. Our novel findings suggest that the presence of DCLK3 in striatal neurons may play a key role in transcription regulation and chromatin remodelling in these brain cells, and show that reduced expression of the kinase in Huntington's disease could render the striatum highly vulnerable to neurodegeneration.


Assuntos
Corpo Estriado/enzimologia , Proteína Huntingtina/genética , Doença de Huntington/terapia , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Quinases Semelhantes a Duplacortina , Regulação para Baixo/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Força da Mão/fisiologia , Doença de Huntington/genética , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora , Neurônios/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Stem Cell Res Ther ; 8(1): 285, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29258610

RESUMO

BACKGROUND: Epidermal grafting using cells derived from pluripotent stem cells will change the face of this side of regenerative cutaneous medicine. To date, the safety of the graft would be the major unmet deal in order to implement long-term skin grafting. In this context, experiments on large animals appear unavoidable to assess this question and possible rejection. Cellular tools for large animal models should be constructed. METHODS: In this study, we generated monkey pluripotent stem cell-derived keratinocytes and evaluated their capacities to reconstruct an epidermis, in vitro as well as in vivo. RESULTS: Monkey pluripotent stem cells were differentiated efficiently into keratinocytes able to reconstruct fully epidermis presenting a low level of major histocompatibility complex class-I antigens, opening the way for autologous or allogeneic epidermal long-term grafting. CONCLUSIONS: Functional keratinocytes generated from nonhuman primate embryonic stem cells and induced pluripotent stem cells reproduce an in-vitro and in-vivo stratified epidermis. These monkey skin grafts will be considered to model autologous or allogeneic epidermal grafting using either embryonic stem cells or induced pluripotent stem cells. This graft model will allow us to further investigate the safety, efficacy and immunogenicity of nonhuman primate PSC-derived epidermis in the perspective of human skin cell therapy.


Assuntos
Queratinócitos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Haplorrinos , Queratinócitos/citologia
16.
Cell Rep ; 20(12): 2980-2991, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28930690

RESUMO

Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases.


Assuntos
Sistemas CRISPR-Cas/genética , Doenças do Sistema Nervoso Central/genética , Edição de Genes , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Sequência de Bases , Células Cultivadas , Córtex Cerebral/citologia , Células HEK293 , Humanos , Proteína Huntingtina/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Cinética , Camundongos , Neurônios/citologia , Neurônios/metabolismo
17.
Mol Ther Methods Clin Dev ; 5: 259-276, 2017 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-28603746

RESUMO

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting from a polyglutamine expansion in the huntingtin (HTT) protein. There is currently no cure for this disease, but recent studies suggest that RNAi to downregulate the expression of both normal and mutant HTT is a promising therapeutic approach. We previously developed a small hairpin RNA (shRNA), vectorized in an HIV-1-derived lentiviral vector (LV), that reduced pathology in an HD rodent model. Here, we modified this vector for preclinical development by using a tat-independent third-generation LV (pCCL) backbone and removing the original reporter genes. We demonstrate that this novel vector efficiently downregulated HTT expression in vitro in striatal neurons derived from induced pluripotent stem cells (iPSCs) of HD patients. It reduced two major pathological HD hallmarks while triggering a minimal inflammatory response, up to 6 weeks after injection, when administered by stereotaxic surgery in the striatum of an in vivo rodent HD model. Further assessment of this shRNA vector in vitro showed proper processing by the endogenous silencing machinery, and we analyzed gene expression changes to identify potential off-targets. These preclinical data suggest that this new shRNA vector fulfills primary biosafety and efficiency requirements for further development in the clinic as a cure for HD.

18.
PLoS One ; 11(2): e0148680, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26863614

RESUMO

Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington's disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150Glued complex interactions.


Assuntos
Mutação , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Adulto , Idade de Início , Alelos , Antígenos Nucleares/análise , Proteínas de Ciclo Celular , Células Cultivadas , Complexo Dinactina , Dineínas/análise , Genes Dominantes , Células-Tronco Embrionárias Humanas/citologia , Humanos , Proteína Huntingtina , Proteínas Associadas aos Microtúbulos/análise , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/fisiologia , Células-Tronco Neurais/ultraestrutura , Proteínas Associadas à Matriz Nuclear/análise , Peptídeos/análise , Células-Tronco Pluripotentes/citologia , Polimorfismo de Nucleotídeo Único , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/genética , Fuso Acromático/ultraestrutura , Frações Subcelulares/química , Expansão das Repetições de Trinucleotídeos
19.
Cell Stem Cell ; 15(5): 653-65, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25517469

RESUMO

Considerable progress has been made in generating fully functional and transplantable dopamine neurons from human embryonic stem cells (hESCs). Before these cells can be used for cell replacement therapy in Parkinson's disease (PD), it is important to verify their functional properties and efficacy in animal models. Here we provide a comprehensive preclinical assessment of hESC-derived midbrain dopamine neurons in a rat model of PD. We show long-term survival and functionality using clinically relevant MRI and PET imaging techniques and demonstrate efficacy in restoration of motor function with a potency comparable to that seen with human fetal dopamine neurons. Furthermore, we show that hESC-derived dopamine neurons can project sufficiently long distances for use in humans, fully regenerate midbrain-to-forebrain projections, and innervate correct target structures. This provides strong preclinical support for clinical translation of hESC-derived dopamine neurons using approaches similar to those established with fetal cells for the treatment of Parkinson's disease.


Assuntos
Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/transplante , Células-Tronco Embrionárias/citologia , Feto/citologia , Doença de Parkinson/terapia , Animais , Axônios/metabolismo , Sobrevivência Celular , Modelos Animais de Doenças , Humanos , Masculino , Mesencéfalo/embriologia , Neostriado/patologia , Fatores de Transcrição Otx/metabolismo , Doença de Parkinson/patologia , Ratos Nus , Substância Negra/patologia , Transmissão Sináptica , Fatores de Tempo
20.
PLoS One ; 9(6): e99341, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24926995

RESUMO

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting from polyglutamine expansion in the huntingtin (HTT) protein and for which there is no cure. Although suppression of both wild type and mutant HTT expression by RNA interference is a promising therapeutic strategy, a selective silencing of mutant HTT represents the safest approach preserving WT HTT expression and functions. We developed small hairpin RNAs (shRNAs) targeting single nucleotide polymorphisms (SNP) present in the HTT gene to selectively target the disease HTT isoform. Most of these shRNAs silenced, efficiently and selectively, mutant HTT in vitro. Lentiviral-mediated infection with the shRNAs led to selective degradation of mutant HTT mRNA and prevented the apparition of neuropathology in HD rat's striatum expressing mutant HTT containing the various SNPs. In transgenic BACHD mice, the mutant HTT allele was also silenced by this approach, further demonstrating the potential for allele-specific silencing. Finally, the allele-specific silencing of mutant HTT in human embryonic stem cells was accompanied by functional recovery of the vesicular transport of BDNF along microtubules. These findings provide evidence of the therapeutic potential of allele-specific RNA interference for HD.


Assuntos
Encéfalo/citologia , Terapia Genética/métodos , Doença de Huntington/terapia , Proteínas Mutantes/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , RNA Interferente Pequeno/genética , Animais , Encéfalo/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Células-Tronco Embrionárias/citologia , Células HEK293 , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Técnicas In Vitro , Masculino , Camundongos , Proteínas Mutantes/genética , Polimorfismo de Nucleotídeo Único , Isoformas de RNA/metabolismo , Estabilidade de RNA , Ratos , Ratos Wistar
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