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1.
FASEB J ; 37(2): e22770, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36688807

RESUMO

The search for reliable human blood-brain barrier (BBB) models represents a challenge for the development/testing of strategies aiming to enhance brain delivery of drugs. Human-induced pluripotent stem cells (hiPSCs) have raised hopes in the development of predictive BBB models. Differentiating strategies are thus required to generate endothelial cells (ECs), a major component of the BBB. Several hiPSC-based protocols have reported the generation of in vitro models with significant differences in barrier properties. We studied in depth the properties of iPSCs byproducts from two protocols that have been established to yield these in vitro barrier models. Our analysis/study reveals that iPSCs derivatives endowed with EC features yield high permeability models while the cells that exhibit outstanding barrier properties show principally epithelial cell-like (EpC) features. We found that models containing EpC-like cells express tight junction proteins, transporters/efflux pumps and display a high functional tightness with very low permeability, which are features commonly shared between BBB and epithelial barriers. Our study demonstrates that hiPSC-based BBB models need extensive characterization beforehand and that a reliable human BBB model containing EC-like cells and displaying low permeability is still needed.


Assuntos
Barreira Hematoencefálica , Células-Tronco Pluripotentes Induzidas , Humanos , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Permeabilidade
3.
FASEB J ; 35(7): e21727, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34117802

RESUMO

We previously discovered the implication of membrane-type 5-matrix metalloproteinase (MT5-MMP) in Alzheimer's disease (AD) pathogenesis. Here, we shed new light on pathogenic mechanisms by which MT5-MMP controls the processing of amyloid precursor protein (APP) and the fate of amyloid beta peptide (Aß) as well as its precursor C99, and C83. We found in human embryonic kidney cells (HEK) carrying the APP Swedish familial mutation (HEKswe) that deleting the C-terminal non-catalytic domains of MT5-MMP hampered its ability to process APP and release the soluble 95 kDa form (sAPP95). Catalytically inactive MT5-MMP variants increased the levels of Aß and promoted APP/C99 sorting in the endolysosomal system, likely through interactions of the proteinase C-terminal portion with C99. Most interestingly, the deletion of the C-terminal domain of MT5-MMP caused a strong degradation of C99 by the proteasome and prevented Aß accumulation. These discoveries reveal new control of MT5-MMP over APP by proteolytic and non-proteolytic mechanisms driven by the C-terminal domains of the proteinase. The targeting of these non-catalytic domains of MT5-MMP could, therefore, provide new insights into the therapeutic regulation of APP-related pathology in AD.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteólise
4.
Mol Psychiatry ; 26(5): 1606-1618, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32327736

RESUMO

Autism spectrum disorders (ASD) are complex neurodevelopmental disorders with a very large number of risk loci detected in the genome. However, at best, each of them explains rare cases, the majority being idiopathic. Genomic data on ASD derive mostly from post-mortem brain analyses or cell lines derived from blood or patient-specific induced pluripotent stem cells (iPSCS). Therefore, the transcriptional and regulatory architecture of the nervous system, particularly during early developmental periods, remains highly incomplete. To access the critical disturbances that may have occurred during pregnancy or early childhood, we recently isolated stem cells from the nasal cavity of anesthetized patients diagnosed for ASD and compared them to stem cells from gender-matched control individuals without neuropsychiatric disorders. This allowed us to discover MOCOS, a non-mutated molybdenum cofactor sulfurase-coding gene that was under-expressed in the stem cells of most ASD patients of our cohort, disturbing redox homeostasis and synaptogenesis. We now report that a divergent transcription upstream of MOCOS generates an antisense long noncoding RNA, to which we coined the name COSMOC. Surprisingly, COSMOC is strongly under-expressed in all ASD patients of our cohort with the exception of a patient affected by Asperger syndrome. Knockdown studies indicate that loss of COSMOC reduces MOCOS expression, destabilizes lipid and energy metabolisms of stem cells, but also affects neuronal maturation and splicing of synaptic genes. Impaired expression of the COSMOC/MOCOS bidirectional unit might shed new lights on the origins of ASD that could be of importance for future translational studies.


Assuntos
Síndrome de Asperger , Transtorno do Espectro Autista , Células-Tronco Pluripotentes Induzidas , Transtornos do Neurodesenvolvimento , Sulfurtransferases/genética , Transtorno do Espectro Autista/genética , Humanos , Sistema Nervoso
5.
Nature ; 491(7425): 603-7, 2012 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-23075850

RESUMO

Nuclear-architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as ageing. It is therefore plausible that diseases whose manifestations correlate with ageing might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of ageing-associated disorders by focusing on a leucine-rich repeat kinase 2 (LRRK2) dominant mutation (G2019S; glycine-to-serine substitution at amino acid 2019), which is associated with familial and sporadic Parkinson's disease as well as impairment of adult neurogenesis in mice. Here we report on the generation of induced pluripotent stem cells (iPSCs) derived from Parkinson's disease patients and the implications of LRRK2(G2019S) mutation in human neural-stem-cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in nuclear-envelope organization, clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in Parkinson's disease iPSCs and were recapitulated after targeted knock-in of the LRRK2(G2019S) mutation in human embryonic stem cells. Analysis of human brain tissue showed nuclear-envelope impairment in clinically diagnosed Parkinson's disease patients. Together, our results identify the nucleus as a previously unknown cellular organelle in Parkinson's disease pathology and may help to open new avenues for Parkinson's disease diagnoses as well as for the potential development of therapeutics targeting this fundamental cell structure.


Assuntos
Proteínas Mutantes/metabolismo , Células-Tronco Neurais/patologia , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Apoptose , Diferenciação Celular , Divisão Celular , Linhagem Celular , Células Clonais/metabolismo , Células Clonais/patologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/patologia , Técnicas de Introdução de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Proteínas Mutantes/genética , Mutação , Células-Tronco Neurais/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Estresse Fisiológico
6.
Circulation ; 131(14): 1278-1290, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25739401

RESUMO

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development. METHODS AND RESULTS: By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs, TERMINATOR, ALIEN, and PUNISHER, specifically expressed in undifferentiated pluripotent stem cells, cardiovascular progenitors, and differentiated endothelial cells, respectively. Functional characterization, including localization studies, dynamic expression analyses, epigenetic modification monitoring, and knockdown experiments in lower vertebrates, as well as murine embryos and human cells, confirmed a critical role for each lncRNA specific for each analyzed developmental stage. CONCLUSIONS: We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development, and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development, mesodermal commitment, and cardiovascular specification.


Assuntos
Sistema Cardiovascular/crescimento & desenvolvimento , Células Endoteliais/química , Regulação da Expressão Gênica no Desenvolvimento/genética , Miócitos Cardíacos/química , Células-Tronco Pluripotentes/química , RNA Longo não Codificante/isolamento & purificação , Vertebrados/genética , Animais , Sistema Cardiovascular/metabolismo , Diferenciação Celular , Linhagem da Célula , Mapeamento Cromossômico , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Coração Fetal/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Dados de Sequência Molecular , Morfolinos/farmacocinética , Miócitos Cardíacos/citologia , RNA Longo não Codificante/fisiologia , Análise de Sequência de RNA , Transcriptoma , Vertebrados/crescimento & desenvolvimento , Peixe-Zebra/embriologia
7.
Nat Methods ; 10(1): 77-83, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23202434

RESUMO

Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct, in the absence of proliferation and multipotent progenitor generation, or indirect, by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state, induced by brief exposure to reprogramming factors, followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells, including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies.


Assuntos
Diferenciação Celular , Linhagem da Célula , Reprogramação Celular , Endotélio Vascular/citologia , Fibroblastos/citologia , Miócitos de Músculo Liso/citologia , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Western Blotting , Movimento Celular , Proliferação de Células , Células Cultivadas , Endotélio Vascular/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , Miócitos de Músculo Liso/metabolismo , Neovascularização Fisiológica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo
8.
Stem Cells ; 32(11): 2923-2938, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25175072

RESUMO

Reprogramming technologies have emerged as a promising approach for future regenerative medicine. Here, we report on the establishment of a novel methodology allowing for the conversion of human fibroblasts into hematopoietic progenitor-like cells with macrophage differentiation potential. SOX2 overexpression in human fibroblasts, a gene found to be upregulated during hematopoietic reconstitution in mice, induced the rapid appearance of CD34+ cells with a concomitant upregulation of mesoderm-related markers. Profiling of cord blood hematopoietic progenitor cell populations identified miR-125b as a factor facilitating commitment of SOX2-generated CD34+ cells to immature hematopoietic-like progenitor cells with grafting potential. Further differentiation toward the monocytic lineage resulted in the appearance of CD14+ cells with functional phagocytic capacity. In vivo transplantation of SOX2/miR-125b-generated CD34+ cells facilitated the maturation of the engrafted cells toward CD45+ cells and ultimately the monocytic/macrophage lineage. Altogether, our results indicate that strategies combining lineage conversion and further lineage specification by in vivo or in vitro approaches could help to circumvent long-standing obstacles for the reprogramming of human cells into hematopoietic cells with clinical potential.


Assuntos
Diferenciação Celular/fisiologia , Fibroblastos/citologia , Monócitos/citologia , Células-Tronco/citologia , Animais , Antígenos CD34/metabolismo , Linhagem da Célula/fisiologia , Células Cultivadas , Humanos , Antígenos Comuns de Leucócito/metabolismo , Camundongos
9.
J Biol Chem ; 287(29): 24131-8, 2012 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-22613719

RESUMO

The tubular epithelium of the kidney is susceptible to injury from a number of different causes, including inflammatory and immune disorders, oxidative stress, and nephrotoxins, among others. Primary renal epithelial cells remain one of the few tools for studying the biochemical and physiological characteristics of the renal tubular system. Nevertheless, differentiated primary cells are not suitable for recapitulation of disease properties that might arise during embryonic kidney formation and further maturation. Thus, cellular systems resembling kidney characteristics are in urgent need to model disease as well as to establish reliable drug-testing platforms. Induced pluripotent stem cells (iPSCs) bear the capacity to differentiate into every cell lineage comprising the adult organism. Thus, iPSCs bring the possibility for recapitulating embryonic development by directed differentiation into specific lineages. iPSC differentiation ultimately allows for both disease modeling in vitro and the production of cellular products with potential for regenerative medicine. Here, we describe the rapid, reproducible, and highly efficient generation of iPSCs derived from endogenous kidney tubular renal epithelial cells with only two transcriptional factors, OCT4 and SOX2. Kidney-derived iPSCs may provide a reliable cellular platform for the development of kidney differentiation protocols allowing drug discovery studies and the study of kidney pathology.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética
10.
Cell Rep ; 40(7): 111200, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35977506

RESUMO

Apolipoprotein E4 (APOEε4) is the major allelic risk factor for late-onset sporadic Alzheimer's disease (sAD). Inflammation is increasingly considered as critical in sAD initiation and progression. Identifying brain molecular mechanisms that could bridge these two risk factors remain unelucidated. Leveraging induced pluripotent stem cell (iPSC)-based strategies, we demonstrate that APOE controls inflammation in human astrocytes by regulating Transgelin 3 (TAGLN3) expression and, ultimately, nuclear factor κB (NF-κB) activation. We uncover that APOE4 specifically downregulates TAGLN3, involving histone deacetylases activity, which results in low-grade chronic inflammation and hyperactivated inflammatory responses. We show that APOE4 exerts a dominant negative effect to prime astrocytes toward a pro-inflammatory state that is pharmacologically reversible by TAGLN3 supplementation. We further confirm that TAGLN3 is downregulated in the brain of patients with sAD. Our findings highlight the APOE-TAGLN3-NF-κB axis regulating neuroinflammation in human astrocytes and reveal TAGLN3 as a molecular target to modulate neuroinflammation, as well as a potential biomarker for AD.


Assuntos
Doença de Alzheimer , Apolipoproteína E4 , Apolipoproteínas E/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doença de Alzheimer/metabolismo , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Apolipoproteínas E/genética , Astrócitos/metabolismo , Humanos , Inflamação/metabolismo , NF-kappa B/metabolismo
11.
Cells ; 10(7)2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34359875

RESUMO

For some time, it has been accepted that the ß-site APP cleaving enzyme 1 (BACE1) and the γ-secretase are two main players in the amyloidogenic processing of the ß-amyloid precursor protein (APP). Recently, the membrane-type 5 matrix metalloproteinase (MT5-MMP/MMP-24), mainly expressed in the nervous system, has been highlighted as a new key player in APP-processing, able to stimulate amyloidogenesis and also to generate a neurotoxic APP derivative. In addition, the loss of MT5-MMP has been demonstrated to abrogate pathological hallmarks in a mouse model of Alzheimer's disease (AD), thus shedding light on MT5-MMP as an attractive new therapeutic target. However, a more comprehensive analysis of the role of MT5-MMP is necessary to evaluate how its targeting affects neurons and glia in pathological and physiological situations. In this study, leveraging on CRISPR-Cas9 genome editing strategy, we established cultures of human-induced pluripotent stem cells (hiPSC)-derived neurons and astrocytes to investigate the impact of MT5-MMP deficiency on their phenotypes. We found that MT5-MMP-deficient neurons exhibited an increased number of primary and secondary neurites, as compared to isogenic hiPSC-derived neurons. Moreover, MT5-MMP-deficient astrocytes displayed higher surface area and volume compared to control astrocytes. The MT5-MMP-deficient astrocytes also exhibited decreased GLAST and S100ß expression. These findings provide novel insights into the physiological role of MT5-MMP in human neurons and astrocytes, suggesting that therapeutic strategies targeting MT5-MMP should be controlled for potential side effects on astrocytic physiology and neuronal morphology.


Assuntos
Astrócitos/metabolismo , Transportador 1 de Aminoácido Excitatório/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/genética , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Potenciais de Ação/fisiologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Astrócitos/citologia , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Transportador 1 de Aminoácido Excitatório/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Metaloproteinases da Matriz Associadas à Membrana/deficiência , Células-Tronco Neurais/citologia , Neurônios/citologia , Técnicas de Patch-Clamp , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Transdução de Sinais
12.
Stem Cells Int ; 2019: 2945435, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236114

RESUMO

The discovery of novel drugs for neurodegenerative diseases has been a real challenge over the last decades. The development of patient- and/or disease-specific in vitro models represents a powerful strategy for the development and validation of lead candidates in preclinical settings. The implementation of a reliable platform modeling dopaminergic neurons will be an asset in the study of dopamine-associated pathologies such as Parkinson's disease. Disease models based on cell reprogramming strategies, using either human-induced pluripotent stem cells or transcription factor-mediated transdifferentiation, are among the most investigated strategies. However, multipotent adult stem cells remain of high interest to devise direct conversion protocols and establish in vitro models that could bypass certain limitations associated with reprogramming strategies. Here, we report the development of a six-step chemically defined protocol that drives the transdifferentiation of human nasal olfactory stem cells into dopaminergic neurons. Morphological changes were progressively accompanied by modifications matching transcript and protein dopaminergic signatures such as LIM homeobox transcription factor 1 alpha (LMX1A), LMX1B, and tyrosine hydroxylase (TH) expression, within 42 days of differentiation. Phenotypic changes were confirmed by the production of dopamine from differentiated neurons. This new strategy paves the way to develop more disease-relevant models by establishing reprogramming-free patient-specific dopaminergic cell models for drug screening and/or target validation for neurodegenerative diseases.

14.
Front Mol Neurosci ; 11: 452, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30618604

RESUMO

Age-related neurosensory deficit of the inner ear is mostly due to a loss of hair cells (HCs). Development of stem cell-based therapy requires a better understanding of factors and signals that drive stem cells into otic sensory progenitor cells (OSPCs) to replace lost HCs. Human induced pluripotent stem cells (hiPSCs) theoretically represent an unlimited supply for the generation of human OSPCs in vitro. In this study, we developed a monolayer-based differentiation system to generate an enriched population of OSPCs via a stepwise differentiation of hiPSCs. Gene and protein expression analyses revealed the efficient induction of a comprehensive panel of otic/placodal and late otic markers over the course of the differentiation. Furthermore, whole transcriptome analysis confirmed a developmental path of OSPC differentiation from hiPSCs. We found that modulation of WNT and transforming growth factor-ß (TGF-ß) signaling combined with fibroblast growth factor 3 (FGF3) and FGF10 treatment over a 6-day period drives the expression of early otic/placodal markers followed by late otic sensory markers within 13 days, indicative of a differentiation into embryonic-like HCs. In summary, we report a rapid and efficient strategy to generate an enriched population of OSPCs from hiPSCs, thereby establishing the value of this approach for disease modeling and cell-based therapies of the inner ear.

15.
PLoS One ; 13(6): e0198954, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29902227

RESUMO

The inner ear represents a promising system to develop cell-based therapies from human induced pluripotent stem cells (hiPSCs). In the developing ear, Notch signaling plays multiple roles in otic region specification and for cell fate determination. Optimizing hiPSC induction for the generation of appropriate numbers of otic progenitors and derivatives, such as hair cells, may provide an unlimited supply of cells for research and cell-based therapy. In this study, we used monolayer cultures, otic-inducing agents, Notch modulation, and marker expression to track early and otic sensory lineages during hiPSC differentiation. Otic/placodal progenitors were derived from hiPSC cultures in medium supplemented with FGF3/FGF10 for 13 days. These progenitor cells were then treated for 7 days with retinoic acid (RA) and epidermal growth factor (EGF) or a Notch inhibitor. The differentiated cultures were analyzed in parallel by qPCR and immunocytochemistry. After the 13 day induction, hiPSC-derived cells displayed an upregulated expression of a panel of otic/placodal markers. Strikingly, a subset of these induced progenitor cells displayed key-otic sensory markers, the percentage of which was increased in cultures under Notch inhibition as compared to RA/EGF-treated cultures. Our results show that modulating Notch pathway during in vitro differentiation of hiPSC-derived otic/placodal progenitors is a valuable strategy to promote the expression of human otic sensory lineage genes.


Assuntos
Orelha Interna/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular , Linhagem da Célula , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores Notch/metabolismo
16.
Stem Cells Int ; 2017: 1478606, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28698717

RESUMO

Stem cell-based therapies critically rely on selective cell migration toward pathological or injured areas. We previously demonstrated that human olfactory ectomesenchymal stem cells (OE-MSCs), derived from an adult olfactory lamina propria, migrate specifically toward an injured mouse hippocampus after transplantation in the cerebrospinal fluid and promote functional recoveries. However, the mechanisms controlling their recruitment and homing remain elusive. Using an in vitro model of blood-brain barrier (BBB) and secretome analysis, we observed that OE-MSCs produce numerous proteins allowing them to cross the endothelial wall. Then, pan-genomic DNA microarrays identified signaling molecules that lesioned mouse hippocampus overexpressed. Among the most upregulated cytokines, both recombinant SPP1/osteopontin and CCL2/MCP-1 stimulate OE-MSC migration whereas only CCL2 exerts a chemotactic effect. Additionally, OE-MSCs express SPP1 receptors but not the CCL2 cognate receptor, suggesting a CCR2-independent pathway through other CCR receptors. These results confirm that OE-MSCs can be attracted by chemotactic cytokines overexpressed in inflamed areas and demonstrate that CCL2 is an important factor that could promote OE-MSC engraftment, suggesting improvement for future clinical trials.

18.
Nat Commun ; 7: 10743, 2016 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-26899176

RESUMO

Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently, the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed, respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last, screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together, our results highlight the potential of hiPSCs for studying human tumourigenesis.


Assuntos
Transformação Celular Neoplásica , Glioma/etiologia , Células-Tronco Pluripotentes Induzidas , Células-Tronco Neoplásicas/fisiologia , Células-Tronco Neurais/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Fatores de Transcrição SOXB1/metabolismo , Ensaio Tumoral de Célula-Tronco
19.
Nat Protoc ; 9(11): 2693-704, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25340442

RESUMO

This protocol presents recently developed methodologies for the differentiation of human pluripotent stem cells (hPSCs) into ureteric bud (UB) progenitor-like cells. Differentiation of human PSCs to UB progenitor-like cells allows for the generation of chimeric kidney cultures in which the human cells can self-assemble into chimeric 3D structures in combination with embryonic mouse kidney cells over a period of 18 d. UB progenitor-like cells are generated by a two-step process that combines in vitro commitment of human PSCs, whether embryonic stem cells (ESCs) or induced PSCs (iPSCs), under chemically defined culture conditions, with ex vivo cultures for the induction of 3D organogenesis. The models described here provide new opportunities for investigating human kidney development, modeling disease, evaluating regenerative medicine strategies, as well as for toxicology studies.


Assuntos
Técnicas de Cultura de Células , Rim/citologia , Técnicas de Cultura de Órgãos/métodos , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Feminino , Humanos , Rim/embriologia , Masculino , Camundongos Endogâmicos , Organogênese
20.
Cell Stem Cell ; 15(5): 589-604, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25517466

RESUMO

Heart failure is a leading cause of mortality and morbidity in the developed world, partly because mammals lack the ability to regenerate heart tissue. Whether this is due to evolutionary loss of regenerative mechanisms present in other organisms or to an inability to activate such mechanisms is currently unclear. Here we decipher mechanisms underlying heart regeneration in adult zebrafish and show that the molecular regulators of this response are conserved in mammals. We identified miR-99/100 and Let-7a/c and their protein targets smarca5 and fntb as critical regulators of cardiomyocyte dedifferentiation and heart regeneration in zebrafish. Although human and murine adult cardiomyocytes fail to elicit an endogenous regenerative response after myocardial infarction, we show that in vivo manipulation of this molecular machinery in mice results in cardiomyocyte dedifferentiation and improved heart functionality after injury. These data provide a proof of concept for identifying and activating conserved molecular programs to regenerate the damaged heart.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/fisiologia , Mamíferos/genética , MicroRNAs/genética , Regeneração/genética , Animais , Desdiferenciação Celular/genética , Proliferação de Células , Regulação para Baixo/genética , Inativação Gênica , Genoma , Humanos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Peixe-Zebra/genética
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