RESUMO
TDP-43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP, the gene encoding TDP-43, are one of the common causes of familial ALS. In this study, we investigate TDP-43 protein behavior in induced pluripotent stem cell (iPSC)-derived motor neurons from three ALS patients with different TARDBP mutations, three healthy controls and an isogenic control. TARDPB mutations induce several TDP-43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP-43, C-terminal fragments, and phospho-TDP-43. By generating iPSC lines with allele-specific tagging of TDP-43, we find that mutant TDP-43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry. Our findings also indicate that TDP-43 proteinopathy results in a defect in mitochondrial transport. Lastly, we show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP-43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP-43 pathology.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Transporte Axonal , Proteínas de Ligação a DNA/genética , Desacetilase 6 de Histona/metabolismo , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/genética , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Mutação de Sentido IncorretoRESUMO
Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy caused by a 1.5 megabase tandem duplication of chromosome 17 harboring the PMP22 gene. This dose-dependent overexpression of PMP22 results in disrupted Schwann cell myelination of peripheral nerves. To get better insights into the underlying pathogenic mechanisms in CMT1A, we investigated the role of PMP22 duplication on cellular homeostasis in CMT1A mouse models and in patient-derived induced pluripotent stem cells differentiated into Schwann cell precursors (iPSC-SCPs). We performed lipidomic profiling and bulk RNA sequencing on sciatic nerves of two developing CMT1A mouse models and on CMT1A patient derived iPSC-SCPs. For the sciatic nerves of the CMT1A mice, cholesterol and lipid metabolism was dose-dependently downregulated throughout development. For the CMT1A iPSC-SCPs, transcriptional analysis unveiled a strong suppression of genes related to autophagy and lipid metabolism. Gene ontology enrichment analysis identified disturbances in pathways related to plasma membrane components and cell receptor signaling. Lipidomic analysis confirmed the severe dysregulation in plasma membrane lipids, particularly sphingolipids, in CMT1A iPSC-SCPs. Furthermore, we identified reduced lipid raft dynamics, disturbed plasma membrane fluidity, and impaired cholesterol incorporation and storage, all of which could result from altered lipid storage homeostasis in the patient-derived CMT1A iPSC-SCPs. Importantly, this phenotype could be rescued by stimulating autophagy and lipolysis. We conclude that PMP22 duplication disturbs intracellular lipid storage and leads to a more disordered plasma membrane due to an alteration in the lipid composition, which ultimately may lead to impaired axo-glial interactions. Moreover, targeting lipid handling and metabolism could hold promise for the treatment of CMT1A patients.
RESUMO
Parkinson's disease (PD) is a progressive, neurodegenerative disease affecting over 1% of the population beyond 65 years of age. Although some PD cases are inheritable, the majority of PD cases occur in a sporadic manner. Risk factors comprise next to heredity, age, and gender also exposure to neurotoxins from for instance pesticides and herbicides. As PD is characterized by a loss of dopaminergic neurons in the substantia nigra, it is nearly impossible to access and extract these cells from patients for investigating disease mechanisms. The emergence of induced pluripotent stem (iPSC) technology allows differentiating and growing human dopaminergic neurons, which can be used for in vitro disease modeling. Here, we differentiated human iPSCs into dopaminergic neurons, and subsequently exposed the cells to increasing concentrations of the neurotoxin MPP+. Temporal transcriptomics analysis revealed a strong time- and dose-dependent response with genes over-represented across pathways involved in PD etiology such as "Parkinson's Disease", "Dopaminergic signaling" and "calcium signaling". Moreover, we validated this disease model by showing robust overlap with a meta-analysis of transcriptomics data from substantia nigra from post-mortem PD patients. The overlap included genes linked to e.g. mitochondrial dysfunction, neuron differentiation, apoptosis and inflammation. Our data shows, that MPP+-induced, human iPSC-derived dopaminergic neurons present molecular perturbations as observed in the etiology of PD. Therefore we propose iPSC-derived dopaminergic neurons as a foundation for a novel sporadic PD model to study the pathomolecular mechanisms of PD, but also to screen for novel anti-PD drugs and to develop and test new treatment strategies.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Doença de Parkinson , Humanos , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Doenças Neurodegenerativas/metabolismo , Transcriptoma/genéticaRESUMO
Loss-of-function mutations in the PRKN, PINK1 and PARK7 genes (encoding parkin, PINK1 and DJ-1, respectively) cause autosomal recessive forms of Parkinson's disease. PINK1 and parkin jointly mediate selective autophagy of damaged mitochondria (mitophagy), but the mechanisms by which loss of DJ-1 induces Parkinson's disease are not well understood. Here, we investigated PINK1/parkin-mediated mitophagy in cultured human fibroblasts and induced pluripotent stem cell-derived neurons with homozygous PARK7 mutations. We found that DJ-1 is essential for PINK1/parkin-mediated mitophagy. Loss of DJ-1 did not interfere with PINK1 or parkin activation after mitochondrial depolarization but blocked mitophagy further downstream by inhibiting recruitment of the selective autophagy receptor optineurin to depolarized mitochondria. By contrast, starvation-induced, non-selective autophagy was not affected by loss of DJ-1. In wild-type fibroblasts and induced pluripotent stem cell-derived dopaminergic neurons, endogenous DJ-1 translocated to depolarized mitochondria in close proximity to optineurin. DJ-1 translocation to depolarized mitochondria was dependent on PINK1 and parkin and did not require oxidation of cysteine residue 106 of DJ-1. Overexpression of DJ-1 did not rescue the mitophagy defect of PINK1- or parkin-deficient cells. These findings position DJ-1 downstream of PINK1 and parkin in the same pathway and suggest that disruption of PINK1/parkin/DJ-1-mediated mitophagy is a common pathogenic mechanism in autosomal recessive Parkinson's disease.
Assuntos
Mitofagia , Doença de Parkinson , Proteínas Quinases , Humanos , Mitocôndrias/metabolismo , Mitofagia/genética , Mitofagia/fisiologia , Doença de Parkinson/metabolismo , Proteínas Quinases/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Axonal Charcot-Marie-Tooth neuropathies (CMT type 2) are caused by inherited mutations in various genes functioning in different pathways. The types of genes and multiplicity of mutations reflect the clinical and genetic heterogeneity in CMT2 disease, which complicates its diagnosis and has inhibited the development of therapies. Here, we used CMT2 patient-derived pluripotent stem cells (iPSCs) to identify common hallmarks of axonal degeneration shared by different CMT2 subtypes. We compared the cellular phenotypes of neurons differentiated from CMT2 patient iPSCs with those from healthy controls and a CRISPR/Cas9-corrected isogenic line. Our results demonstrated neurite network alterations along with extracellular electrophysiological abnormalities in the differentiated motor neurons. Progressive deficits in mitochondrial and lysosomal trafficking, as well as in mitochondrial morphology, were observed in all CMT2 patient lines. Differentiation of the same CMT2 iPSC lines into peripheral sensory neurons only gave rise to cellular phenotypes in subtypes with sensory involvement, supporting the notion that some gene mutations predominantly affect motor neurons. We revealed a common mitochondrial dysfunction in CMT2-derived motor neurons, supported by alterations in the expression pattern and oxidative phosphorylation, which could be recapitulated in the sciatic nerve tissue of a symptomatic mouse model. Inhibition of a dual leucine zipper kinase could partially ameliorate the mitochondrial disease phenotypes in CMT2 subtypes. Altogether, our data reveal shared cellular phenotypes across different CMT2 subtypes and suggests that targeting such common pathomechanisms could allow the development of a uniform treatment for CMT2.
Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Mitocôndrias/metabolismo , Neurônios Motores/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Genótipo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Mitocôndrias/patologia , Neurônios Motores/patologia , Mutação , LinhagemRESUMO
INTRODUCTION: Tauopathies are neurodegenerative diseases characterized by TAU protein-related pathology, including frontotemporal dementia and Alzheimer's disease among others. Mutant TAU animal models are available, but none of them faithfully recapitulates human pathology and are not suitable for drug screening. METHODS: To create a new in vitro tauopathy model, we generated a footprint-free triple MAPT-mutant human induced pluripotent stem cell line (N279K, P301L, and E10+16 mutations) using clustered regularly interspaced short palindromic repeats-FokI and piggyBac transposase technology. RESULTS: Mutant neurons expressed pathogenic 4R and phosphorylated TAU, endogenously triggered TAU aggregation, and had increased electrophysiological activity. TAU-mutant cells presented deficiencies in neurite outgrowth, aberrant sequence of differentiation to cortical neurons, and a significant activation of stress response pathways. RNA sequencing confirmed stress activation, demonstrated a shift toward GABAergic identity, and an upregulation of neurodegenerative pathways. DISCUSSION: In summary, we generated a novel in vitro human induced pluripotent stem cell TAU-mutant model displaying neurodegenerative disease phenotypes that could be used for disease modeling and drug screening.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Tauopatias/metabolismo , Proteínas tau/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Potenciais da Membrana/fisiologia , Mutação , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurogênese/fisiologia , Crescimento Neuronal/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Tauopatias/genética , Tauopatias/patologia , Transcriptoma , Proteínas tau/genéticaRESUMO
Recent studies described the association between hematopoietic stem/progenitor cell (HSPC) expansion in the bone marrow (BM), leukocytosis in the peripheral blood, and accelerated atherosclerosis. We hypothesized that circulating HSPC may home to inflamed vessels, where they might contribute to inflammation and neointima formation. We demonstrated that Lin(-) Sca-1(+) cKit(+) (LSK cells) in BM and peripheral blood of LDLr(-/-) mice on high fat diet expressed significantly more integrin ß2 , which was responsible for LSK cell adhesion and migration toward ICAM-1 in vitro, and homing to injured arteries in vivo, all of which were blocked with an anti-CD18 blocking antibody. When homed LSK cells were isolated from ligated artery and injected to irradiated recipients, they resulted in BM reconstitution. Injection of CD18(+/+) LSK cells to immunodeficient Balb/C Rag2(-) É£C(-/-) recipients resulted in more severe inflammation and reinforced neointima formation in the ligated carotid artery, compared to mice injected with PBS and CD18(-/-) LSK cells. Hypercholesterolemia stimulated ERK phosphorylation (pERK) in LSK cells of LDLr(-/-) mice in vivo. Blockade of pERK reduced ARF1 expression, leading to decreased integrin ß2 function on HSPC. In addition, integrin ß2 function could be regulated via ERK-independent LRP1 pathway. Integrin ß2 expression on HSPC is regulated by hypercholesterolemia, specifically LDL, in pERK-dependent and -independent manners, leading to increased homing and localization of HSPC to injured arteries, which is highly correlated with arteriosclerosis.
Assuntos
Arteriosclerose/metabolismo , Antígenos CD18/biossíntese , Progressão da Doença , Células-Tronco Hematopoéticas/metabolismo , Animais , Arteriosclerose/patologia , Células-Tronco Hematopoéticas/patologia , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Adult stem cells have been investigated increasingly over the past years for multiple applications. Although they have a more favorable safety profile compared to pluripotent stem cells, they are still capable of self-renewal and differentiate into several cell types. We investigated the behavior of Oct4-positive (Oct4(+)) and Oct4-negative (Oct4(-) ) murine or rat bone marrow (BM)-derived stem cells in the healthy brain of syngeneic mice and rats. Engraftment of mouse and rat Oct4-positive BM-derived hypoblast-like stem cells (m/rOct4(+) BM-HypoSCs) resulted in yolk-sac tumor formation in the healthy brain which was monitored longitudinally using magnetic resonance imaging (MRI) and bioluminescence imaging (BLI). Contrast enhanced MRI confirmed the disruption of the blood brain barrier. In contrast, m/r Oct4-negative BM-derived multipotent adult progenitor cells (m/rOct4(-) BM-MAPCs) did not result in mass formation after engraftment into the brain. mOct4(+) BM-HypoSCs and mOct4(-) BM-MAPCs were transduced to express enhanced green fluorescent protein, firefly luciferase (fLuc), and herpes simplex virus-thymidine kinase to follow up suicide gene expression as a potential "safety switch" for tumor-forming stem cells by multimodal imaging. Both cell lines were eradicated efficiently in vivo by ganciclovir administration indicating successful suicide gene expression in vivo, as assessed by MRI, BLI, and histology. The use of suicide genes to prevent tumor formation is in particular of interest for therapeutic approaches where stem cells are used as vehicles to deliver therapeutic genes.
Assuntos
Ganciclovir/metabolismo , Células-Tronco/citologia , Animais , Encéfalo/metabolismo , Linhagem Celular , Proteínas de Fluorescência Verde/metabolismo , Luciferases de Vaga-Lume/metabolismo , Imageamento por Ressonância Magnética , Camundongos , Modelos Animais , Fator 3 de Transcrição de Octâmero/metabolismo , Coelhos , RatosRESUMO
OBJECTIVE: Recently, we demonstrated that scavenger receptor type BI (SR-BI), a high-density lipoprotein (HDL) receptor, was expressed on murine hematopoietic stem/progenitor cells (HSPC) and infusion of reconstituted HDL and purified human apolipoprotein A-I (apoA-I) suppressed HSPC proliferation. We hypothesized that SR-B1 expression is required for the observed antiproliferative effects of HDL on HSPC. APPROACH AND RESULTS: SR-BI-deficient (SR-BI(-/-)) mice and wild-type controls were fed on chow or high-fat diet (HFD) for 8 to 10 weeks. Under chow diet, a significant increase in Lin(-) Sca1(+) cKit(+) cells (LSK cells, so-called HSPC) was found in the bone marrow of SR-BI(-/-) mice when compared with wild-type mice. HFD induced a further expansion of CD150(+)CD48(-) LSK cells (HSC), HSPC, and granulocyte monocyte progenitors in SR-BI(-/-) mice. Injection of reactive oxygen species inhibitor N-acetylcysteine attenuated HFD-induced HSPC expansion, leukocytosis, and atherosclerosis in SR-BI(-/-) mice. ApoA-I infusion inhibited HSPC cell proliferation, Akt phosphorylation and reactive oxygen species production in HSPC and plaque progression in low-density lipoprotein receptor knockout (LDLr(-/-)) apoA-I(-/-) mice on HFD but had no effect on SR-BI(-/-) mice on HFD. Transplantation of SR-BI(-/-) bone marrow cells into irradiated LDLr(-/-) recipients resulted in enhanced white blood cells reconstitution, inflammatory cell production, and plaque development. In patients with coronary heart disease, HDL levels were negatively correlated with white blood cells count and HSPC frequency in the peripheral blood. By flow cytometry, SR-BI expression was detected on human HSPC. CONCLUSIONS: SR-BI plays a critical role in the HDL-mediated regulation HSPC proliferation and differentiation, which is associated with atherosclerosis progression.
Assuntos
Aterosclerose/etiologia , Células-Tronco Hematopoéticas/metabolismo , Lipoproteínas HDL/metabolismo , Receptores Depuradores Classe B/sangue , Receptores Depuradores Classe B/fisiologia , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Animais , Apolipoproteína A-I/farmacologia , Aterosclerose/sangue , Aterosclerose/prevenção & controle , Transplante de Medula Óssea , Divisão Celular/efeitos dos fármacos , Dieta Aterogênica/efeitos adversos , Progressão da Doença , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Hipercolesterolemia/complicações , Hipercolesterolemia/genética , Leucocitose/etiologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quimera por Radiação , Espécies Reativas de Oxigênio/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Receptores Depuradores Classe B/deficiência , Receptores Depuradores Classe B/genéticaRESUMO
Parkinson's disease (PD) is a complex neurodegenerative disorder influenced by genetic factors and environmental exposures. Polychlorinated biphenyls (PCBs), a group of synthetic organic compounds, have been identified as potential environmental risk factors for neurodegenerative diseases, including PD. We explored PCB-induced neurotoxicity mechanisms using iPSC-derived dopaminergic neurons and assessed their transcriptomic responses to varying PCB concentrations (0.01 µM, 0.5 µM, and 10 µM). Specifically, we focused on PCB-180, a congener known for its accumulation in human brains. The exposure durations were 24 h and 74 h, allowing us to capture both short-term and more prolonged effects on gene expression patterns. We observed that PCB exposure led to the suppression of oxidative phosphorylation, synaptic function, and neurotransmitter release, implicating these pathways in PCB-induced neurotoxicity. In our comparative analysis, we noted similarities in PCB-induced changes with other PD-related compounds like MPP+ and rotenone. Our findings also aligned with gene expression changes in human blood derived from a population exposed to PCBs, highlighting broader inflammatory responses. Additionally, molecular patterns seen in iPSC-derived neurons were confirmed in postmortem PD brain tissues, validating our in vitro results. In conclusion, our study offers novel insights into the multifaceted impacts of PCB-induced perturbations on various cellular contexts relevant to PD. The use of iPSC-derived dopaminergic neurons allowed us to decipher intricate transcriptomic alterations, bridging the gap between in vitro and in vivo findings. This work underscores the potential role of PCB exposure in neurodegenerative diseases like PD, emphasizing the need to consider both systemic and cell specific effects.
Assuntos
Neurônios Dopaminérgicos , Doença de Parkinson , Bifenilos Policlorados , Transcriptoma , Bifenilos Policlorados/toxicidade , Neurônios Dopaminérgicos/efeitos dos fármacos , Humanos , Transcriptoma/efeitos dos fármacos , Células Sanguíneas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Poluentes Ambientais/toxicidadeRESUMO
Oligodendrocytes (OLs) are responsible for myelin production and metabolic support of neurons. Defects in OLs are crucial in several neurodegenerative diseases including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). This protocol describes a method to generate oligodendrocyte precursor cells (OPCs) from human pluripotent stem cells (hPSCs) in only ~20 d, which can subsequently myelinate neurons, both in vitro and in vivo. To date, OPCs have been derived from eight different hPSC lines including those derived from patients with spontaneous and familial forms of MS and ALS, respectively. hPSCs, fated for 8 d toward neural progenitors, are transduced with an inducible lentiviral vector encoding for SOX10. The addition of doxycycline for 10 d results in >60% of cells being O4-expressing OPCs, of which 20% co-express the mature OL marker myelin basic protein (MBP). The protocol also describes an alternative for viral transduction, by incorporating an inducible SOX10 in the safe harbor locus AAVS1, yielding ~100% pure OPCs. O4+ OPCs can be purified and either cryopreserved or used for functional studies. As an example of the type of functional study for which the derived cells could be used, O4+ cells can be co-cultured with maturing hPSC-derived neurons in 96/384-well-format plates, allowing the screening of pro-myelinating compounds.
Assuntos
Bainha de Mielina/metabolismo , Células-Tronco Neurais/citologia , Neurogênese , Oligodendroglia/citologia , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células/métodos , Linhagem Celular , Humanos , Proteína Básica da Mielina/análise , Proteína Básica da Mielina/metabolismo , Células-Tronco Neurais/metabolismo , Oligodendroglia/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
Scarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation.
Assuntos
Diferenciação Celular/genética , Oligodendroglia/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXE/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Antígenos de Superfície/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Proteína Básica da Mielina/genética , Neurônios/patologia , Neurônios/transplante , Oligodendroglia/citologia , Oligodendroglia/transplante , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/transplante , Transcriptoma/genéticaRESUMO
Infections with the Zika virus (ZIKV) are responsible for congenital abnormalities and neurological disorders. We here demonstrate that ZIKV productively infects three types of human iPSC (induced pluripotent stem cells)-derived cells from the neural lineage, i.e. cortical and motor neurons as well as astrocytes. ZIKV infection results in all three cell types in the production of infectious virus particles and induces cytopathic effects (CPE). In cortical and motor neurons, an Asian isolate (PRVABC59) produced roughly 10-fold more virus than the prototypic African strain (MR766 strain). Viral replication and CPE is efficiently inhibited by the nucleoside polymerase inhibitor 7-deaza-2'-C-methyladenosine (7DMA). However, ribavirin and favipiravir, two molecules that inhibit ZIKV replication in Vero cells, did not inhibit ZIKV replication in the neuronal cells. These results highlight the need to assess the potential antiviral activity of novel ZIKV inhibitors in stem cell derived neuronal cultures.
Assuntos
Antivirais/farmacologia , Células-Tronco Pluripotentes Induzidas/virologia , Neurônios/virologia , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Zika virus/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Replicação do DNA/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ribavirina/farmacologia , Células VeroRESUMO
Modulation of adult neurogenesis may offer new therapeutic strategies for various brain disorders. In the adult mammalian brain the subventricular zone (SVZ) of the lateral ventricle is a region of continuous neurogenesis. Lentiviral vectors stably integrate into dividing and nondividing cells, in contrast to retroviral vectors, which integrate only into dividing cells. We compared their potential for gene transfer into both quiescent and slowly dividing stem cells as well as into more rapidly dividing progenitor cells. In contrast to retroviral vectors, stereotactic injection of lentiviral vectors into the SVZ of adult mice resulted in efficient and long-term marker gene expression in cells with characteristics of both immature type B cells and migrating precursor cells. After migration along the rostral migratory stream and differentiation, the number of enhanced green fluorescent protein (eGFP)-expressing granular and periglomerular interneurons increased over time in the ipsilateral olfactory bulb. Moreover, the number of eGFP-labeled neuronal progenitor cells in the SVZ increased over time. By intraventricular injection of lentiviral vectors we could restrict gene transfer to ependymal cells and type B astroglial-like stem cells. In conclusion, lentiviral vectors surpass retroviral vectors in efficient long-term in vivo marking of subventricular zone stem cells for basic research and therapeutic applications.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/fisiologia , Interneurônios/fisiologia , Lentivirus/fisiologia , Células-Tronco/fisiologia , Animais , Astrócitos/citologia , Astrócitos/virologia , Movimento Celular/fisiologia , Células Cultivadas , Epêndima/citologia , Epêndima/virologia , Feminino , Genes Reporter , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Injeções Intraventriculares , Interneurônios/citologia , Ventrículos Laterais/citologia , Ventrículos Laterais/virologia , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Proteínas Recombinantes/análise , Células-Tronco/citologia , Células-Tronco/virologia , Fatores de Tempo , Transfecção/métodosRESUMO
Although pluripotent stem cells can be differentiated into the hepatocyte lineages, such cells retain an immature phenotype. As the chromatin state of regulatory regions controls spatiotemporal gene expression during development, we evaluated changes in epigenetic histone marks in lineage-specific genes throughout in vitro hepatocyte differentiation from human embryonic stem cells (hESCs). Active acetylation and methylation marks at promoters and enhancers correlated with progressive changes in gene expression. However, repression-associated H3K27me3 marks at these control regions showed an inverse correlation with gene repression during transition from hepatic endoderm to a hepatocyte-like state. Inhibitor of Enhancer of Zeste Homolog 2 (EZH2) reduced H3K27me3 decoration but did not improve hepatocyte maturation. Thus, H3K27me3 at regulatory regions does not regulate transcription and appears dispensable for hepatocyte lineage differentiation of hESCs in vitro.
Assuntos
Biomarcadores/metabolismo , Linhagem da Célula , Hepatócitos/citologia , Hepatócitos/metabolismo , Histonas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Lisina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Dimetil Sulfóxido/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Metilação , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.
Assuntos
Células-Tronco Embrionárias/metabolismo , Marcação de Genes/métodos , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Recombinases/metabolismo , Transgenes , Células Cultivadas , Metilação de DNA , Dependovirus/genética , Células-Tronco Embrionárias/citologia , Inativação Gênica , Loci Gênicos , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Recombinases/genéticaRESUMO
RNA interference (RNAi) is an evolutionarily conserved mechanism of posttranscriptional gene-specific silencing. For in vivo applications, RNAi has been hampered until recently by inefficient delivery methods and by the transient nature of the gene suppression. Lentiviral vectors (LVs) hold great promise for gene therapeutic applications, pharmaceutical target validation, and functional genomics because stable gene transfer is mediated both in dividing and nondividing cells. We have used a lentiviral vector-based system for RNAi. We produced human immunodeficiency virus type 1-derived LVs encoding a short hairpin RNA specific for enhanced green fluorescent protein (EGFP) mRNA that were capable of inhibiting EGFP expression in mammalian cells. EGFP knockdown persisted after multiple passages of the cells. Of particular interest, our RNAi LVs were equally effective in suppression and prevention of EGFP expression after stereotactic injection in adult mouse brain. Therefore, we believe that the use of LVs for stable RNAi in brain will become a powerful aid to probe gene function in vivo and for gene therapy of diseases of the central nervous system.
Assuntos
Encéfalo , Regulação da Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos , Lentivirus/genética , Interferência de RNA , Animais , Sequência de Bases , Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/terapia , Regulação para Baixo , Proteínas de Fluorescência Verde , HIV-1/genética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Lentiviral vectors are promising tools for gene transfer into the central nervous system. We have characterized in detail transduction with human immunodeficiency virus type 1 (HIV-1)-derived vectors encoding enhanced green fluorescent protein (eGFP) in the adult mouse brain. Different brain regions such as the striatum, hippocampus, and the lateral ventricle were targeted. The eGFP protein was transported anterogradely in the nigrostriatal pathway, but we have found no evidence of transport of the lentiviral vector particle. The performance levels of the different generations of packaging and transfer plasmid were compared. Omission of the accessory genes from the packaging plasmid resulted in a modest decrease in transgene expression. Inclusion of the woodchuck hepatitis posttranscriptional regulatory element, on the one hand, and the central polypurine tract and termination sequences, on the other hand, in the transfer vector each resulted in a 4- to 5-fold increase in transgene expression levels. Combination of both elements enhanced expression levels more than the sum of the individual components, suggesting a synergistic effect. In the serum of mice injected with lentiviral vectors a humoral response to vector proteins was detected, but this did not compromise transgene expression. Immune response to the transgene was found only in a minority of the animals.
Assuntos
Encéfalo/metabolismo , Técnicas de Transferência de Genes , Lentivirus/genética , Animais , Apoptose , Western Blotting , Encéfalo/patologia , Divisão Celular , Vetores Genéticos , Proteínas de Fluorescência Verde , Vírus da Hepatite B da Marmota/genética , Hipocampo/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Microscopia Confocal , Microscopia de Fluorescência , Neuroglia/patologia , Neurônios/patologia , Plasmídeos/metabolismo , Fatores de Tempo , Transcrição Gênica , TransgenesRESUMO
Expression of NKX2-1 is required to specify definitive endoderm to respiratory endoderm. However, the transcriptional regulation of NKX2-1 is not fully understood. Here we demonstrate that aside from specifying undifferentiated human embryonic stem cell (hESC) to definitive endoderm, high concentrations of Activin-A are also necessary and sufficient to induce hESC-derived definitive endodermal progeny to a FOXA2/NKX2-1/GATA6/PAX9 positive respiratory epithelial fate. Activin-A directly mediates the induction of NKX2-1 by interacting with ALK4, leading to phosphorylation of SMAD2, which binds directly to the NKX2-1 promoter and activates its expression. Activin-A can be replaced by GDF11 but not transforming growth factor ß1. Addition of Wnt3a, SHH, FGF2, or BMP4 failed to induce NKX2-1. These results suggest that direct binding of Activin-A-responsive SMAD2 to the NKX2-1 promoter plays essential role during respiratory endoderm specification.