RESUMO
Three-dimensional (3D) in vitro culture systems using human induced pluripotent stem cells (hiPSCs) are useful tools to model neurodegenerative disease biology in physiologically relevant microenvironments. Though many successful biomaterials-based 3D model systems have been established for other neurogenerative diseases, such as Alzheimer's disease, relatively few exist for Parkinson's disease (PD) research. We employed tissue engineering approaches to construct a 3D silk scaffold-based platform for the culture of hiPSC-dopaminergic (DA) neurons derived from healthy individuals and PD patients harboring LRRK2 G2019S or GBA N370S mutations. We then compared results from protein, gene expression, and metabolic analyses obtained from two-dimensional (2D) and 3D culture systems. The 3D platform enabled the formation of dense dopamine neuronal network architectures and developed biological profiles both similar and distinct from 2D culture systems in healthy and PD disease lines. PD cultures developed in 3D platforms showed elevated levels of α-synuclein and alterations in purine metabolite profiles. Furthermore, computational network analysis of transcriptomic networks nominated several novel molecular interactions occurring in neurons from patients with mutations in LRRK2 and GBA. We conclude that the brain-like 3D system presented here is a realistic platform to interrogate molecular mechanisms underlying PD biology.
Assuntos
Neurônios Dopaminérgicos/patologia , Doença de Parkinson/patologia , Bioengenharia , Técnicas de Cultura de Células em Três Dimensões , Células Cultivadas , Neurônios Dopaminérgicos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/patologia , Neurogênese , Seda/química , Alicerces Teciduais/químicaRESUMO
The long-term risk of malignancy associated with stem cell therapies is a significant concern in the clinical application of this exciting technology. We report a cancer-selective strategy to enhance the safety of stem cell therapies. Briefly, using a cell engineering approach, we show that aggressive cancers derived from human or murine induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are strikingly sensitive to temporary MYC blockade. On the other hand, differentiated tissues derived from human or mouse iPSCs can readily tolerate temporary MYC inactivation. In cancer cells, endogenous MYC is required to maintain the metabolic and epigenetic functions of the embryonic and cancer-specific pyruvate kinase M2 isoform (PKM2). In summary, our results implicate PKM2 in cancer's increased MYC dependence and indicate dominant MYC inhibition as a cancer-selective fail-safe for stem cell therapies.
Assuntos
Engenharia Celular , Terapia Baseada em Transplante de Células e Tecidos/normas , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/diagnóstico por imagem , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Imageamento por Ressonância Magnética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/terapia , Neurogênese , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Radiografia , Hormônios Tireóideos/metabolismo , Proteínas de Ligação a Hormônio da TireoideRESUMO
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.
Assuntos
Envelhecimento/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Reprogramação Celular , Senescência Celular , Criança , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Neurônios Dopaminérgicos/transplante , Neurônios Dopaminérgicos/ultraestrutura , Fibroblastos/metabolismo , Humanos , Lamina Tipo A , Mesencéfalo/patologia , Camundongos , Pessoa de Meia-Idade , Doença de Parkinson/patologia , Fenótipo , Doadores de TecidosRESUMO
Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases, including schizophrenia, autism, and epilepsy. Here, we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes, and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology.
Assuntos
Diferenciação Celular , Córtex Cerebral/citologia , Células-Tronco Embrionárias/citologia , Interneurônios/citologia , Animais , Ciclo Celular , Linhagem da Célula , Movimento Celular , Células-Tronco Embrionárias/metabolismo , Potenciais Pós-Sinápticos Excitadores , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Potenciais Pós-Sinápticos Inibidores , Interneurônios/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/metabolismo , Fenótipo , Transdução de Sinais , Sinapses/metabolismo , Fator Nuclear 1 de Tireoide , Fatores de Tempo , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismoRESUMO
Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurr1+ stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.
Assuntos
Neurônios Dopaminérgicos/transplante , Células-Tronco Embrionárias/transplante , Células-Tronco Neurais/transplante , Animais , Diferenciação Celular , Linhagem Celular , Separação Celular/métodos , Sobrevivência Celular , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Genes Reporter , Sobrevivência de Enxerto , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TranscriptomaRESUMO
Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson's disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson's disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson's disease.
Assuntos
Transplante de Tecido Encefálico , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/transplante , Células-Tronco Embrionárias/citologia , Doença de Parkinson/terapia , Animais , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Feminino , Humanos , Macaca mulatta , Mesencéfalo/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ratos , Ratos Sprague-DawleyRESUMO
The isolation of human induced pluripotent stem cells (iPSCs) offers a new strategy for modelling human disease. Recent studies have reported the derivation and differentiation of disease-specific human iPSCs. However, a key challenge in the field is the demonstration of disease-related phenotypes and the ability to model pathogenesis and treatment of disease in iPSCs. Familial dysautonomia (FD) is a rare but fatal peripheral neuropathy, caused by a point mutation in the IKBKAP gene involved in transcriptional elongation. The disease is characterized by the depletion of autonomic and sensory neurons. The specificity to the peripheral nervous system and the mechanism of neuron loss in FD are poorly understood owing to the lack of an appropriate model system. Here we report the derivation of patient-specific FD-iPSCs and the directed differentiation into cells of all three germ layers including peripheral neurons. Gene expression analysis in purified FD-iPSC-derived lineages demonstrates tissue-specific mis-splicing of IKBKAP in vitro. Patient-specific neural crest precursors express particularly low levels of normal IKBKAP transcript, suggesting a mechanism for disease specificity. FD pathogenesis is further characterized by transcriptome analysis and cell-based assays revealing marked defects in neurogenic differentiation and migration behaviour. Furthermore, we use FD-iPSCs for validating the potency of candidate drugs in reversing aberrant splicing and ameliorating neuronal differentiation and migration. Our study illustrates the promise of iPSC technology for gaining new insights into human disease pathogenesis and treatment.
Assuntos
Disautonomia Familiar/patologia , Disautonomia Familiar/terapia , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/transplante , Adolescente , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Proteínas de Transporte/genética , Desdiferenciação Celular , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Células Cultivadas , Criança , Disautonomia Familiar/tratamento farmacológico , Disautonomia Familiar/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Cinetina/farmacologia , Cinetina/uso terapêutico , Masculino , Camundongos , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Especificidade de Órgãos , Fenótipo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Fatores de Elongação da TranscriçãoRESUMO
Neural stem or progenitor cells (NSC/NPCs) able to generate the different neuron and glial cell types of the cerebellum have been isolated in vitro, but their identity and location in the intact cerebellum are unclear. Here, we use inducible Cre recombination in GFAPCreER(T2) mice to irreversibly activate reporter gene expression at P2 (postnatal day 2), P5, and P12 in cells with GFAP (glial fibrillary acidic protein) promoter activity and analyze the fate of genetically tagged cells in vivo. We show that cells tagged at P2-P5 with beta-galactosidase or enhanced green fluorescent proteins reporter genes generate at least 30% of basket and stellate GABAergic interneurons in the molecular layer (ML) and that they lose their neurogenic potential by P12, after which they generate only glia. Tagged cells in the cerebellar white matter (WM) were initially GFAP/S100beta+ and expressed the NSC/NPCs proteins LeX, Musashi1, and Sox2 in vivo. One week after tagging, reporter+ cells in the WM upregulated the neuronal progenitor markers Mash1, Pax2, and Gad-67. These Pax2+ progenitors migrated throughout the cerebellar cortex, populating the ML and leaving the WM by P18. These data suggest that a pool of GFAP/S100beta+ glial cells located in the cerebellar WM generate a large fraction of cerebellar interneurons for the ML within the first postnatal 12 days of cerebellar development. This restricted critical period implies that powerful inhibitory factors may restrict their fate potential in vivo at later stages of development.
Assuntos
Cerebelo/metabolismo , Proteína Glial Fibrilar Ácida/genética , Interneurônios/metabolismo , Regiões Promotoras Genéticas/genética , Células-Tronco/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Biomarcadores/metabolismo , Linhagem da Célula/efeitos dos fármacos , Cerebelo/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Integrases/metabolismo , Interneurônios/citologia , Interneurônios/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Recombinação Genética/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Tamoxifeno/farmacologia , Fatores de TempoRESUMO
To identify the fates that astroglial cells can attain in the postnatal brain, we generated mice carrying an inducible Cre recombinase (Cre-ER(T2)) controlled by the human GFAP promoter (hGFAP). In mice carrying the GCE (hGFAP-Cre-ER(T2)) transgene, OHT (4-hydroxy-tamoxifen) injections induced Cre recombination in astroglial cells at postnatal day 5 and allowed us to permanently tag these cells with reporter genes. Three days after recombination, reporter-tagged cells were quiescent astroglial cells that expressed the stem cell marker LeX in the subventricular zone (SVZ) and dentate gyrus (DG). After 2-4 weeks, the tagged GFAP lineage included proliferating progenitors expressing the neuronal marker Dcx (Doublecortin) in the SVZ and the DG. After 4 weeks, the GFAP lineage generated mature neurons in the olfactory bulb (OB), DG, and, strikingly, also in the cerebral cortex. A major portion of all neurons in the DG and OB born at the end of the first postnatal week were generated from GFAP+ cells. In addition to neurons, mature oligodendrocytes and astrocytes populating the cerebral cortex and white matter were also the progeny of GFAP+ astroglial ancestors. Thus, genetic fate mapping of postnatal GFAP+ cells reveals that they seed the postnatal brain with neural progenitors/stem cells that in turn give rise to neural precursors and their mature neuronal and oligodendrocytic progeny in many CNS regions, including the cerebral cortex.