RESUMO
Significant efforts are ongoing to develop refined differentiation protocols to generate midbrain dopamine (DA) neurons from pluripotent stem cells for application in disease modeling, diagnostics, drug screening and cell-based therapies for Parkinson's disease. An increased understanding of the timing and molecular mechanisms that promote the generation of distinct subtypes of human midbrain DA during development will be essential for guiding future efforts to generate molecularly defined and subtype-specific DA neurons from pluripotent stem cells. Here, we use droplet-based single-cell RNA sequencing to transcriptionally profile the developing human ventral midbrain (VM) when the DA neurons are generated (6-11â weeks post-conception) and their subsequent differentiation into functional mature DA neurons in primary fetal 3D organoid-like cultures. This approach reveals that 3D cultures are superior to monolayer conditions for their ability to generate and maintain mature DA neurons; hence, they have the potential to be used for studying human VM development. These results provide a unique transcriptional profile of the developing human fetal VM and functionally mature human DA neurons that can be used to guide stem cell-based therapies and disease modeling approaches in Parkinson's disease.
Assuntos
Doença de Parkinson , Células-Tronco Pluripotentes , Humanos , Doença de Parkinson/genética , Doença de Parkinson/terapia , Neurônios Dopaminérgicos , Mesencéfalo , Diferenciação Celular/genéticaRESUMO
Two-dimensional neuronal cultures have a limited ability to recapitulate the in vivo environment of the brain. Here, we introduce a three-dimensional in vitro model for human glia-to-neuron conversion, surpassing the spatial and temporal constrains of two-dimensional cultures. Focused on direct conversion to induced dopamine neurons (iDANs) relevant to Parkinson disease, the model generates functionally mature iDANs in 2 weeks and allows long-term survival. As proof of concept, we use single-nucleus RNA sequencing and molecular lineage tracing during iDAN generation and find that all glial subtypes generate neurons and that conversion relies on the coordinated expression of three neural conversion factors. We also show the formation of mature and functional iDANs over time. The model facilitates molecular investigations of the conversion process to enhance understanding of conversion outcomes and offers a system for in vitro reprogramming studies aimed at advancing alternative therapeutic strategies in the diseased brain.
Assuntos
Neurônios Dopaminérgicos , Neuroglia , Humanos , Neurônios Dopaminérgicos/metabolismo , Neuroglia/metabolismo , Diferenciação Celular , Células CultivadasRESUMO
Tissue clearing is commonly used for whole-brain imaging but seldom used for brain slices. Here, we present a simple protocol to slice, immunostain, and clear sections of adult rat brains for subsequent high-resolution confocal imaging. The protocol does not require toxic reagents or specialized equipment. We also provide instructions for culturing of rat brain slices free floating on permeable culture inserts, maintained in regular CO2 incubators, and handled only at media change.
Assuntos
Encéfalo , Ratos , Animais , Encéfalo/diagnóstico por imagem , Microscopia ConfocalRESUMO
Human pluripotent stem cells (hPSCs) are intrinsically able to self-organize into cerebral organoids that mimic features of developing human brain tissue. These three-dimensional structures provide a unique opportunity to generate cytoarchitecture and cell-cell interactions reminiscent of human brain complexity in a dish. However, current in vitro brain organoid methodologies often result in intra-organoid variability, limiting their use in recapitulating later developmental stages as well as in disease modeling and drug discovery. In addition, cell stress and hypoxia resulting from long-term culture lead to incomplete maturation and cell death within the inner core. Here, we used a recombinant silk microfiber network as a scaffold to drive hPSCs to self-arrange into engineered cerebral organoids. Silk scaffolding promoted neuroectoderm formation and reduced heterogeneity of cellular organization within individual organoids. Bulk and single cell transcriptomics confirmed that silk cerebral organoids display more homogeneous and functionally mature neuronal properties than organoids grown in the absence of silk scaffold. Furthermore, oxygen sensing analysis showed that silk scaffolds create more favorable growth and differentiation conditions by facilitating the delivery of oxygen and nutrients. The silk scaffolding strategy appears to reduce intra-organoid variability and enhances self-organization into functionally mature human brain organoids.
RESUMO
We have developed an efficient approach to generate functional induced dopaminergic (DA) neurons from adult human dermal fibroblasts. When performing DA neuronal conversion of patient fibroblasts with idiopathic Parkinson's disease (PD), we could specifically detect disease-relevant pathology in these cells. We show that the patient-derived neurons maintain age-related properties of the donor and exhibit lower basal chaperone-mediated autophagy compared with healthy donors. Furthermore, stress-induced autophagy resulted in an age-dependent accumulation of macroautophagic structures. Finally, we show that these impairments in patient-derived DA neurons leads to an accumulation of phosphorylated alpha-synuclein, the classical hallmark of PD pathology. This pathological phenotype is absent in neurons generated from induced pluripotent stem cells from the same patients. Taken together, our results show that direct neural reprogramming can be used for obtaining patient-derived DA neurons, which uniquely function as a cellular model to study age-related pathology relevant to idiopathic PD.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Adulto , Autofagia/fisiologia , Neurônios Dopaminérgicos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Doença de Parkinson/genética , alfa-Sinucleína/genéticaRESUMO
Direct reprogramming is an emerging research field where you can generate neurons from a somatic cell, such as a skin or glial cell by overexpressing neurogenic transcription factors. This technique allows fast generation of subtype-specific and functional neurons from both human and mouse cells. Despite the fact that neurons have been successfully generated both in vitro and in vivo, a more extensive analysis of the induced neurons including phenotypic functional identity or gradual maturity is still lacking. This is an important step for a further development of induced neurons towards cell therapy or disease modeling of neurological diseases. In this protocol, we describe a method for functional assessment of direct reprogrammed neuronal cells both in vitro and in vivo. Using a synapsin-driven reporter, our protocol allows for a direct identification of the reprogrammed neurons that permits functional assessment using patch-clamp electrophysiology. For in vitro reprogramming we further provide an optimized coating condition that allows a long-term maturation of human induced neurons in vitro.
Assuntos
Diferenciação Celular/genética , Reprogramação Celular , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Reprogramação Celular/genética , Técnicas de Reprogramação Celular , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação , Humanos , Lentivirus/genética , Camundongos , Técnicas de Patch-Clamp , Fatores de Transcrição/genética , Transdução GenéticaRESUMO
BACKGROUND: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson's disease (PD) and they provide the option of using the patient's own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. OBJECTIVE: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. METHODS: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. RESULTS: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. CONCLUSION: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.
Assuntos
Células-Tronco Pluripotentes Induzidas , Oxidopamina/farmacologia , Doença de Parkinson , Sinucleinopatias , alfa-Sinucleína/química , Animais , Neurônios Dopaminérgicos/metabolismo , Humanos , Oxidopamina/química , Doença de Parkinson/terapia , Ratos , alfa-Sinucleína/genéticaRESUMO
Three-dimensional brain organoids have emerged as a valuable model system for studies of human brain development and pathology. Here we establish a midbrain organoid culture system to study the developmental trajectory from pluripotent stem cells to mature dopamine neurons. Using single cell RNA sequencing, we identify the presence of three molecularly distinct subtypes of human dopamine neurons with high similarity to those in developing and adult human midbrain. However, despite significant advancements in the field, the use of brain organoids can be limited by issues of reproducibility and incomplete maturation which was also observed in this study. We therefore designed bioengineered ventral midbrain organoids supported by recombinant spider-silk microfibers functionalized with full-length human laminin. We show that silk organoids reproduce key molecular aspects of dopamine neurogenesis and reduce inter-organoid variability in terms of cell type composition and dopamine neuron formation.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Dopamina/metabolismo , Neurônios/metabolismo , Organoides/crescimento & desenvolvimento , Encéfalo/citologia , Humanos , Neurogênese , Neurônios/citologia , Organoides/citologia , Organoides/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , TranscriptomaRESUMO
Human glial progenitor cells (hGPCs) are promising cellular substrates to explore for the in situ production of new neurons for brain repair. Proof of concept for direct neuronal reprogramming of glial progenitors has been obtained in mouse models in vivo, but conversion using human cells has not yet been demonstrated. Such studies have been difficult to perform since hGPCs are born late during human fetal development, with limited accessibility for in vitro culture. In this study, we show proof of concept of hGPC conversion using fetal cells and also establish a renewable and reproducible stem cell-based hGPC system for direct neural conversion in vitro. Using this system, we have identified optimal combinations of fate determinants for the efficient dopaminergic (DA) conversion of hGPCs, thereby yielding a therapeutically relevant cell type that selectively degenerates in Parkinson's disease. The induced DA neurons show a progressive, subtype-specific phenotypic maturation and acquire functional electrophysiological properties indicative of DA phenotype.
Assuntos
Reprogramação Celular , Neurônios Dopaminérgicos/citologia , Células-Tronco Fetais/citologia , Mesencéfalo/citologia , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Fetais/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Modelos Biológicos , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Converting resident glia into functional and subtype-specific neurons in vivo by delivering reprogramming genes directly to the brain provides a step forward toward the possibility of treating brain injuries or diseases. To date, it has been possible to obtain GABAergic and glutamatergic neurons via in vivo conversion, but the precise phenotype of these cells has not yet been analyzed in detail. Here, we show that neurons reprogrammed using Ascl1, Lmx1a, and Nurr1 functionally mature and integrate into existing brain circuitry and that the majority of the reprogrammed neurons have properties of fast-spiking, parvalbumin-containing interneurons. When testing different combinations of genes for neural conversion with a focus on pro-neural genes and dopamine fate determinants, we found that functional neurons can be generated using different gene combinations and in different brain regions and that most of the reprogrammed neurons become interneurons, independently of the combination of reprogramming factors used.
Assuntos
Potenciais de Ação , Reprogramação Celular , Interneurônios/metabolismo , Neuroglia/metabolismo , Parvalbuminas/metabolismo , Animais , Diferenciação Celular , Denervação , Dopamina/metabolismo , Regulação da Expressão Gênica , Camundongos , Neostriado/citologia , Fenótipo , Fatores de TempoRESUMO
Stem cell treatments for neurodegenerative diseases are expected to reach clinical trials soon. Most of the approaches currently under development involve transplantation of immature progenitors that subsequently undergo phenotypic and functional maturation in vivo, and predicting the long-term graft outcome already at the progenitor stage remains a challenge. Here, we took an unbiased approach to identify predictive markers expressed in dopamine neuron progenitors that correlate with graft outcome in an animal model of Parkinson's disease through gene expression analysis of >30 batches of grafted human embryonic stem cell (hESC)-derived progenitors. We found that many of the commonly used markers did not accurately predict in vivo subtype-specific maturation. Instead, we identified a specific set of markers associated with the caudal midbrain that correlate with high dopaminergic yield after transplantation in vivo. Using these markers, we developed a good manufacturing practice (GMP) differentiation protocol for highly efficient and reproducible production of transplantable dopamine progenitors from hESCs.
Assuntos
Biomarcadores/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/transplante , Doença de Parkinson/terapia , Transplante de Células-Tronco , Pesquisa Translacional Biomédica , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Células Cultivadas , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Fator 8 de Crescimento de Fibroblasto/metabolismo , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Laminina/farmacologia , Mesencéfalo/metabolismo , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Núcleo Subtalâmico/citologia , Núcleo Subtalâmico/metabolismo , Fatores de Tempo , Resultado do TratamentoRESUMO
Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , Humanos , Fatores de Transcrição/genéticaRESUMO
The possibility of directly converting non-neuronal cells into neurons in situ in the brain would open therapeutic avenues aimed at repairing the brain after injury or degenerative disease. We have developed an adeno-associated virus (AAV)-based reporter system that allows selective GFP labeling of reprogrammed neurons. In this system, GFP is turned on only in reprogrammed neurons where it is stable and maintained for long time periods, allowing for histological and functional characterization of mature neurons. When combined with a modified rabies virus-based trans-synaptic tracing methodology, the system allows mapping of 3D circuitry integration into local and distal brain regions and shows that the newly reprogrammed neurons are integrated into host brain.