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BACKGROUND: Cell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including tunneling nanotubes, gap junctions, and extracellular vesicles (EV). Depending on local and systemic conditions, EVs may contain cargoes that promote survival, neuroprotection, or pathology. Our understanding of pathologic intercellular signaling has been bolstered by disease models using neurons derived from human pluripotent stems cells (hPSC). METHODS: Here, we used hPSC-derived retinal ganglion cells (hRGC) and the mouse visual system to investigate the influence of modulating EV generation on intercellular trafficking and cell survival. We probed the impact of EV modulation on cell survival by decreasing the catabolism of sphingomyelin into ceramide through inhibition of neutral sphingomyelinase (nSMase), using GW4869. We assayed for cell survival in vitro by probing for annexin A5, phosphatidylserine, viable mitochondria, and mitochondrial reactive oxygen species. In vivo, we performed intraocular injections of GW4869 and measured RGC and superior colliculus neuron density and RGC anterograde axon transport. RESULTS: Following twenty-four hours of dosing hRGCs with GW4869, we found that inhibition of nSMase decreased ceramide and enhanced GM1 ganglioside accumulation. This inhibition also reduced the density of small EVs, increased the density of large EVs, and enriched the pro-apoptotic protein, annexin A5. Reducing nSMase activity increased hRGC apoptosis initiation due to enhanced density and uptake of apoptotic particles, as identified by the annexin A5 binding phospholipid, phosphatidylserine. We assayed intercellular trafficking of mitochondria by developing a coculture system of GW4869-treated and naïve hRGCs. In treated cells, inhibition of nSMase reduced the number of viable mitochondria, while driving mitochondrial reactive oxygen species not only in treated, but also in naive hRGCs added in coculture. In mice, 20 days following a single intravitreal injection of GW4869, we found a significant loss of RGCs and their axonal recipient neurons in the superior colliculus. This followed a more dramatic reduction in anterograde RGC axon transport to the colliculus. CONCLUSION: Overall, our data suggest that perturbing the physiologic catabolism of sphingomyelin by inhibiting nSMase reorganizes plasma membrane associated sphingolipids, alters the profile of neuron-generated EVs, and promotes neurodegeneration in vitro and in vivo by shifting the balance of pro-survival versus -degenerative EVs. Video Abstract.
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Esfingomielina Fosfodiesterase , Esfingomielinas , Camundongos , Animais , Humanos , Esfingomielina Fosfodiesterase/metabolismo , Anexina A5 , Espécies Reativas de Oxigênio/metabolismo , Fosfatidilserinas , Ceramidas/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.
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Organoides/citologia , Células-Tronco Pluripotentes/citologia , Retina/citologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Forma Celular , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Humanos , Interneurônios/citologia , Interneurônios/metabolismo , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/ultraestrutura , Reprodutibilidade dos Testes , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Sinapses/metabolismo , Tomografia de Coerência ÓpticaRESUMO
Mutations in BBS6 cause two clinically distinct syndromes, Bardet-Biedl syndrome (BBS), a syndrome caused by defects in cilia transport and function, as well as McKusick-Kaufman syndrome, a genetic disorder characterized by congenital heart defects. Congenital heart defects are rare in BBS, and McKusick-Kaufman syndrome patients do not develop retinitis pigmentosa. Therefore, the McKusick-Kaufman syndrome allele may highlight cellular functions of BBS6 distinct from the presently understood functions in the cilia. In support, we find that the McKusick-Kaufman syndrome disease-associated allele, BBS6H84Y; A242S, maintains cilia function. We demonstrate that BBS6 is actively transported between the cytoplasm and nucleus, and that BBS6H84Y; A242S, is defective in this transport. We developed a transgenic zebrafish with inducible bbs6 to identify novel binding partners of BBS6, and we find interaction with the SWI/SNF chromatin remodeling protein Smarcc1a (SMARCC1 in humans). We demonstrate that through this interaction, BBS6 modulates the sub-cellular localization of SMARCC1 and find, by transcriptional profiling, similar transcriptional changes following smarcc1a and bbs6 manipulation. Our work identifies a new function for BBS6 in nuclear-cytoplasmic transport, and provides insight into the disease mechanism underlying the congenital heart defects in McKusick-Kaufman syndrome patients.
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Anormalidades Múltiplas/genética , Síndrome de Bardet-Biedl/genética , Chaperoninas do Grupo II/genética , Cardiopatias Congênitas/genética , Hidrocolpos/genética , Polidactilia/genética , Fatores de Transcrição/genética , Doenças Uterinas/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Transporte Ativo do Núcleo Celular/genética , Animais , Animais Geneticamente Modificados/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patologia , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Cílios/metabolismo , Cílios/patologia , Citoplasma/metabolismo , Modelos Animais de Doenças , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Humanos , Hidrocolpos/metabolismo , Hidrocolpos/patologia , Camundongos , Mutação , Polidactilia/metabolismo , Polidactilia/patologia , Transporte Proteico/genética , Fatores de Transcrição/biossíntese , Doenças Uterinas/metabolismo , Doenças Uterinas/patologia , Peixe-Zebra/genéticaRESUMO
Bardet-Biedl syndrome (BBS) is a well-known ciliopathy with mutations reported in 18 different genes. Most of the protein products of the BBS genes localize at or near the primary cilium and the centrosome. Near the centrosome, BBS proteins interact with centriolar satellite proteins, and the BBSome (a complex of seven BBS proteins) is believed to play a role in transporting ciliary membrane proteins. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. Here, we show that a centriolar satellite protein, AZI1 (also known as CEP131), interacts with the BBSome and regulates BBSome ciliary trafficking activity. Furthermore, we show that AZI1 interacts with the BBSome through BBS4. AZI1 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon AZI1 depletion. Under conditions in which the BBSome does not normally enter cilia, such as in BBS3 or BBS5 depleted cells, knock down of AZI1 with siRNA restores BBSome trafficking to cilia. Finally, we show that azi1 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer's vesicle abnormalities and melanosome transport delay. These findings associate AZI1 with the BBS pathway. Our findings provide further insight into the regulation of BBSome ciliary trafficking and identify AZI1 as a novel BBS candidate gene.
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Síndrome de Bardet-Biedl/genética , Centríolos/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Fatores de Ribosilação do ADP/genética , Animais , Síndrome de Bardet-Biedl/patologia , Proteínas de Ciclo Celular/genética , Centríolos/metabolismo , Centrossomo/metabolismo , Cílios/genética , Proteínas do Citoesqueleto , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas dos Microtúbulos/genética , Mutação , Transporte Proteico/genética , Proteínas/genética , RNA Interferente Pequeno , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Purpose: Axons depend on long-range transport of proteins and organelles which increases susceptibility to metabolic stress in disease. The axon initial segment (AIS) is particularly vulnerable due to the high bioenergetic demand of action potential generation. Here, we prepared retinal ganglion cells derived from human embryonic stem cells (hRGCs) to probe how axonal stress alters AIS morphology. Methods: hRGCs were cultured on coverslips or microfluidic platforms. We assayed AIS specification and morphology by immunolabeling against ankyrin G (ankG), an axon-specific protein, and postsynaptic density 95 (PSD-95), a dendrite-specific protein. Using microfluidic platforms that enable fluidic isolation, we added colchicine to the axon compartment to lesion axons. We verified axonopathy by measuring the anterograde axon transport of cholera toxin subunit B and immunolabeling against cleaved caspase 3 (CC3) and phosphorylated neurofilament H (SMI-34). We determined the influence of axon injury on AIS morphology by immunolabeling samples against ankG and measuring AIS distance from soma and length. Results: Based on measurements of ankG and PSD-95 immunolabeling, microfluidic platforms promote the formation and separation of distinct somatic-dendritic versus axonal compartments in hRGCs compared to coverslip cultures. Chemical lesioning of axons by colchicine reduced hRGC anterograde axon transport, increased varicosity density, and enhanced expression of CC3 and SMI-34. Interestingly, we found that colchicine selectively affected hRGCs with axon-carrying dendrites by reducing AIS distance from somas and increasing length, thus suggesting reduced capacity to maintain excitability. Conclusions: Thus, microfluidic platforms promote polarized hRGCs that enable modeling of axonopathy. Translational Relevance: Microfluidic platforms may be used to assay compartmentalized degeneration that occurs during glaucoma.
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Microfluídica , Células Ganglionares da Retina , Humanos , Axônios/metabolismo , Potenciais de Ação/fisiologiaRESUMO
Brain microphysiological systems (bMPS), which recapitulate human brain cellular architecture and functionality more closely than traditional monolayer cultures, have become a practical, non-invasive, and increasingly relevant platform for the study of neurological function in health and disease. These models include 3D spheroids and organoids as well as organ-on-chip models. Currently, however, existing 3D brain models vary in reflecting the relative populations of the different cell types present in the human brain. Most of the models consist mainly of neurons, while glial cells represent a smaller portion of the cell populations. Here, by means of a chemically defined glial-enriched medium (GEM), we present an improved method to expand the population of astrocytes and oligodendrocytes without compromising neuronal differentiation in bMPS. An important finding is that astrocytes not only increased in number but also changed in morphology when cultured in GEM, more closely recapitulating primary culture astrocytes. We demonstrate oligodendrocyte and astrocyte enrichment in GEM bMPS using a variety of complementary methods. We found that GEM bMPS are electro-chemically active and showed different patterns of Ca +2 staining and flux. Synaptic vesicles and terminals observed by electron microscopy were also present. No significant changes in neuronal differentiation were observed by gene expression, however, GEM enhanced neurite outgrowth and cell migration, and differentially modulated neuronal maturation in two different iPSC lines. Our results have the potential to significantly improve in vivo-like functionality of bMPS for the study of neurological diseases and drug discovery, contributing to the unmet need for safe human models.
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Brain microphysiological systems (bMPS) recapitulate human brain cellular architecture and functionality more closely than traditional monolayer cultures and have become increasingly relevant for the study of neurological function in health and disease. Existing 3D brain models vary in reflecting the relative populations of different cell types present in the human brain. Most models consist mainly of neurons, while glial cells represent a smaller portion of the cell populations. Here, by means of a chemically defined glial-enriched medium (GEM), an improved method to expand the population of astrocytes and oligodendrocytes without compromising neuronal differentiation in bMPS, is presented. An important finding is that astrocytes also change in morphology when cultured in GEM, more closely recapitulating primary culture astrocytes. GEM bMPS are electro-chemically active and show different patterns of calcium staining and flux. Synaptic vesicles and terminals observed by electron microscopy are also present. No significant changes in neuronal differentiation are observed by gene expression, however, GEM enhanced neurite outgrowth and cell migration, and differentially modulated neuronal maturation in two different cell lines. These results have the potential to significantly improve functionality of bMPS for the study of neurological diseases and drug discovery, contributing to the unmet need for safe human models.
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Promoting myelination capacity of endogenous oligodendrocyte precursor cells (OPCs) is a promising therapeutic approach for CNS demyelinating disorders such as Multiple Sclerosis (MS). To aid in the discovery of myelination-promoting compounds, we generated a genome-engineered human pluripotent stem cell (hPSC) line that consists of three reporters: identification-and-purification tag, GFP, and secreted-NanoLuc, driven by the endogenous PDGFRA, PLP1, and MBP genes, respectively. Using this cell line, we established a high-throughput drug screening platform and performed a small-molecule screen, which identified at least two myelination-promoting small-molecule (Ro1138452 and SR2211) that target prostacyclin (IP) receptor and retinoic acid receptor-related orphan receptor γ (RORγ), respectively. Single-cell-transcriptomic analysis of differentiating OPCs treated with these molecules further confirmed that they promote oligodendrocyte differentiation and revealed several pathways that are potentially modulated by them. The molecules and their target pathways provide promising targets for the possible development of remyelination-based therapy for MS and other demyelinating disorders.
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Introduction: Oligodendrocytes (OLs) are the myelin-forming cells of the central nervous system (CNS). Although OLs can be differentiated from human-induced pluripotent stem cells (hiPSCs), the in vitro modeling of axon myelination in human cells remains challenging. Brain microphysiological systems (bMPS, e.g. organoids) are complex three-dimensional (3D) cultures that offer an ideal system to study this process as OLs differentiate in a more in vivo-like environment; surrounded by neurons and astrocytes, which support the myelination of axons. Methods: Here, we take advantage of CRISPR/Cas9 technology to generate a hiPSC line in which proteolipid protein 1 (PLP1), an OLs marker, is tagged with super-fold GFP (sfGFP). While generating the PLP1-sfGFP reporter, we used reverse transfection and obtained higher Knock-In (KI) efficiency compared to forward transfection (61-72 vs. 46%). Results: After validation of the KI and quality control of the PLP1-sfGFP line, selected clones were differentiated into bMPS, and the fidelity, specificity, and function of the tagged PLP protein were verified in this model. We tracked different stages of oligodendrogenesis in the verified lines based on PLP1-sfGFP+ cells' morphology, and the presence of PLP1-sfGFP surrounding axons during bMPS' differentiation. Finally, we challenged the bMPS with cuprizone and quantified changes in both the percentage of PLP1-sfGFP expressing cells and the intensity of GFP expression. Discussion: This work demonstrates an efficient method for generating hiPSC KI lines and the description of a new 3D model to study OL differentiation, migration, and maturation both during in vitro neurodevelopment as well as in response to environmental chemicals or disease-associated stressors.
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Purpose: To investigate the effect of an active integrated stress response (ISR) on human corneal epithelial cell motility and cytokine production. Methods: ISR agonists tunicamycin (TUN) and SAL003 (SAL) were used to stimulate the ISR in immortalized corneal epithelial cell lines, primary human limbal epithelial stem cells, and ex vivo human corneas. Reporter lines for ISR-associated transcription factors activating transcription factor 4 (ATF4) and XBP1 activity were generated to visualize pathway activity in response to kinase-specific agonists. Scratch assays and multiplex magnetic bead arrays were used to investigate the effects of an active ISR on scratch wounds and cytokine production. A C/EBP homologous protein (CHOP) knockout cell line was generated to investigate the effects of ISR ablation. Finally, an ISR antagonist was assayed for its ability to rescue negative phenotypic changes associated with an active ISR. Results: ISR stimulation, mediated through CHOP, inhibited cell motility in both immortalized and primary human limbal epithelial cells. Scratch wounding of ex vivo corneas elicited an increase in the ISR mediators phosphorylated-eIF2α and ATF4. ISR stimulation also increased the production of vascular endothelial growth factor (VEGF) and proinflammatory cytokines. ISR ablation, through CHOP knockout or inhibition with integrated stress response inhibitor (ISRIB) rescued epithelia migration ability and reduced VEGF secretion. Conclusions: We demonstrate that the ISR has dramatic effects on the ability of corneal epithelial cells to respond to wounding models and increases the production of proinflammatory and angiogenic factors. Inhibition of the ISR may provide a new therapeutic option for corneal diseases in which the ISR is implicated.
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Estresse Fisiológico , Fator A de Crescimento do Endotélio Vascular , Fator 4 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/farmacologia , Citocinas/metabolismo , Células Epiteliais/metabolismo , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cell replacement therapies may be key in achieving functional recovery in neurodegenerative optic neuropathies diseases such as glaucoma. One strategy that holds promise in this regard is the use of human embryonic stem cell and induced pluripotent stem-derived retinal ganglion cells (hRGCs). Previous hRGC transplantation studies have shown modest success. This is in part due to the low survival and integration of the transplanted cells in the host retina. The field is further challenged by mixed assays and outcome measurements that probe and determine transplantation success. Thefore, we have devised a transplantation assay involving hRGCs and mouse retina explants that bypasses physical barriers imposed by retinal membranes. We show that hRGC neurites and somas are capable of invading mouse explants with a subset of hRGC neurites being guided by mouse RGC axons. Neonatal mouse retina explants, and to a lesser extent, adult explants, promote hRGC integrity and neurite outgrowth. Using this assay, we tested whether suppmenting cultures with brain derived neurotrophic factor (BDNF) and the adenylate cyclase activator, forskolin, enhances hRGC neurite integration, neurite outgrowth, and integrity. We show that supplementing cultures with a combination BDNF and forskolin strongly favors hRGC integrity, increasing neurite outgrowth and complexity as well as the invasion of mouse explants. The transplantation assay presented here is a practical tool for investigating strategies for testing and optimizing the integration of donor cells into host tissues.
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Células-Tronco Neurais , Retina , Células Ganglionares da Retina , Animais , Humanos , Camundongos , Adenilil Ciclases , Fator Neurotrófico Derivado do Encéfalo , Colforsina/farmacologia , Retina/cirurgia , Células Ganglionares da Retina/transplante , Células-Tronco Neurais/transplanteRESUMO
Astrocytes are instrumental in maintaining central nervous system (CNS) homeostasis and responding to injury. A major limitation of studying neurodegenerative diseases like multiple sclerosis (MS) is lack of human pathological specimens obtained during the acute stages, thereby relegating research to post-mortem specimens obtained years after the initiation of pathology. Rodent reactive astrocytes have been shown to be cytotoxic to neurons and oligodendrocytes but may differ from human cells, especially in diseases with genetic susceptibility. Herein, we purified human CD49f+ astrocytes from induced pluripotent stem cells derived from individual patient and control peripheral leukocytes. We compared TNF and IL1α stimulated human reactive astrocytes from seven persons with MS and six non-MS controls and show their transcriptomes are remarkably similar to those described in rodents. The functional effect of astrocyte conditioned media (ACM) was examined in a human oligodendrocyte precursor cell (OPC) line differentiation assay. ACM was not cytotoxic to the OPCs but robustly inhibited the myelin basic protein (MBP) reporter. No differences were seen between MS and control stimulated astrocytes at either the transcript level or in ACM mediated OPC suppression assays. We next used RNAseq to interrogate differentially expressed genes in the OPC lines that had suppressed differentiation from the human ACM. Remarkably, not only was OPC differentiation and myelin gene expression suppressed, but we observed induction of several immune pathways in OPCs exposed to the ACM. These data support the notion that reactive astrocytes can inhibit OPC differentiation thereby limiting their remyelination capacity, and that OPCs take on an immune profile in the context of inflammatory cues.
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Purpose: Human retinal ganglion cells (hRGC) derived from human pluripotent stem cells are promising candidates to model, protect, and replace degenerating RGCs. Here, we examined intrinsic morphologic and physiologic development of hRGCs. Methods: We used CRISPR-Cas9 to selectively express tdTomato under the RGC-specific promoter, BRN3B. Human pluripotent stem cells were chemically differentiated into hRGCs and cultured up to 7 weeks. We measured soma area, neurite complexity, synaptic protein, axon-related messenger RNA and protein, and voltage-dependent responses. Results: Soma area, neurite complexity, and postsynaptic density protein 95 increased over time. Soma area and neurite complexity increased proportionally week to week, and this relationship was dynamic, strengthening between 2 and 3 weeks and diminishing by 4 weeks. Postsynaptic density 95 localization was dependent on culture duration. After 1 to 2 weeks, postsynaptic density 95 localized within somas but redistributed along neurites after 3 to 4 weeks. Axon initial segment scaffolding protein, Ankyrin G, expression also increased over time, and by 7 weeks, Ankyrin G often localized within putative axons. Voltage-gated inward currents progressively developed, but outward currents matured by 4 weeks. Current-induced spike generation increased over time but limited by depolarization block. Conclusions: Human RGCs develop up to 7 weeks after culture. Thus, the state of hRGC maturation should be accounted for in designing models and treatments for optic neuropathies. Translational Relevance: We characterized hRGC morphologic and physiologic development towards identifying key time points when hRGCs express mechanisms that may be harnessed to enhance the efficacy of neuroprotective and cell replacement therapies.
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Células-Tronco Pluripotentes , Células Ganglionares da Retina , Axônios , Diferenciação Celular , Humanos , NeuritosRESUMO
Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of human oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, here we describe time-course single-cell-transcriptomic analysis of developing human stem cell-derived oligodendrocyte-lineage-cells (hOLLCs). The study includes hOLLCs derived from both genome engineered embryonic stem cell (ESC) reporter cells containing an Identification-and-Purification tag driven by the endogenous PDGFRα promoter and from unmodified induced pluripotent (iPS) cells. Our analysis uncovers substantial transcriptional heterogeneity of PDGFRα-lineage hOLLCs. We discover sub-populations of human oligodendrocyte progenitor cells (hOPCs) including a potential cytokine-responsive hOPC subset, and identify candidate regulatory genes/networks that define the identity of these sub-populations. Pseudotime trajectory analysis defines developmental pathways of oligodendrocytes vs astrocytes from PDGFRα-expressing hOPCs and predicts differentially expressed genes between the two lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of these pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs.
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Heterogeneidade Genética , Variação Genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Análise de Célula Única/métodos , Transcriptoma/genética , Astrócitos/citologia , Astrócitos/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Colesterol/biossíntese , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Redes Reguladoras de Genes/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células Precursoras de Oligodendrócitos/citologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Retinal ganglion cell axons are heavily myelinated (98%) and myelin damage in the optic nerve (ON) severely affects vision. Understanding the molecular mechanism of oligodendrocyte progenitor cell (OPC) differentiation into mature oligodendrocytes will be essential for developing new therapeutic approaches for ON demyelinating diseases. To this end, we developed a new method for isolation and culture of ON-derived oligodendrocyte lineage cells and used it to study OPC differentiation. A critical aspect of cellular differentiation is macroautophagy/autophagy, a catabolic process that allows for cell remodeling by degradation of excess or damaged cellular molecules and organelles. Knockdown of ATG9A and BECN1 (pro-autophagic proteins involved in the early stages of autophagosome formation) led to a significant reduction in proliferation and survival of OPCs. We also found that autophagy flux (a measure of autophagic degradation activity) is significantly increased during progression of oligodendrocyte differentiation. Additionally, we demonstrate a significant change in mitochondrial dynamics during oligodendrocyte differentiation, which is associated with a significant increase in programmed mitophagy (selective autophagic clearance of mitochondria). This process is mediated by the mitophagy receptor BNIP3L (BCL2/adenovirus E1B interacting protein 3-like). BNIP3L-mediated mitophagy plays a crucial role in the regulation of mitochondrial network formation, mitochondrial function and the viability of newly differentiated oligodendrocytes. Our studies provide novel evidence that proper mitochondrial dynamics is required for establishment of functional mitochondria in mature oligodendrocytes. These findings are significant because targeting BNIP3L-mediated programmed mitophagy may provide a novel therapeutic approach for stimulating myelin repair in ON demyelinating diseases.Abbreviations: A2B5: a surface antigen of oligodendrocytes precursor cells, A2B5 clone 105; ACTB: actin, beta; APC: an antibody to label mature oligodendrocytes, anti-adenomatous polyposis coli clone CC1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9A: autophagy related 9A; AU: arbitrary units; BafA1: bafilomycin A1; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CNP: 2',3'-cyclic nucleotide 3'-phosphodiesterase; Ctl: control; COX8: cytochrome c oxidase subunit; CSPG4/NG2: chondroitin sulfate proteoglycan 4; DAPI: 4'6-diamino-2-phenylindole; DNM1L: dynamin 1-like; EGFP: enhanced green fluorescent protein; FACS: fluorescence-activated cell sorting; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; GFP: green fluorescent protein; HsESC: human embryonic stem cell; IEM: immunoelectron microscopy; LAMP1: lysosomal-associated membrane protein 1; LC3B: microtubule-associated protein 1 light chain 3; MBP: myelin basic protein; MFN2: mitofusin 2; Mito-Keima: mitochondria-targeted monomeric keima-red; Mito-GFP: mitochondria-green fluorescent protein; Mito-RFP: mitochondria-red fluorescent protein; MitoSOX: red mitochondrial superoxide probe; MKI67: antigen identified by monoclonal antibody Ki 67; MMP: mitochondrial membrane potential; O4: oligodendrocyte marker O4; OLIG2: oligodendrocyte transcription factor 2; ON: optic nerve; OPA1: OPA1, mitochondrial dynamin like GTPase; OPC: oligodendrocyte progenitor cell; PDL: poly-D-lysine; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; RGC: retinal ganglion cell; ROS: reactive oxygen species; RT-PCR: real time polymerase chain reaction; SEM: standard error of the mean; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; TUBB3: tubulin, beta 3 class III.
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Autofagia , Mitofagia , Autofagia/fisiologia , Diferenciação Celular/fisiologia , Mitocôndrias/metabolismo , Mitofagia/fisiologia , Oligodendroglia/metabolismo , Nervo Óptico/metabolismoRESUMO
Purpose: To determine whether priming of bone marrow mesenchymal stem cells (MSCs) by signals from injured retina, particularly tumor necrosis factor α (TNFα), increase their exosomes' neuroprotective efficacy on retinal ganglion cells (RGCs). Methods: MSCs were primed with retinal cell culture conditioned medium, with or without the TNFα blocker etanercept or TNFα prior to isolation of exosomes. MSC conditioned medium or exosomes were added to rat retinal cultures or human stem cell-derived retinal ganglion cell (hRGC) cultures, and RGC neuroprotective effects were quantified. Luminex assays were used to compare primed versus unprimed exosomes. Results: MSC conditioned medium and exosomes exerted a significant neuroprotective effect on injured rat and hRGC. This effect was significantly increased after MSCs were primed with retinal conditioned medium or TNFα. Blocking of TNFα signaling with etanercept prevented priming-induced RGC neuroprotective efficacy. Priming increased PEDF and VEGF-AA exosomal abundance. Conclusions: MSC exosomes promote RGC survival not just in rodent retinal cultures but also with hRGC. Their efficacy can be further enhanced through TNFα priming with the mechanism of action potentially mediated, at least in part, through increased levels of PEDF and VEGF-AA.
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Células-Tronco Mesenquimais/fisiologia , Fármacos Neuroprotetores/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células Cultivadas , Etanercepte/farmacologia , Exossomos , Proteínas do Olho/metabolismo , Feminino , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Fatores de Crescimento Neural/metabolismo , Neuroproteção/efeitos dos fármacos , Ratos Sprague-Dawley , Serpinas/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Pluripotent stem cells (PSCs) edited with genetic reporters are useful tools for differentiation analysis and for isolation of specific cell populations for study. Reporter integration into the genome is now commonly achieved by targeted DNA nuclease-enhanced homology directed repair (HDR). However, human PSCs are known to have a low frequency of gene knock-in (KI) by HDR, making reporter line generation an arduous process. Here, we report a methodology for scarless KI of large fluorescent reporter genes into PSCs by transient selection with puromycin or zeocin. With this method, we can perform targeted KI of a single reporter gene with up to 65% efficiency, as well as simultaneous KI of two reporter genes into different loci with up to 11% efficiency. Additionally, we demonstrate that this method also works in mouse PSCs.
Assuntos
Técnicas de Introdução de Genes/métodos , Células-Tronco Pluripotentes/metabolismo , Animais , Linhagem Celular , Genes Reporter , Humanos , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos , Células-Tronco Pluripotentes/citologia , TransgenesRESUMO
The mechanisms underlying specification of neuronal subtypes within the human nervous system are largely unknown. The blue (S), green (M), and red (L) cones of the retina enable high-acuity daytime and color vision. To determine the mechanism that controls S versus L/M fates, we studied the differentiation of human retinal organoids. Organoids and retinas have similar distributions, expression profiles, and morphologies of cone subtypes. S cones are specified first, followed by L/M cones, and thyroid hormone signaling controls this temporal switch. Dynamic expression of thyroid hormone-degrading and -activating proteins within the retina ensures low signaling early to specify S cones and high signaling late to produce L/M cones. This work establishes organoids as a model for determining mechanisms of human development with promising utility for therapeutics and vision repair.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Organoides/crescimento & desenvolvimento , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/classificação , Hormônios Tireóideos/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Humanos , Mutação , Organoides/metabolismo , Proteólise , Retina/citologiaRESUMO
We used single cell sequencing technology to characterize the transcriptomes of 1,174 human embryonic stem cell-derived retinal ganglion cells (RGCs) at the single cell level. The human embryonic stem cell line BRN3B-mCherry (A81-H7), was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and prepared for single cell RNA sequencing. Our data indicates the presence of three distinct subpopulations of cells, with various degrees of maturity. One cluster of 288 cells showed increased expression of genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature RGC phenotype.
Assuntos
Células-Tronco Embrionárias , RNA/genética , Células Ganglionares da Retina , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Humanos , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/fisiologia , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularly-interspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for large-scale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. Stem Cells Translational Medicine 2017;6:1972-1986.