RESUMEN
The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , Linaje de la Célula , Desarrollo Embrionario , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Análisis de la Célula Individual , Vía de Señalización WntRESUMEN
Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.
Asunto(s)
COVID-19/inmunología , Caspasa 8/metabolismo , Interferón gamma/metabolismo , Linfohistiocitosis Hemofagocítica/inmunología , Macrófagos/inmunología , Mitocondrias/metabolismo , SARS-CoV-2/fisiología , Animales , Caspasa 8/genética , Células Cultivadas , Citotoxicidad Inmunológica , Humanos , Interferón gamma/genética , Activación de Macrófagos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Transducción de Señal , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development1-6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.
Asunto(s)
Reprogramación Celular/genética , Regulación de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismo , Adulto , Cromatina/genética , Cromatina/metabolismo , Ectodermo/citología , Ectodermo/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Transcripción GenéticaRESUMEN
The diversity of COVID-19 disease in otherwise healthy people, from seemingly asymptomatic infection to severe life-threatening disease, is not clearly understood. We passaged a naturally occurring near-ancestral SARS-CoV-2 variant, capable of infecting wild-type mice, and identified viral genomic mutations coinciding with the acquisition of severe disease in young adult mice and lethality in aged animals. Transcriptomic analysis of lung tissues from mice with severe disease elucidated a host antiviral response dominated mainly by interferon and IL-6 pathway activation in young mice, while in aged animals, a fatal outcome was dominated by TNF and TGF-ß signaling. Congruent with our pathway analysis, we showed that young TNF-deficient mice had mild disease compared to controls and aged TNF-deficient animals were more likely to survive infection. Emerging clinical correlates of disease are consistent with our preclinical studies, and our model may provide value in defining aberrant host responses that are causative of severe COVID-19.
Asunto(s)
COVID-19 , SARS-CoV-2 , Adulto Joven , Humanos , Ratones , Animales , Anciano , SARS-CoV-2/genética , COVID-19/genética , Virulencia/genética , Mutación , Modelos Animales de EnfermedadRESUMEN
The canonical Wnt/ß-catenin pathway is crucial for early embryonic patterning, tissue homeostasis, and regeneration. While canonical Wnt/ß-catenin stimulation has been used extensively to modulate pluripotency and differentiation of pluripotent stem cells (PSCs), the mechanism of these two seemingly opposing roles has not been fully characterized and is currently largely attributed to activation of nuclear Wnt target genes. Here, we show that low levels of Wnt stimulation via ectopic expression of Wnt1 or administration of glycogen synthase kinase-3 inhibitor CHIR99021 significantly increases PSC differentiation into neurons, cardiomyocytes and early endodermal intermediates. Our data indicate that enhanced differentiation outcomes are not mediated through activation of traditional Wnt target genes but by ß-catenin's secondary role as a binding partner of membrane bound cadherins ultimately leading to the activation of developmental genes. In summary, fine-tuning of Wnt signaling to subthreshold levels for detectable nuclear ß-catenin function appears to act as a switch to enhance differentiation of PSCs into multiple lineages. Our observations highlight a mechanism by which Wnt/ß-catenin signaling can achieve dosage dependent dual roles in regulating self-renewal and differentiation. Stem Cells 2018;36:822-833.
Asunto(s)
Células Madre Pluripotentes/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/genética , Animales , Diferenciación Celular , Humanos , Ratones , Transducción de SeñalRESUMEN
In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/ß-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/ß-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of ß-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/ß-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/ß-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.
Asunto(s)
Diferenciación Celular , Autorrenovación de las Células , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Vía de Señalización Wnt , Apoptosis , Benzotiazoles/farmacología , Biomarcadores , Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/genética , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Compuestos Heterocíclicos con 3 Anillos/farmacología , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Modelos Biológicos , Proteómica/métodos , ARN Interferente Pequeño/genética , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
In the mouse, naïve pluripotent stem cells (PSCs) are thought to represent the cell culture equivalent of the late epiblast in the pre-implantation embryo, with which they share a unique defining set of features. Recent studies have focused on the identification and propagation of a similar cell state in human. Although the capture of an exact human equivalent of the mouse naïve PSC remains an elusive goal, comparative studies spurred on by this quest are lighting the path to a deeper understanding of pluripotent state regulation in early mammalian development.
Asunto(s)
Blastocisto/fisiología , Linaje de la Célula/fisiología , Desarrollo Embrionario/fisiología , Epigénesis Genética/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Estratos Germinativos/citología , Células Madre Pluripotentes/citología , Animales , Blastocisto/citología , Humanos , Ratones , Especificidad de la EspecieRESUMEN
Fibrosis of vital organs is a major public health problem with limited therapeutic options. Mesenchymal cells including microvascular mural cells (pericytes) are major progenitors of scar-forming myofibroblasts in kidney and other organs. Here we show pericytes in healthy kidneys have active WNT/ß-catenin signaling responses that are markedly up-regulated following kidney injury. Dickkopf-related protein 1 (DKK-1), a ligand for the WNT coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP-5 and LRP-6) and an inhibitor of WNT/ß-catenin signaling, effectively inhibits pericyte activation, detachment, and transition to myofibroblasts in vivo in response to kidney injury, resulting in attenuated fibrogenesis, capillary rarefaction, and inflammation. DKK-1 blocks activation and proliferation of established myofibroblasts in vitro and blocks pericyte proliferation to PDGF, pericyte migration, gene activation, and cytoskeletal reorganization to TGF-ß or connective tissue growth factor. These effects are largely independent of inhibition of downstream ß-catenin signaling. DKK-1 acts predominantly by inhibiting PDGF-, TGF-ß-, and connective tissue growth factor-activated MAPK and JNK signaling cascades, acting via LRP-6 with associated WNT ligand. Biochemically, LRP-6 interacts closely with PDGF receptor ß and TGF-ß receptor 1 at the cell membrane, suggesting that it may have roles in pathways other than WNT/ß-catenin. In summary, DKK-1 blocks many of the changes in pericytes required for myofibroblast transition and attenuates established myofibroblast proliferation/activation by mechanisms dependent on LRP-6 and WNT ligands but not the downstream ß-catenin pathway.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patología , Pericitos/metabolismo , Pericitos/patología , Animales , Becaplermina , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/farmacología , Fibrosis , Puntos de Control de la Fase G1 del Ciclo Celular , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pericitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-sis/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal , Factor de Crecimiento Transformador beta/farmacología , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
Facioscapulohumeral muscular dystrophy is a dominantly inherited myopathy associated with chromatin relaxation of the D4Z4 macrosatellite array on chromosome 4. DUX4 is encoded within each unit of the D4Z4 array where it is normally transcriptionally silenced and packaged as constitutive heterochromatin. Truncation of the array to less than 11 D4Z4 units (FSHD1) or mutations in SMCHD1 (FSHD2) results in chromatin relaxation and a small percentage of cultured myoblasts from these individuals exhibit infrequent bursts of DUX4 expression. There are no cellular or animal models to determine the trigger of the DUX4 producing transcriptional bursts and there has been a failure to date to detect the protein in significant numbers of cells from FSHD-affected individuals. Here, we demonstrate for the first time that myotubes generated from FSHD patients express sufficient amounts of DUX4 to undergo DUX4-dependent apoptosis. We show that activation of the Wnt/ß-catenin signaling pathway suppresses DUX4 transcription in FSHD1 and FSHD2 myotubes and can rescue DUX4-mediated myotube apoptosis. In addition, reduction of mRNA transcripts from Wnt pathway genes ß-catenin, Wnt3A and Wnt9B results in DUX4 activation. We propose that Wnt/ß-catenin signaling is important for transcriptional repression of DUX4 and identify a novel group of therapeutic targets for the treatment of FSHD.
Asunto(s)
Apoptosis , Proteínas de Homeodominio/metabolismo , Fibras Musculares Esqueléticas/fisiología , Distrofia Muscular Facioescapulohumeral/genética , Vía de Señalización Wnt , Animales , Línea Celular , Células Cultivadas , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Humanos , Ratones , ARN Interferente Pequeño/genéticaRESUMEN
Signal transduction pathways play diverse, context-dependent roles in vertebrate development. In studies of human embryonic stem cells (hESCs), conflicting reports claim Wnt/ß-catenin signaling promotes either self-renewal or differentiation. We use a sensitive reporter to establish that Wnt/ß-catenin signaling is not active during hESC self-renewal. Inhibiting this pathway over multiple passages has no detrimental effect on hESC maintenance, whereas activating signaling results in loss of self-renewal and induction of mesoderm lineage genes. Following exposure to pathway agonists, hESCs exhibit a delay in activation of ß-catenin signaling, which led us to postulate that Wnt/ß-catenin signaling is actively repressed during self-renewal. In support of this hypothesis, we demonstrate that OCT4 represses ß-catenin signaling during self-renewal and that targeted knockdown of OCT4 activates ß-catenin signaling in hESCs. Using a fluorescent reporter of ß-catenin signaling in live hESCs, we observe that the reporter is activated in a very heterogeneous manner in response to stimulation with Wnt ligand. Sorting cells on the basis of their fluorescence reveals that hESCs with elevated ß-catenin signaling express higher levels of differentiation markers. Together these data support a dominant role for Wnt/ß-catenin signaling in the differentiation rather than self-renewal of hESCs.
Asunto(s)
Células Madre Embrionarias/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Técnicas de Cocultivo , Genes Reporteros , Humanos , Ratones , Modelos Biológicos , Transducción de SeñalRESUMEN
Age-related macular degeneration (AMD) is a leading cause of severe vision loss in the Western world and is increasing exponentially as the population ages. Despite enormous worldwide efforts, the earliest pathogenic pathways involved in AMD are still not fully understood. It is essential to develop research tools for effective modeling of AMD pathogenesis and for subsequent drug discovery and cell or molecular therapies. This review will focus on the current progress in human pluripotent stem cells for understanding and treating AMD.
Asunto(s)
Degeneración Macular/terapia , Células Madre Pluripotentes/trasplante , Trasplante de Células Madre , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Degeneración Macular/etiologíaRESUMEN
Necroptosis, a type of lytic cell death executed by the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) has been implicated in the detrimental inflammation caused by SARS-CoV-2 infection. We minimally and extensively passaged a single clinical SARS-CoV-2 isolate to create models of mild and severe disease in mice allowing us to dissect the role of necroptosis in SARS-CoV-2 disease pathogenesis. We infected wild-type and MLKL-deficient mice and found no significant differences in viral loads or lung pathology. In our model of severe COVID-19, MLKL-deficiency did not alter the host response, ameliorate weight loss, diminish systemic pro-inflammatory cytokines levels, or prevent lethality in aged animals. Our in vivo models indicate that necroptosis is dispensable in the pathogenesis of mild and severe COVID-19.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Ratones , SARS-CoV-2/metabolismo , Necroptosis/fisiología , Proteínas Quinasas/metabolismo , Modelos Animales de Enfermedad , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover, TP63, KRT15, CXCL14, and ITGß4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs), which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGß4 could be used to enrich human corneal epithelial cell progenitors, which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1.
Asunto(s)
Epitelio Corneal , Limbo de la Córnea , Humanos , Nicho de Células Madre , Células Madre Limbares , Córnea , Epitelio Corneal/metabolismo , Perfilación de la Expresión GénicaRESUMEN
BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.
Asunto(s)
COVID-19 , Proteínas Portadoras , Cricetinae , Humanos , Ratones , Ratas , Animales , Vacunas contra la COVID-19 , SARS-CoV-2 , Subunidades de Proteína , COVID-19/prevención & control , Australia , Adyuvantes Inmunológicos , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralizing capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralizers with enhanced ability to control viral escape mutants.
RESUMEN
Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general.
Asunto(s)
Sitios Genéticos , Células Madre Embrionarias Humanas/metabolismo , Complejos Multiproteicos/metabolismo , Proteína Homeótica Nanog/metabolismo , Proteínas Co-Represoras/metabolismo , Células Madre Embrionarias Humanas/citología , HumanosRESUMEN
Dysfunction of retinal glial cells, particularly Müller cells, has been implicated in several retinal diseases. Despite their important contribution to retinal homeostasis, a specific way to differentiate retinal glial cells from human pluripotent stem cells has not yet been described. Here, we report a method to differentiate retinal glial cells from human embryonic stem cells (hESCs) through promoting the Notch signaling pathway. We first generated retinal progenitor cells (RPCs) from hESCs then promoted the Notch signaling pathway using Notch ligands, including Delta-like ligand 4 and Jagged-1. We validated glial cell differentiation with qRT-PCR, immunocytochemistry, western blots and fluorescence-activated cell sorting as we promoted Notch signaling in RPCs. We found that promoting Notch signaling in RPCs for 2 weeks led to upregulation of glial cell markers, including glial fibrillary acidic protein (GFAP), glutamine synthetase, vimentin and cellular retinaldehyde-binding protein (CRALBP). Of these markers, we found the greatest increase in expression of the pan glial cell marker, GFAP. Conversely, we also found that inhibition of Notch signaling in RPCs led to upregulation of retinal neuronal markers including cone-rod homeobox (CRX) and orthodenticle homeobox 2 (OTX2) but with little expression of GFAP. This retinal glial differentiation method will help advance the generation of stem cell disease models to study the pathogenesis of retinal diseases associated with glial dysfunction such as macular telangiectasia type 2. This method may also be useful for the development of future therapeutics such as drug screening and gene editing using patient-derived retinal glial cells.
RESUMEN
UNLABELLED: Our current understanding of the induction of pluripotency by defined factors indicates that this process occurs in discrete stages characterized by specific alterations in the cellular transcriptome and epigenome. However, the final phase of the reprogramming process is incompletely understood. We sought to generate tools to characterize the transition to a fully reprogramed state. We used combinations of stem cell surface markers to isolate colonies emerging after transfection of human fibroblasts with reprogramming factors and then analyzed their expression of genes associated with pluripotency and early germ lineage specification. We found that expression of a subset of these genes, including the cell-cell adhesion molecule CDH3, characterized a late stage in the reprogramming process. Combined live-cell staining with the antibody GCTM-2 and anti-CDH3 during reprogramming identified colonies of cells that showed gene expression patterns very similar to those of embryonic stem cell or established induced pluripotent stem cell lines, and gave rise to stable induced pluripotent stem cell lines at high frequency. Our findings will facilitate studies of the final stages of reprogramming of human cells to pluripotency and will provide a simple means for prospective identification of fully reprogrammed cells. SIGNIFICANCE: Reprogramming of differentiated cells back to an embryonic pluripotent state has wide ranging applications in understanding and treating human disease. However, how cells traverse the barriers on the journey to pluripotency still is not fully understood. This report describes tools to study the late stages of cellular reprogramming. The findings enable a more precise approach to dissecting the final phases of conversion to pluripotency, a process that is particularly poorly defined. The results of this study also provide a simple new method for the selection of fully reprogrammed cells, which could enhance the efficiency of derivation of cell lines for research and therapy.
Asunto(s)
Reprogramación Celular/genética , Fibroblastos/citología , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/genética , Transcriptoma/fisiología , Biomarcadores/metabolismo , Células Cultivadas , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/fisiología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Proteínas de la Membrana/metabolismo , Análisis por MicromatricesRESUMEN
We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.
Asunto(s)
Medios de Cultivo Condicionados/metabolismo , Células Madre Pluripotentes/citología , Retina/citología , Epitelio Pigmentado de la Retina/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Células Cultivadas , Células Epiteliales/citología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Fagocitosis/fisiología , Células Fotorreceptoras/citología , Células Fotorreceptoras/metabolismo , Pigmentación/fisiología , Células Madre Pluripotentes/metabolismo , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
We assessed structural elements of the retina in individuals with Friedreich ataxia (FRDA) and in mouse models of FRDA, as well as functions of the retinal pigment epithelium (RPE) in FRDA using induced pluripotent stem cells (iPSCs). We analyzed the retina of the FRDA mouse models YG22R and YG8R containing a human FRATAXIN (FXN) transgene by histology. We complemented this work with post-mortem evaluation of eyes from FRDA patients. Finally, we derived RPE cells from patient FRDA-iPSCs to assess oxidative phosphorylation (OXPHOS) and phagocytosis. We showed that whilst the YG22R and YG8R mouse models display elements of retinal degeneration, they do not recapitulate the loss of retinal ganglion cells (RGCs) found in the human disease. Further, RPE cells differentiated from human FRDA-iPSCs showed normal OXPHOS and we did not observe functional impairment of the RPE in Humans.