RESUMEN
Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the developed world. Vision loss in the advanced stages of the disease is caused by atrophy of retinal photoreceptors, overlying retinal pigment epithelium (RPE) and choroidal endothelial cells. The molecular events that underline the development of these cell types from in utero to adult as well as the progression to intermediate and advanced stages AMD are not yet fully understood. We performed single-cell RNA-sequencing (RNA-Seq) of human fetal and adult RPE-choroidal tissues, profiling in detail all the cell types and elucidating cell type-specific proliferation, differentiation and immunomodulation events that occur up to midgestation. Our data demonstrate that progression from the fetal to adult state is characterized by an increase in expression of genes involved in the oxidative stress response and detoxification from heavy metals, suggesting a better defence against oxidative stress in the adult RPE-choroid tissue. Single-cell comparative transcriptional analysis between a patient with intermediate AMD and an unaffected subject revealed a reduction in the number of RPE cells and melanocytes in the macular region of the AMD patient. Together these findings may suggest a macular loss of RPE cells and melanocytes in the AMD patients, but given the complex processing of tissues required for single-cell RNA-Seq that is prone to technical artefacts, these findings need to be validated by additional techniques in a larger number of AMD patients and controls.
Asunto(s)
Degeneración Macular , Epitelio Pigmentado de la Retina , Humanos , Adulto , Epitelio Pigmentado de la Retina/metabolismo , Células Endoteliales/metabolismo , Coroides/metabolismo , Degeneración Macular/genética , Degeneración Macular/metabolismo , Desarrollo Fetal , Análisis de Secuencia de ARNRESUMEN
Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co-culture system containing retinal organoids and microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Microglía , Retina , Organoides , Diferenciación CelularRESUMEN
The demand for induced pluripotent stem cells (iPSC)-derived retinal organoid and retinal pigment epithelium (RPE) models for the modelling of inherited retinopathies has increased significantly in the last decade. These models are comparable with foetal retinas up until the later stages of retinogenesis, expressing all of the key neuronal markers necessary for retinal function. These models have proven to be invaluable in the understanding of retinogenesis, particular in the context of patient-specific diseases. Inherited retinopathies are infamously described as clinically and phenotypically heterogeneous, such that developing gene/mutation-specific animal models in each instance of retinal disease is not financially or ethically feasible. Further to this, many animal models are insufficient in the study of disease pathogenesis due to anatomical differences and failure to recapitulate human disease phenotypes. In contrast, iPSC-derived retinal models provide a high throughput platform which is physiologically relevant for studying human health and disease. They also serve as a platform for drug screening, gene therapy approaches and in vitro toxicology of novel therapeutics in pre-clinical studies. One unique characteristic of stem cell-derived retinal models is the ability to mimic in vivo retinogenesis, providing unparalleled insights into the effects of pathogenic mutations in cells of the developing retina, in a highly accessible way. This review aims to give the reader an overview of iPSC-derived retinal organoids and/or RPE in the context of disease modelling of several inherited retinopathies including Retinitis Pigmentosa, Stargardt disease and Retinoblastoma. We describe the ability of each model to recapitulate in vivo disease phenotypes, validate previous findings from animal models and identify novel pathomechanisms that underpin individual IRDs.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedades de la Retina , Animales , Humanos , Retina , Epitelio Pigmentado de la Retina/patología , Organoides , Enfermedades de la Retina/genética , Enfermedades de la Retina/patologíaRESUMEN
Retinoblastoma (Rb) is a rare malignant disorder affecting the developing retina of children under the age of five. Chemotherapeutic agents used for treating Rb have been associated with defects of the retinal pigment epithelium (RPE), such as hyperplasia, gliosis, and mottling. Herein, we have developed two pluripotent stem cell (PSC)-RPE models to assess the cytotoxicity of known Rb chemotherapeutics such as Melphalan, Topotecan and TW-37. Our findings demonstrate that these drugs alter the RPE by decreasing the monolayer barrier's trans-epithelial resistance and affecting the cells' phagocytic activity. Transcriptional analyses demonstrate an altered expression of genes involved in melanin and retinol processing, tight junction and apical-basal polarity pathways in both models. When applied within the clinical range, none of the drug treatments caused significant cytotoxic effects, changes to the apical-basal polarity, tight junction network or cell cycle. Together, our results demonstrate that although the most commonly used Rb chemotherapeutic drugs do not cause cytotoxicity in RPE, their application in vitro leads to compromised phagocytosis and strength of the barrier function, in addition to changes in gene expression that could alter the visual cycle in vivo. Our data demonstrate that widely used Rb chemotherapeutic drugs can have a deleterious impact on RPE cells and thus great care has to be exercised with regard to their delivery so the adjacent healthy RPE is not damaged during the course of tumor eradication.
Asunto(s)
Neoplasias de la Retina , Retinoblastoma , Niño , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retina , Neoplasias de la Retina/tratamiento farmacológico , Neoplasias de la Retina/genética , Neoplasias de la Retina/metabolismo , Expresión Génica , Diferenciación CelularRESUMEN
The airway epithelium represents the main barrier between inhaled air and the tissues of the respiratory tract and is therefore an important point of contact with xenobiotic substances into the human body. Several studies have recently shown that in vitro models of the airway grown at an air-liquid interface (ALI) can be particularly useful to obtain mechanistic information about the toxicity of chemical compounds. However, such methods are not very amenable to high throughput since the primary cells cannot be expanded indefinitely in culture to obtain a sustainable number of cells. Induced pluripotent stem cells (iPSCs) have become a popular option in the recent years for modelling the airways of the lung, but despite progress in the field, such models have so far not been assessed for their ability to metabolise xenobiotic compounds and how they compare to the primary bronchial airway model (pBAE). Here, we report a comparative analysis by TempoSeq (oligo-directed sequencing) of an iPSC-derived airway model (iBAE) with a primary bronchial airway model (pBAE). The iBAE and pBAE were differentiated at an ALI and then evaluated in a 5-compound screen with exposure to a sub-lethal concentration of each compound for 24 h. We found that despite lower expression of xenobiotic metabolism genes, the iBAE similarly predicted the toxic pathways when compared to the pBAE model. Our results show that iPSC airway models at ALI show promise for inhalation toxicity assessments with further development.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Transcriptoma , Xenobióticos/toxicidad , Xenobióticos/metabolismo , Mucosa Respiratoria/metabolismo , Epitelio , Células Epiteliales/metabolismoRESUMEN
The scarcity of embryonic/foetal material as a resource for direct study means that there is still limited understanding of human retina development. Here, we present an integrated transcriptome analysis combined with immunohistochemistry in human eye and retinal samples from 4 to 19 post-conception weeks. This analysis reveals three developmental windows with specific gene expression patterns that informed the sequential emergence of retinal cell types and enabled identification of stage-specific cellular and biological processes, and transcriptional regulators. Each stage is characterised by a specific set of alternatively spliced transcripts that code for proteins involved in the formation of the photoreceptor connecting cilium, pre-mRNA splicing and epigenetic modifiers. Importantly, our data show that the transition from foetal to adult retina is characterised by a large increase in the percentage of mutually exclusive exons that code for proteins involved in photoreceptor maintenance. The circular RNA population is also defined and shown to increase during retinal development. Collectively, these data increase our understanding of human retinal development and the pre-mRNA splicing process, and help to identify new candidate disease genes.
Asunto(s)
Perfilación de la Expresión Génica , Retina/embriología , Retina/metabolismo , Empalme Alternativo/genética , Animales , Biomarcadores/metabolismo , Cilios/metabolismo , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Organogénesis/genética , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/metabolismo , Análisis de Componente Principal , ARN/genética , ARN/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Circular , Retina/citología , Retina/ultraestructura , Transcriptoma/genéticaRESUMEN
Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.
Asunto(s)
Proteínas de Unión al ADN/deficiencia , Mutación de Línea Germinal , Mutación con Pérdida de Función , Trastornos Linfoproliferativos/genética , Proteínas Proto-Oncogénicas/deficiencia , Inmunodeficiencia Combinada Grave/genética , Aloinjertos , Apoptosis , Subgrupos de Linfocitos B/patología , Técnicas de Reprogramación Celular , Codón sin Sentido , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Dioxigenasas , Resultado Fatal , Femenino , Trasplante de Células Madre Hematopoyéticas , Humanos , Células Madre Pluripotentes Inducidas/patología , Recién Nacido , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células T Periférico/genética , Linfoma de Células T Periférico/patología , Masculino , Mutación Missense , Neoplasias Primarias Múltiples/genética , Linaje , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Inmunodeficiencia Combinada Grave/patología , Subgrupos de Linfocitos T/patología , Secuenciación del ExomaRESUMEN
Retinal dystrophies often lead to blindness. Developing therapeutic interventions to restore vision is therefore of paramount importance. Here we demonstrate the ability of pluripotent stem cell-derived cone precursors to engraft and restore light responses in the Pde6brd1 mouse, an end-stage photoreceptor degeneration model. Our data show that up to 1.5% of precursors integrate into the host retina, differentiate into cones, and engraft in close apposition to the host bipolar cells. Half of the transplanted mice exhibited visual behavior and of these 33% showed binocular light sensitivity. The majority of retinal ganglion cells exhibited contrast-sensitive ON, OFF or ON-OFF light responses and even motion sensitivity; however, quite a few exhibited unusual responses (eg, light-induced suppression), presumably reflecting remodeling of the neural retina. Our data indicate that despite relatively low engraftment yield, pluripotent stem cell-derived cone precursors can elicit light responsiveness even at advanced degeneration stages. Further work is needed to improve engraftment yield and counteract retinal remodeling to achieve useful clinical applications.
Asunto(s)
Células Madre Pluripotentes , Células Fotorreceptoras Retinianas Conos , Degeneración Retiniana , Trasplante de Células Madre , Animales , Ratones , Células Madre Pluripotentes/trasplante , Degeneración Retiniana/terapia , Células Ganglionares de la Retina/patologíaRESUMEN
Development of the retina is regulated by growth factors, such as insulin-like growth factors 1 and 2 (IGF-1/2), which coordinate proliferation, differentiation, and maturation of the neuroepithelial precursors cells. In the circulation, IGF-1/2 are transported by the insulin growth factor binding proteins (IGFBPs) family members. IGFBPs can impact positively and negatively on IGF-1, by making it available or sequestering IGF-1 to or from its receptor. In this study, we investigated the expression of IGFBPs and their role in the generation of human retinal organoids from human pluripotent stem cells, showing a dynamic expression pattern suggestive of different IGFBPs being used in a stage-specific manner to mediate IGF-1 functions. Our data show that IGF-1 addition to culture media facilitated the generation of retinal organoids displaying the typical laminated structure and photoreceptor maturation. The organoids cultured in the absence of IGF-1, lacked the typical laminated structure at the early stages of differentiation and contained significantly less photoreceptors and more retinal ganglion cells at the later stages of differentiation, confirming the positive effects of IGF-1 on retinal lamination and photoreceptor development. The organoids cultured with the IGFBP inhibitor (NBI-31772) and IGF-1 showed lack of retinal lamination at the early stages of differentiation, an increased propensity to generate horizontal cells at mid-stages of differentiation and reduced photoreceptor development at the later stages of differentiation. Together these data suggest that IGFBPs enable IGF-1's role in retinal lamination and photoreceptor development in a stage-specific manner.
Asunto(s)
Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Organoides/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Madre Pluripotentes/metabolismo , Catecoles/farmacología , Diferenciación Celular/efectos de los fármacos , Proteína 3 Similar a ELAV/genética , Proteína 3 Similar a ELAV/metabolismo , Proteína 4 Similar a ELAV/genética , Proteína 4 Similar a ELAV/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/antagonistas & inhibidores , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor II del Crecimiento Similar a la Insulina/metabolismo , Isoquinolinas/farmacología , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Organoides/citología , Organoides/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Recoverina/genética , Recoverina/metabolismo , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , gamma-Sinucleína/genética , gamma-Sinucleína/metabolismoRESUMEN
As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal-like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal-like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air-liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS-CoV-2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs.
Asunto(s)
COVID-19/patología , COVID-19/virología , Células Madre Pluripotentes Inducidas/patología , Pulmón/patología , Pulmón/virología , Modelos Biológicos , SARS-CoV-2/fisiología , Línea Celular , Citocinas/metabolismo , Células Epiteliales/patología , Células Epiteliales/virología , Humanos , Mediadores de Inflamación/metabolismo , Replicación Viral/fisiologíaRESUMEN
Induced pluripotent stem cell (iPSC)-derived retinal organoids provide a platform to study human retinogenesis, disease modeling, and compound screening. Although retinal organoids may represent tissue structures with greater physiological relevance to the in vivo human retina, their generation is not without limitations. Various protocols have been developed to enable development of organoids with all major retinal cell types; however, variability across iPSC lines is often reported. Modulating signaling pathways important for eye formation, such as those involving bone morphogenetic protein 4 (BMP4) and insulin-like growth factor 1 (IGF1), is a common approach used for the generation of retinal tissue in vitro. We used three human iPSC lines to generate retinal organoids by activating either BMP4 or IGF1 signaling and assessed differentiation efficiency by monitoring morphological changes, gene and protein expression, and function. Our results showed that the ability of iPSC to give rise to retinal organoids in response to IGF1 and BMP4 activation was line- and method-dependent. This demonstrates that careful consideration is needed when choosing a differentiation approach, which would also depend on overall project aims.
RESUMEN
Cell replacement therapy is a promising treatment for irreversible retinal cell death in diverse diseases such as Stargardt's disease, age-related macular degeneration, and retinitis pigmentosa. The final impact of all retinal dystrophies is the loss of photoreceptors; hence, there is a pressing need for research into replacement. Seminal work has shown that a simple three-dimensional culture system enables differentiation of human pluripotent stem cells to retinal organoids containing large numbers of photoreceptors developing alongside retinal neurons and Müller glia cells in a laminated structure that resembles the native retina. Despite these promising developments, current protocols show different efficiencies across pluripotent stem cells and result in retinal organoids with a mixture of photoreceptor cells at varying maturation states, along with nonphotoreceptor cell types. In this study, we investigated the impact of stage-specific addition of retinoic acid (RA), 9-cis-retinal, 11-cis-retinal, levodopa (l-DOPA), triiodothyronine (T3), and γ-secretase inhibitor ((2S)-N-[(3,5-Difluorophenyl)acetyl]-l-alanyl-2-phenyl]glycine1,1-dimethylethyl ester2L [DAPT]) in the generation of cone and rod photoreceptors. Our results indicate that addition of RA + T3 during days 90 to 120 of differentiation enhanced the generation of rod and S-cone photoreceptor formation, while the combined addition of DAPT from days 28 to 42 with RA during days 30 to 120 of differentiation led to enhanced generation of L/M-cones at the expense of rods. l-DOPA when added together with RA during days 90 to 120 of differentiation also promoted the emergence of S-cones at the expense of rod photoreceptors. Collectively, these data represent an advance in our ability to direct generation of rod and cone photoreceptors in vitro.
Asunto(s)
Organoides/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Madre Pluripotentes/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Diferenciación Celular , Humanos , Organoides/citología , Células Fotorreceptoras de Vertebrados/citología , Retina/citología , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Bastones/citologíaRESUMEN
Increased pollution by plastics has become a serious global environmental problem, but the concerns for human health have been raised after reported presence of microplastics (MPs) and nanoplastics (NPs) in food and beverages. Unfortunately, few studies have investigate the potentially harmful effects of MPs/NPs on early human development and human health. Therefore, we used a new platform to study possible effects of polystyrene NPs (PSNPs) on the transcription profile of preimplantation human embryos and human induced pluripotent stem cells (hiPSCs). Two pluripotency genes, LEFTY1 and LEFTY2, which encode secreted ligands of the transforming growth factor-beta, were downregulated, while CA4 and OCLM, which are related to eye development, were upregulated in both samples. The gene set enrichment analysis showed that the development of atrioventricular heart valves and the dysfunction of cellular components, including extracellular matrix, were significantly affected after exposure of hiPSCs to PSNPs. Finally, using the HiPathia method, which uncovers disease mechanisms and predicts clinical outcomes, we determined the APOC3 circuit, which is responsible for increased risk for ischemic cardiovascular disease. These results clearly demonstrate that better understanding of NPs bioactivities and its implications for human health is of extreme importance. Thus, the presented platform opens further aspects to study interactions between different environmental and intracellular pollutions with the aim to decipher the mechanism and origin of human diseases.
Asunto(s)
Contaminación Ambiental/análisis , Nanopartículas/química , Plásticos/análisis , Poliestirenos/química , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Espacio Intracelular , Transcriptoma/genética , Resultado del TratamientoRESUMEN
Direct reprogramming of human somatic cells toward induced pluripotent stem cells holds great promise for regenerative medicine and basic biology. We used a high-throughput small interfering RNA screening assay in the initiation phase of reprogramming for 784 genes belonging to kinase and phosphatase families and identified 68 repressors and 22 effectors. Six new candidates belonging to the family of the G protein-coupled receptors (GPCRs) were identified, suggesting an important role for this key signaling pathway during somatic cell-induced reprogramming. Downregulation of one of the key GPCR effectors, endothelial differentiation GPCR5 (EDG5), impacted the maintenance of pluripotency, actin cytoskeleton organization, colony integrity, and focal adhesions in human embryonic stem cells, which were associated with the alteration in the RhoA-ROCK-Cofilin-PAXILLIN-actin signaling pathway. Similarly, downregulation of EDG5 during the initiation stage of somatic cell-induced reprogramming resulted in alteration of cytoskeleton, loss of human-induced pluripotent stem cell colony integrity, and a significant reduction in partially and fully reprogrammed cells as well as the number of alkaline phosphatase positive colonies at the end of the reprogramming process. Together, these data point to an important role of EDG5 in the maintenance and acquisition of pluripotency. Stem Cells 2019;37:318-331.
Asunto(s)
Reprogramación Celular , Regulación hacia Abajo , Células Madre Embrionarias Humanas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Transducción de Señal , Receptores de Esfingosina-1-Fosfato/metabolismo , Línea Celular , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Receptores de Esfingosina-1-Fosfato/genéticaRESUMEN
The rapid improvements in single cell sequencing technologies and analyses afford greater scope for dissecting organoid cultures composed of multiple cell types and create an opportunity to interrogate these models to understand tissue biology, cellular behavior and interactions. To this end, retinal organoids generated from human embryonic stem cells (hESCs) were analyzed by single cell RNA-sequencing (scRNA-Seq) at three time points of differentiation. Combinatorial data from all time points revealed the presence of nine clusters, five of which corresponded to key retinal cell types: retinal pigment epithelium (RPE), retinal ganglion cells (RGCs), cone and rod photoreceptors, and Müller glia. The remaining four clusters expressed genes typical of mitotic cells, extracellular matrix components and those involved in homeostasis. The cell clustering analysis revealed the decreasing presence of mitotic cells and RGCs, formation of a distinct RPE cluster, the emergence of cone and rod photoreceptors from photoreceptor precursors, and an increasing number of Müller glia cells over time. Pseudo-time analysis resembled the order of cell birth during retinal development, with the mitotic cluster commencing the trajectory and the large majority of Müller glia completing the time line. Together, these data demonstrate the feasibility and potential of scRNA-Seq to dissect the inherent complexity of retinal organoids and the orderly birth of key retinal cell types. Stem Cells 2019;37:593-598.
Asunto(s)
Diferenciación Celular/genética , Organoides/citología , Células Madre Pluripotentes/citología , Retina/crecimiento & desarrollo , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Células Madre Embrionarias Humanas/citología , Humanos , RNA-Seq/métodos , Retina/citología , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Epitelio Pigmentado de la Retina/crecimiento & desarrollo , Epitelio Pigmentado de la Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Análisis de la Célula Individual/métodosRESUMEN
Nerve growth factor (NGF) has demonstrated great benefit in the treatment of neurotrophic corneal ulcers. There is evidence for multiple modes of action in promoting corneal healing, but only indirect evidence exists for NGF's effects on limbal stem cells (LSCs). Understanding the role of NGF in LSC biology will improve our understanding of paracrine regulation of the limbal niche and the design of stem cell-based therapies for conditions such as LSC deficiency. In this article, we studied the regulation of NGF signaling components during LSC differentiation and the role of NGF in LSC proliferation and maintenance of the stem cell phenotype. LSC differentiation was induced by prolonged (40 day) culture which resulted in a significant increase in cell size, decrease in colony-forming efficiency and expression of putative LSC markers. A protein microarray measuring expression of 248 signaling proteins indicated the low affinity NGF receptor p75NTR to be the most downregulated protein upon differentiation. Further confirmation by Western blotting and real-time quantitative polymerase chain reaction indicated that NGF and p75NTR are expressed in early LSC cultures and downregulated upon differentiation. LSC cultures grown in the presence of anti-NGF antibody showed decreased colony-forming efficiency, DNA replication and expression of putative LSC markers ABCG2 and C/EBPδ. Supplementation of LSC culture medium with NGF extended the life span of LSC cultures in vitro and increased the expression of putative LSC markers ΔNp63α and ABCG2. Taken together, our data indicate that NGF signaling is a key promoter of LSC proliferation, colony-forming efficiency, and a maintainer of the LSC phenotype. Stem Cells 2019;37:139-149.
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Limbo de la Córnea/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Células Madre/metabolismo , Diferenciación Celular , Humanos , FenotipoRESUMEN
Death of photoreceptors is a common cause of age-related and inherited retinal dystrophies, and thus their replenishment from renewable stem cell sources is a highly desirable therapeutic goal. Human pluripotent stem cells provide a useful cell source in view of their limitless self-renewal capacity and potential to not only differentiate into cells of the retina but also self-organize into tissue with structure akin to the human retina as part of three-dimensional retinal organoids. Photoreceptor precursors have been isolated from differentiating human pluripotent stem cells through application of cell surface markers or fluorescent reporter approaches and shown to have a similar transcriptome to fetal photoreceptors. In this study, we investigated the transcriptional profile of CRX-expressing photoreceptor precursors derived from human pluripotent stem cells and their engraftment capacity in an animal model of retinitis pigmentosa (Pde6brd1), which is characterized by rapid photoreceptor degeneration. Single cell RNA-Seq analysis revealed the presence of a dominant cell cluster comprising 72% of the cells, which displayed the hallmarks of early cone photoreceptor expression. When transplanted subretinally into the Pde6brd1 mice, the CRX+ cells settled next to the inner nuclear layer and made connections with the inner neurons of the host retina, and approximately one-third of them expressed the pan cone marker, Arrestin 3, indicating further maturation upon integration into the host retina. Together, our data provide valuable molecular insights into the transcriptional profile of human pluripotent stem cells-derived CRX+ photoreceptor precursors and indicate their usefulness as a source of transplantable cone photoreceptors. Stem Cells 2019;37:609-622.
Asunto(s)
Diferenciación Celular/genética , Retina/crecimiento & desarrollo , Células Fotorreceptoras Retinianas Conos/trasplante , Degeneración Retiniana/terapia , Animales , Linaje de la Célula/genética , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Ratones , Organoides/trasplante , Células Madre Pluripotentes/trasplante , Células Fotorreceptoras Retinianas Conos/citología , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/trasplante , Transcriptoma/genéticaRESUMEN
Hypoplastic left heart syndrome (HLHS) is among the most severe forms of congenital heart disease. Although the consensus view is that reduced flow through the left heart during development is a key factor in the development of the condition, the molecular mechanisms leading to hypoplasia of left heart structures are unknown. We have generated induced pluripotent stem cells (iPSC) from five HLHS patients and two unaffected controls, differentiated these to cardiomyocytes and identified reproducible in vitro cellular and functional correlates of the HLHS phenotype. Our data indicate that HLHS-iPSC have a reduced ability to give rise to mesodermal, cardiac progenitors and mature cardiomyocytes and an enhanced ability to differentiate to smooth muscle cells. HLHS-iPSC-derived cardiomyocytes are characterised by a lower beating rate, disorganised sarcomeres and sarcoplasmic reticulum and a blunted response to isoprenaline. Whole exome sequencing of HLHS fibroblasts identified deleterious variants in NOTCH receptors and other genes involved in the NOTCH signalling pathway. Our data indicate that the expression of NOTCH receptors was significantly downregulated in HLHS-iPSC-derived cardiomyocytes alongside NOTCH target genes confirming downregulation of NOTCH signalling activity. Activation of NOTCH signalling via addition of Jagged peptide ligand during the differentiation of HLHS-iPSC restored their cardiomyocyte differentiation capacity and beating rate and suppressed the smooth muscle cell formation. Together, our data provide firm evidence for involvement of NOTCH signalling in HLHS pathogenesis, reveal novel genetic insights important for HLHS pathology and shed new insights into the role of this pathway during human cardiac development.
Asunto(s)
Síndrome del Corazón Izquierdo Hipoplásico/metabolismo , Síndrome del Corazón Izquierdo Hipoplásico/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Receptor Notch1/metabolismo , Estudios de Casos y Controles , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Recién Nacido/metabolismo , Masculino , Miocitos del Músculo Liso/metabolismo , Organogénesis , Transducción de Señal/fisiologíaRESUMEN
Hematopoietic stem cells derived from pluripotent stem cells could be used as an alternative to bone marrow transplants. Deriving these has been a long-term goal for researchers. However, the success of these efforts has been limited with the cells produced able to engraft in the bone marrow of recipient animals only in very low numbers. There is evidence that defects in the migratory and homing capacity of the cells are due to mis-regulation of miRNA expression and are responsible for their failure to engraft. We compared the miRNA expression profile of hematopoietic progenitors derived from pluripotent stem cells to those derived from bone marrow and found that numerous miRNAs are too highly expressed in hematopoietic progenitors derived from pluripotent stem cells, and that most of these are inhibitors of epithelial-mesenchymal transition or metastasis (including miR-200b, miR-200c, miR-205, miR-148a, and miR-424). We hypothesize that the high expression of these factors, which promote an adherent phenotype, may be causing the defect in hematopoietic differentiation. However, inhibiting these miRNAs, individually or in multiplex, was insufficient to improve hematopoietic differentiation in vitro, suggesting that other miRNAs and/or genes may be involved in this process. Stem Cells 2018;36:55-64.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Células Madre Hematopoyéticas/metabolismo , MicroARNs/genética , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , Regulación hacia Abajo , HumanosRESUMEN
The purpose of this study is to investigate the outcomes of penetrating keratoplasty (PKP) following autologous cultivated limbal epithelial stem cell transplantation (CLET). A prospective, single center, interventional cohort study investigating patients with unilateral total limbal stem cell deficiency (LSCD) treated with CLET who underwent PKP. Patients with confirmed corneal re-epithelialization > 6 months post-CLET, and with best-corrected visual acuity (BCVA) <0.3 logMAR were offered PKP. CLET survival assessed by slit lamp, corneal impression cytology (CIC), and in vivo confocal microscopy. Confirmation of corneal re-epithelialization by histological and immunocytochemical (ICC) examination of trephined corneal buttons. Mean change in best-corrected visual acuity (logMAR) following PKP and PKP survival at 12 months were calculated. Twenty patients underwent PKP. Mean time of PKP was 19 months (range 11-41 months, SD 7.26) post-CLET. Median follow-up time post-PKP was 15 months (range 1-32, SD 10.2). CIC and ICC of all corneas confirmed corneal re-epithelialization before PKP. Mean pre-PKP BCVA was 1.46 (range 0.3-2.7, SD 0.94) improving to a mean post-PKP BCVA of 0.74 (range 0-2.7, SD 0.87); mean improvement in BCVA post-PKP of 36 letters (95% CI 15.0-57.1, p = .002). Kaplan-Meier mean graft survival was 90.9% (95% CI 50.8-98.7) at 12 months. We recommend a two-stage approach with CLET followed by PKP >12 months later. Patients experienced a significant improvement in BCVA following PKP. PKP did not have a detrimental effect on CLET survival. PKP survival post-CLET is better than that reported for high risk PKP. Stem Cells 2018;36:925-931.