RESUMEN
Brain development is regulated by conserved transcriptional programs across species, but little is known about the divergent mechanisms that create species-specific characteristics. Among brain regions, human cerebellar histogenesis differs in complexity compared with nonhuman primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. We report a rapid protocol for the derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSCs). Upon transplantation into juvenile mice, hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification-seq, we identified an unexpected temporal shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, in the human outer external granule layer. This molecular divergence may enable the protracted development of the human cerebellum compared to mice.
Asunto(s)
Antígenos Nucleares/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cerebelo/metabolismo , Proteínas del Tejido Nervioso/genética , Animales , Antígenos Nucleares/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Comparative transcriptomics between differentiating human pluripotent stem cells (hPSCs) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate human pluripotent stem cell-derived Purkinje cells (hPSC-PCs) that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we used Tg(Pcp2-L10a-Egfp) TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenic Pcp2-L10a-Egfp TRAP hPSC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides a direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders.
Asunto(s)
Diferenciación Celular , Células de Purkinje/metabolismo , Transcriptoma , Animales , Humanos , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteínas/genética , Proteínas/metabolismo , Células de Purkinje/citologíaRESUMEN
Accumulating evidence suggests that cerebellar dysfunction early in life is associated with autism spectrum disorder (ASD), but the molecular mechanisms underlying the cerebellar deficits at the cellular level are unclear. Tuberous sclerosis complex (TSC) is a neurocutaneous disorder that often presents with ASD. Here, we developed a cerebellar Purkinje cell (PC) model of TSC with patient-derived human induced pluripotent stem cells (hiPSCs) to characterize the molecular mechanisms underlying cerebellar abnormalities in ASD and TSC. Our results show that hiPSC-derived PCs from patients with pathogenic TSC2 mutations displayed mTORC1 pathway hyperactivation, defects in neuronal differentiation and RNA regulation, hypoexcitability and reduced synaptic activity when compared with those derived from controls. Our gene expression analyses revealed downregulation of several components of fragile X mental retardation protein (FMRP) targets in TSC2-deficient hiPSC-PCs. We detected decreased expression of FMRP, glutamate receptor δ2 (GRID2), and pre- and post-synaptic markers such as synaptophysin and PSD95 in the TSC2-deficient hiPSC-PCs. The mTOR inhibitor rapamycin rescued the deficits in differentiation, synaptic dysfunction, and hypoexcitability of TSC2 mutant hiPSC-PCs in vitro. Our findings suggest that these gene expression changes and cellular abnormalities contribute to aberrant PC function during development in TSC affected individuals.
Asunto(s)
Células de Purkinje/metabolismo , Esclerosis Tuberosa/metabolismo , Adulto , Trastorno del Espectro Autista/complicaciones , Trastorno del Espectro Autista/metabolismo , Enfermedades Cerebelosas/metabolismo , Cerebelo/metabolismo , Niño , Preescolar , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/efectos de los fármacos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Modelos Biológicos , Células de Purkinje/patología , Sirolimus/farmacología , Sinapsis/metabolismo , Sinapsis/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Esclerosis Tuberosa/fisiopatología , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
PURPOSE: The purpose of this study was to better understand the role canonical/ß-catenin Wnt signaling plays in the differentiation of human embryonic stem cells (hESCs) into retinal pigmented epithelium (RPE), with the goal of improving methods for derivation. METHODS: Fluorescent reporters were generated to monitor RPE differentiating from hESCs by using a previously described 14-day derivation protocol. Reporters were used to test the effects of the canonical/ß-catenin Wnt pathway agonist CHIR99021 on differentiating RPE. Cells derived from differentiation studies were characterized by lineage-specific transcription factor expression, morphology, pigmentation, and function. The RPE derivation efficiency was determined from percentage positive PMEL17 expression. RESULTS: Fluorescent reporters mimicked expression of endogenous genes during 14-day differentiation to RPE. Analysis of Wnt pathway gene expression showed that the pathway components are expressed in differentiating RPE cells. Addition of CHIR99021 improved RPE derivation based on morphology, expression of RPE-specific lineage markers, and genes involved in melanogenesis. Additionally, expression of the neural retina marker CHX10 was suppressed during differentiation with CHIR99021. Addition of soluble WNT3A, but not WNT5A, had the same result. The CHIR99021-modified protocol yielded cell populations that were 97.77% ± 0.1% positive for the RPE marker PMEL17 at day 14. After cells were expanded to passage 3, they were shown to express RPE markers, carry out phagocytosis of rod outer segments, and secrete pigment epithelium-derived factor apically and vascular endothelial growth factor basally. CONCLUSIONS: Our findings demonstrated the importance of canonical/ß-catenin Wnt signaling in RPE differentiation and showed that manipulating the pathway significantly improves RPE derivation from hESC.
Asunto(s)
Células Madre Embrionarias/citología , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Degeneración Macular/genética , Factores de Crecimiento Nervioso/metabolismo , ARN/genética , Epitelio Pigmentado de la Retina/metabolismo , Serpinas/metabolismo , beta Catenina/genética , Diferenciación Celular , Células Cultivadas , Citometría de Flujo , Humanos , Inmunohistoquímica , Degeneración Macular/metabolismo , Degeneración Macular/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/embriología , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
Human embryonic stem cells (hESCs) offer a potentially unlimited supply of cells for emerging cell-based therapies. Unfortunately, the process of deriving distinct cell types can be time consuming and expensive. In the developed world, age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with more than 7.2 million people afflicted in the U.S. alone. Both hESC-derived retinal pigmented epithelium (hESC-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE) are being developed for AMD therapies by multiple groups, but their potential for expansion in culture is limited. To attempt to overcome this passage limitation, we examined the involvement of Rho-associated, coiled-coil protein kinase (ROCK) in hESC-RPE and iPSC-RPE culture. We report that inhibiting ROCK1/2 with Y-27632 allows extended passage of hESC-RPE and iPSC-RPE. Microarray analysis suggests that ROCK inhibition could be suppressing an epithelial-to-mesenchymal transition through various pathways. These include inhibition of key ligands of the transforming growth factor-ß pathway (TGFB1 and GDF6) and Wnt signaling. Two important processes are affected, allowing for an increase in hESC-RPE expansion. First, ROCK inhibition promotes proliferation by inducing multiple components that are involved in cell cycle progression. Second, ROCK inhibition affects many pathways that could be converging to suppress RPE-to-mesenchymal transition. This allows hESC-RPE to remain functional for an extended but finite period in culture.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Células Madre Pluripotentes/citología , Epitelio Pigmentado de la Retina/citología , Quinasas Asociadas a rho/antagonistas & inhibidores , Amidas/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Madre Embrionarias/enzimología , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Madre Pluripotentes/enzimología , Piridinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/enzimologíaRESUMEN
Controlling the differentiation of human pluripotent stem cells is the goal of many laboratories, both to study normal human development and to generate cells for transplantation. One important cell type under investigation is the retinal pigmented epithelium (RPE). Age-related macular degeneration (AMD), the leading cause of blindness in the Western world, is caused by dysfunction and death of the RPE. Currently, RPE derived from human embryonic stem cells are in clinical trials for the treatment of AMD. Although protocols to generate RPE from human pluripotent stem cells have become more efficient since the first report in 2004, they are still time-consuming and relatively inefficient. We have found that the addition of defined factors at specific times leads to conversion of approximately 80% of the cells to an RPE phenotype in only 14 days. This protocol should be useful for rapidly generating RPE for transplantation as well as for studying RPE development in vitro.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes/citología , Epitelio Pigmentado de la Retina/citología , Activinas/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Niacinamida/farmacología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Pirroles/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Factores de Tiempo , Péptido Intestinal Vasoactivo/farmacología , Campos Visuales/efectos de los fármacosRESUMEN
A potential application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is the generation of retinal pigmented epithelium (RPE) to treat age-related macular degeneration (AMD), a common but incurable retinal disease. RPE cells derived from hESCs (hESC-RPEs) and iPSCs (iPSC-RPEs) express essential RPE markers and can rescue visual function in animal models. However, standard differentiation protocols yield RPE cells at low frequency, especially from iPSC lines, and the common use of Matrigel and xenogeneic feeder cells is not compatible with clinical applications. The extracellular matrix (ECM) can affect differentiation, and therefore changes in ECM composition may improve the frequency of stem cell-RPE differentiation. We selected several purified ECM proteins and substrates, based on the in vivo RPE ECM environment, and tested their ability to support iPSC-RPE differentiation and maintenance. iPSCs differentiated on nearly all tested substrates developed pigmented regions, with Matrigel and mouse laminin-111 supporting the highest pigmentation frequencies. Although iPSC-RPEs cultured on the majority of the tested substrates expressed key RPE genes, only six substrates supported development of confluent monolayers with normal RPE morphology, including Matrigel and mouse laminin-111. iPSCs differentiated on mouse laminin-111 produced iPSC-RPEs expressing RPE proteins, and hESCs differentiated on mouse laminin-111 resulted in high yields of functional hESC-RPEs. This identification of key ECM proteins may assist with future scaffold designs and provide peptide sequences for use in synthetic, xeno-free, GMP-compliant generation of RPE from human pluripotent stem cells relevant to clinical translation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Laminina/farmacología , Epitelio Pigmentado de la Retina/metabolismo , Animales , Línea Celular , Técnicas de Cocultivo , Células Nutrientes/citología , Células Nutrientes/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/trasplante , Laminina/química , Laminina/aislamiento & purificación , Degeneración Macular/metabolismo , Degeneración Macular/terapia , Ratones , Epitelio Pigmentado de la Retina/citologíaRESUMEN
Despite advancements made in our understanding of ocular biology, therapeutic options for many debilitating retinal diseases remain limited. Stem cell-based therapies are a potential avenue for treatment of retinal disease, and this mini-review will focus on current research in this area. Cellular therapies to replace retinal pigmented epithelium (RPE) and/or photoreceptors to treat age-related macular degeneration (AMD), Stargardt's macular dystrophy, and retinitis pigmentosa are currently being developed. Over the past decade, significant advancements have been made using different types of human stem cells with varying capacities to differentiate into these target retinal cell types. We review and evaluate pluripotent stem cells, both human embryonic stem cells and human induced pluripotent stem cells, as well as protocols for differentiation of ocular cells, and culture and transplant techniques that might be used to deliver cells to patients.
Asunto(s)
Células Madre Embrionarias/trasplante , Células Madre Pluripotentes/trasplante , Enfermedades de la Retina/terapia , Trasplante de Células Madre , Diferenciación Celular , Células Madre Embrionarias/citología , Humanos , Células Madre Pluripotentes/citologíaRESUMEN
Reprogramming methodologies have provided multiple routes for achieving pluripotency. However, pluripotency is generally considered to be an almost singular state, with subtle differences described between induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). We profiled miRNA expression levels across 49 human cell lines, including ESCs, iPSCs, differentiated cells, and cancer cell lines. We found that the resulting miRNA profiles divided the iPSCs and hESCs examined into two distinct categories irrespective of the cell line origin. The miRNAs that defined these two pluripotency categories also distinguished cancer cells from differentiated cells. Transcriptome analysis suggested that several gene sets related to p53 distinguished these categories, and overexpression of the p53-targeting miRNAs miR-92 and miR-141 in iPSCs was sufficient to change their classification status. Thus, our results suggest a subdivision of pluripotent stem cell states that is independent of their origin but related to p53 network status.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/metabolismo , Células Madre Pluripotentes/clasificación , Células Madre Pluripotentes/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , MicroARNs/clasificación , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Madre Pluripotentes/citologíaRESUMEN
Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Enfermedades de la Retina/terapia , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/trasplante , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Polaridad Celular , Forma de la Célula , Supervivencia Celular , Células Epiteliales/citología , Células Epiteliales/trasplante , Humanos , Inmunohistoquímica , Macrófagos/citología , Fagocitosis , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/ultraestructura , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Enfermedades de la Retina/patología , Enfermedades de la Retina/fisiopatología , Epitelio Pigmentado de la Retina/ultraestructura , Visión Ocular/fisiologíaRESUMEN
Human induced pluripotent stem cells (iPSCs) have great promise for cellular therapy, but it is unclear if they have the same potential as human embryonic stem cells (hESCs) to differentiate into specialized cell types. Ocular cells such as the retinal pigmented epithelium (RPE) are of particular interest because they could be used to treat degenerative eye diseases, including age-related macular degeneration and retinitis pigmentosa. We show here that iPSCs generated using Oct4, Sox2, Nanog, and Lin28 can spontaneously differentiate into RPE cells, which can then be isolated and cultured to form highly differentiated RPE monolayers. RPE derived from iPSCs (iPS-RPE) were analyzed with respect to gene expression, protein expression, and rod outer segment phagocytosis, and compared with cultured fetal human RPE (fRPE) and RPE derived from hESCs (hESC-RPE). iPS-RPE expression of marker mRNAs was quantitatively similar to that of fRPE and hESC-RPE, and marker proteins were appropriately expressed and localized in polarized monolayers. Levels of rod outer segment phagocytosis by iPS-RPE, fRPE, and hESC-RPE were likewise similar and dependent on integrin alpha v beta 5. This work shows that iPSCs can differentiate into functional RPE that are quantitatively similar to fRPE and hESC-RPE and further supports the finding that iPSCs are similar to hESCs in their differentiation potential.