RESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
A three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy. We previously demonstrated that embryonic stem cells (ESCs) can generate 3D-retina in vitro using a self-organizing stem cell culture technique known as SFEBq. Here we show an optimized culture method for 3D-retina generation from feeder-free human pluripotent stem cells (hPSCs). Although feeder-free hPSC-maintenance culture was suitable for cell therapy, feeder-free hPSC-derived aggregates tended to collapse during 3D-xdifferentiation culture. We found that the initial hPSC state was a key factor and that preconditioning of the hPSC state by modulating TGF-beta and Shh signaling improved self-formation of 3D-neuroepithelium. Using the preconditioning method, several feeder-free hPSC lines robustly differentiated into 3D-retina. In addition, changing preconditioning stimuli in undifferentiated hPSCs altered the proportions of neural retina and retinal pigment epithelium, important quality factors for 3D-retina. We demonstrated that the feeder-free hiPSC-derived 3D-retina differentiated into rod and cone photoreceptors in vitro and in vivo. Thus, preconditioning is a useful culture methodology for cell therapy to direct the initial hPSC state toward self-organizing 3D-neuroepithelium.
Asunto(s)
Técnicas de Cultivo de Célula , Diferenciación Celular , Células Madre Pluripotentes , Retina , Transducción de Señal , Línea Celular , Humanos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Retina/citología , Retina/metabolismoRESUMEN
The neuroectoderm is patterned along a rostral-caudal axis in response to localized factors in the embryo, but exactly how these factors act as positional information for this patterning is not yet fully understood. Here, using the self-organizing properties of mouse embryonic stem cell (ESC), we report that ESC-derived neuroectoderm self-generates a Six3+ rostral and a Irx3+ caudal bipolarized patterning. In this instance, localized Fgf signaling performs dual roles, as it regulates Six3+ rostral polarization at an earlier stage and promotes Wnt signaling at a later stage. The Wnt signaling components are differentially expressed in the polarized tissues, leading to genome-wide Irx3+ caudal-polarization signals. Surprisingly, differentially expressed Wnt agonists and antagonists have essential roles in orchestrating the formation of a balanced rostral-caudal neuroectoderm pattern. Together, our findings provide key processes for dynamic self-patterning and evidence that a temporally and locally regulated interaction between Fgf and Wnt signaling controls self-patterning in ESC-derived neuroectoderm.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Placa Neural/metabolismo , Proteínas Wnt/metabolismo , Animales , Tipificación del Cuerpo , Células Madre Embrionarias , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Factor 5 de Crecimiento de Fibroblastos/genética , Factor 5 de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos ICR , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Placa Neural/fisiología , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Wnt/genética , Proteína Homeobox SIX3RESUMEN
Recent advances in self-organizing, 3-dimensional tissue cultures of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provided an in vitro model that recapitulates many aspects of the in vivo developmental steps. Using Rax-GFP-expressing ESCs, newly generated Six3-/- iPSCs, and conditional null Six3delta/f;Rax-Cre ESCs, we identified Six3 repression of R-spondin 2 (Rspo2) as a required step during optic vesicle morphogenesis and neuroretina differentiation. We validated these results in vivo by showing that transient ectopic expression of Rspo2 in the anterior neural plate of transgenic mouse embryos was sufficient to inhibit neuroretina differentiation. Additionally, using a chimeric eye organoid assay, we determined that Six3 null cells exert a non-cell-autonomous repressive effect during optic vesicle formation and neuroretina differentiation. Our results further validate the organoid culture system as a reliable and fast alternative to identify and evaluate genes involved in eye morphogenesis and neuroretina differentiation in vivo.
Asunto(s)
Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Retina/metabolismo , Trombospondinas/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteína Axina/genética , Proteína Axina/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Hibridación Fluorescente in Situ , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Placa Neural/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Retina/citología , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Trombospondinas/genética , Factores de Transcripción/genética , Proteínas Wnt , Proteína Homeobox SIX3RESUMEN
Pluripotent stem cells can undergo repeated self-renewal while retaining genetic integrity, but they occasionally acquire aneuploidy during long-term culture, which is a practical obstacle for medical applications of human pluripotent stem cells. In this study, we explored the biological roles of ABR, a regulator of RHO family small GTPases, and found that it has pivotal roles during mitotic processes in human embryonic stem cells (hESCs). Although ABR has been shown to be involved in dissociation-induced hESC apoptosis, it does not appear to have direct effects on cell survival unless cell-cell contact is impaired. Instead, we found that it is important for faithful hESC division. Mechanistically, ABR depletion compromised centrosome dynamics and predisposed the cell to chromosome misalignment and missegregation, which raised the frequency of aneuploidy. These results provide insights into the mechanisms that support the genetic integrity of self-renewing hESCs.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Células Madre Embrionarias Humanas/citología , Mitosis , Aneuploidia , Apoptosis , Comunicación Celular , Línea Celular , Puntos de Control de la Fase G2 del Ciclo Celular , Proteínas Activadoras de GTPasa/genética , Eliminación de Gen , Células Madre Embrionarias Humanas/metabolismo , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The thalamus is a diencephalic structure that plays crucial roles in relaying and modulating sensory and motor information to the neocortex. The thalamus develops in the dorsal part of the neural tube at the level of the caudal forebrain. However, the molecular mechanisms that are essential for thalamic differentiation are still unknown. Here, we have succeeded in generating thalamic neurons from mouse embryonic stem cells (mESCs) by modifying the default method that induces the most-anterior neural type in self-organizing culture. A low concentration of the caudalizing factor insulin and a MAPK/ERK kinase inhibitor enhanced the expression of the caudal forebrain markers Otx2 and Pax6. BMP7 promoted an increase in thalamic precursors such as Tcf7l2+/Gbx2+ and Tcf7l2+/Olig3+ cells. mESC thalamic precursors began to express the glutamate transporter vGlut2 and the axon-specific marker VGF, similar to mature projection neurons. The mESC thalamic neurons extended their axons to cortical layers in both organotypic culture and subcortical transplantation. Thus, we have identified the minimum elements sufficient for in vitro generation of thalamic neurons. These findings expand our knowledge of thalamic development.
Asunto(s)
Células Madre Embrionarias de Ratones/citología , Neuronas/citología , Tálamo/citología , Animales , Proteína Morfogenética Ósea 7/farmacología , Agregación Celular/efectos de los fármacos , Células Cultivadas , Cuerpos Embrioides/citología , Cuerpos Embrioides/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuropéptidos/metabolismo , Técnicas de Cultivo de Órganos , Inhibidores de Proteínas Quinasas/farmacología , Ratas Sprague-Dawley , Proteína 2 Similar al Factor de Transcripción 7/metabolismoRESUMEN
Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease characterized by loss of Purkinje cells in the cerebellum. SCA6 is caused by CAG trinucleotide repeat expansion in CACNA1A, which encodes Cav2.1, α1A subunit of P/Q-type calcium channel. However, the pathogenic mechanism and effective therapeutic treatments are still unknown. Here, we have succeeded in generating differentiated Purkinje cells that carry patient genes by combining disease-specific iPSCs and self-organizing culture technologies. Patient-derived Purkinje cells exhibit increased levels of full-length Cav2.1 protein but decreased levels of its C-terminal fragment and downregulation of the transcriptional targets TAF1 and BTG1. We further demonstrate that SCA6 Purkinje cells exhibit thyroid hormone depletion-dependent degeneration, which can be suppressed by two compounds, thyroid releasing hormone and Riluzole. Thus, we have constructed an in vitro disease model recapitulating both ontogenesis and pathogenesis. This model may be useful for pathogenic investigation and drug screening.
Asunto(s)
Células Madre Pluripotentes Inducidas/patología , Células de Purkinje/patología , Ataxias Espinocerebelosas/patología , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/metabolismo , Diferenciación Celular/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Dominios Proteicos , Células de Purkinje/efectos de los fármacos , Células de Purkinje/metabolismo , Riluzol/farmacología , Tirotropina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
We previously demonstrated that mouse embryonic stem cell (mESC)-derived retinal epithelium self-forms an optic cup-like structure. In the developing retina, the dorsal and ventral sides differ in terms of local gene expression and morphological features. This aspect has not yet been shown in vitro Here, we demonstrate that mESC-derived retinal tissue spontaneously acquires polarity reminiscent of the dorsal-ventral (D-V) patterning of the embryonic retina. Tbx5 and Vax2 were expressed in a mutually exclusive manner, as seen in vivo Three-dimensional morphometric analysis showed that the in vitro-formed optic cup often contains cleft structures resembling the embryonic optic fissure. To elucidate the mechanisms underlying the spontaneous D-V polarization of mESC-derived retina, we examined the effects of patterning factors, and found that endogenous BMP signaling plays a predominant role in the dorsal specification. Further analysis revealed that canonical Wnt signaling, which was spontaneously activated at the proximal region, acts upstream of BMP signaling for dorsal specification. These observations suggest that D-V polarity could be established within the self-formed retinal neuroepithelium by intrinsic mechanisms involving the spatiotemporal regulation of canonical Wnt and BMP signals.
Asunto(s)
Tipificación del Cuerpo/fisiología , Polaridad Celular/fisiología , Células Madre Embrionarias/fisiología , Organogénesis/fisiología , Retina/embriología , Animales , Células Cultivadas , Embrión de Mamíferos , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , Técnicas de Cultivo de Órganos , Retina/citología , Transducción de Señal/genéticaRESUMEN
Insulin-like growth factors (IGFs) are involved in growth and tissue development, including diseases such as type-2 diabetes and cancers. However, their roles in lineage specification, especially in early mammalian neural development, are poorly understood. Here, we analyzed the protein expression of IGF-2 in early mouse embryo, and it was preferentially detected in anterior mesodermal tissue, adjacent to the neural plate. We utilized a self-organizing neural tissue culture system and analyzed the direct effect of IGF-2 on the general neural marker Sox1. Interestingly, using recombinant IGF-2 and a chemical inhibitor of its receptor (IGF-1R), we found that the IGF-2/IGF-1R pathway positively regulated Sox1 expression in embryonic stem (ES) cell-derived neural tissue. Furthermore, to visualize the expression patterns of other neural markers, we used reporter ES cell lines and we found that the IGF-2/IGF-1R signaling upregulated the expression of the posterior neural marker Irx3. In contrast, the anterior neural marker Six3 was downregulated by IGF-2/IGF-1R signaling. Together, our results demonstrate that IGF-2/IGF-1R signaling has different effects on neural marker expression, which may influence the early regional identity of ES cell-derived neural tissues.
Asunto(s)
Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Factor II del Crecimiento Similar a la Insulina/fisiología , Proteínas del Tejido Nervioso/metabolismo , Placa Neural/crecimiento & desarrollo , Receptor IGF Tipo 1/fisiología , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Madre Embrionarias/metabolismo , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Placa Neural/metabolismo , Receptor IGF Tipo 1/metabolismo , Factores de Transcripción SOXB1/genética , Transducción de Señal , Factores de Transcripción/genética , Proteína Homeobox SIX3RESUMEN
The epiblast (foremost embryonic ectoderm) generates all three germ layers and therefore has crucial roles in the formation of all mammalian body cells. However, regulation of epiblast gene expression is poorly understood because of the difficulty of manipulating epiblast tissues in vivo. In the present study, using the self-organizing properties of mouse embryonic stem cell (ESC), we generated and characterized epiblast-like tissue in three-dimensional culture. We identified significant genome-wide gene expression changes in this epiblast-like tissue by transcriptomic analysis. In addition, we identified the particular significance of the Erk/Mapk and integrin-linked kinase pathways, and genes related to ectoderm/epithelial formation, using the bioinformatics resources IPA and DAVID. Here, we focused on Fgf5, which ranked in the top 10 among the discovered genes. To develop a functional analysis of Fgf5, we created an efficient method combining CRISPR/Cas9-mediated genome engineering and RNA interference (RNAi). Notably, we show one-step generation of various Fgf5 reporter lines including heterozygous and homozygous knockins (the GET method). For time- and dose-dependent depletion of fgf5 over the course of development, we generated an ESC line harboring Tol2 transposon-mediated integration of an inducible short hairpin RNA interference system (pdiRNAi). Our findings raised the possibility that Fgf/Erk signaling and apicobasal epithelial integrity are important factors in epiblast development. In addition, our methods provide a framework for a broad array of applications in the areas of mammalian genetics and molecular biology to understand development and to improve future therapeutics.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Factor 5 de Crecimiento de Fibroblastos/deficiencia , Factor 5 de Crecimiento de Fibroblastos/genética , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Estratos Germinativos/metabolismo , Interferencia de ARN , Animales , Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Genoma , Estratos Germinativos/citología , Ratones , Ratones Noqueados , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismoRESUMEN
Anterior pituitary is critical for endocrine systems. Its hormonal responses to positive and negative regulators are indispensable for homeostasis. For this reason, generating human anterior pituitary tissue that retains regulatory hormonal control in vitro is an important step for the development of cell transplantation therapy for pituitary diseases. Here we achieve this by recapitulating mouse pituitary development using human embryonic stem cells. We find that anterior pituitary self-forms in vitro following the co-induction of hypothalamic and oral ectoderm. The juxtaposition of these tissues facilitated the formation of pituitary placode, which subsequently differentiated into pituitary hormone-producing cells. They responded normally to both releasing and feedback signals. In addition, after transplantation into hypopituitary mice, the in vitro-generated corticotrophs rescued physical activity levels and survival of the hosts. Thus, we report a useful methodology for the production of regulator-responsive human pituitary tissue that may benefit future studies in regenerative medicine.
Asunto(s)
Corticotrofos/trasplante , Células Madre Embrionarias/fisiología , Hipopituitarismo/terapia , Adenohipófisis/embriología , Ingeniería de Tejidos/métodos , Hormona Adrenocorticotrópica/metabolismo , Animales , Técnicas de Cultivo de Célula , Desarrollo Embrionario , Hormona del Crecimiento/metabolismo , Humanos , Ratones , Adenohipófisis/metabolismoRESUMEN
Morphogenesis in multicellular organisms is accompanied by apoptotic cell behaviors: cell shrinkage and cell disappearance. The mechanical effects of these behaviors are spatiotemporally regulated within multicellular dynamics to achieve proper tissue sizes and shapes in three-dimensional (3D) space. To analyze 3D multicellular dynamics, 3D vertex models have been suggested, in which a reversible network reconnection (RNR) model has successfully expressed 3D cell rearrangements during large deformations. To analyze the effects of apoptotic cell behaviors on 3D multicellular morphogenesis, we modeled cell apoptosis based on the RNR model framework. Cell shrinkage was modeled by the potential energy as a function of individual cell times during the apoptotic phase. Cell disappearance was modeled by merging neighboring polyhedrons at their boundary surface according to the topological rules of the RNR model. To establish that the apoptotic cell behaviors could be expressed as modeled, we simulated morphogenesis driven by cell apoptosis in two types of tissue topology: 3D monolayer cell sheet and 3D compacted cell aggregate. In both types of tissue topology, the numerical simulations successfully illustrated that cell aggregates gradually shrank because of successive cell apoptosis. During tissue shrinkage, the number of cells in aggregates decreased while maintaining individual cell size and shape. Moreover, in case of localizing apoptotic cells within a part of the 3D monolayer cell aggregate, the cell apoptosis caused the global tissue bending by pulling on surrounding cells. In case of localizing apoptotic cells on the surface of the 3D compacted cell aggregate, the cell apoptosis caused successive, directional cell rearrangements from the inside to the surface. Thus, the proposed model successfully provided a basis for expressing apoptotic cell behaviors during 3D multicellular morphogenesis based on an RNR model framework.
Asunto(s)
Apoptosis , Simulación por Computador , Modelos Biológicos , Morfogénesis , Tamaño de la CélulaRESUMEN
Wnt/ß-catenin signaling is crucial for maintenance of pluripotent state of embryonic stem cell (ESC). However, it is unclear how Wnt/ß-catenin signaling affects the differentiation ability of ESC, especially with regard to rostral forebrain cells. Here, using Rax, rostral forebrain marker, and Wnt/ß-catenin reporter lines, we report ratio of Rax(+) and Wnt responding tissue (Wnt(+)) patterns, which were affected by seeding number of ESC in three-dimensional culture system. Surprisingly, we found ß-catenin level and localization are heterogeneous in ESC colony by immunostaining and time-laps imaging of ß-catenin-mEGFP signals. Moreover, activation of Wnt signaling in ESC promoted expression level and nuclear localization of ß-catenin, and mRNA levels of Wnt antagonists, axin2 and dkk1, leading to upregulating Wnt/ß-catenin reporter in ESC state and Rax expression at differentiation culture day 7. Together, our results suggest that activation of Wnt signaling in ESC promotes the differentiation efficacy of rostral forebrain cells. Wnt-priming culture method may provide a useful tool for applications in the areas of basic science and molecular therapeutics for regenerative medicine.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Prosencéfalo/citología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Proteína Axina/genética , Linaje de la Célula/genética , Células Cultivadas , Activación Enzimática , Proteínas del Ojo/metabolismo , Citometría de Flujo , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , ARN Mensajero/genética , ARN no Traducido/genética , Factores de Transcripción/metabolismo , Proteínas Wnt/antagonistas & inhibidores , Vía de Señalización WntRESUMEN
Retinal transplantation therapy for retinitis pigmentosa is increasingly of interest due to accumulating evidence of transplantation efficacy from animal studies and development of techniques for the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells into retinal tissues or cells. In this study, we aimed to assess the potential clinical utility of hESC-derived retinal tissues (hESC-retina) using newly developed primate models of retinal degeneration to obtain preparatory information regarding the potential clinical utility of these hESC-retinas in transplantation therapy. hESC-retinas were first transplanted subretinally into nude rats with or without retinal degeneration to confirm their competency as a graft to mature to form highly specified outer segment structure and to integrate after transplantation. Two focal selective photoreceptor degeneration models were then developed in monkeys by subretinal injection of cobalt chloride or 577-nm optically pumped semiconductor laser photocoagulation. The utility of the developed models and a practicality of visual acuity test developed for monkeys were evaluated. Finally, feasibility of hESC-retina transplantation was assessed in the developed monkey models under practical surgical procedure and postoperational examinations. Grafted hESC-retina was observed differentiating into a range of retinal cell types, including rod and cone photoreceptors that developed structured outer nuclear layers after transplantation. Further, immunohistochemical analyses suggested the formation of host-graft synaptic connections. The findings of this study demonstrate the clinical feasibility of hESC-retina transplantation and provide the practical tools for the optimization of transplantation strategies for future clinical applications.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Retina/citología , Retina/trasplante , Degeneración Retiniana/cirugía , Animales , Diferenciación Celular , Cobalto/toxicidad , Modelos Animales de Enfermedad , Haplorrinos , Humanos , Células Fotorreceptoras/patología , Primates , Ratas , Degeneración Retiniana/inducido químicamente , Degeneración Retiniana/patologíaRESUMEN
The developing dorsomedial telencephalon includes the medial pallium, which goes on to form the hippocampus. Generating a reliable source of human hippocampal tissue is an important step for cell-based research into hippocampus-related diseases. Here we show the generation of functional hippocampal granule- and pyramidal-like neurons from self-organizing dorsomedial telencephalic tissue using human embryonic stem cells (hESCs). First, we develop a hESC culture method that utilizes bone morphogenetic protein (BMP) and Wnt signalling to induce choroid plexus, the most dorsomedial portion of the telencephalon. Then, we find that titrating BMP and Wnt exposure allowed the self-organization of medial pallium tissues. Following long-term dissociation culture, these dorsomedial telencephalic tissues give rise to Zbtb20(+)/Prox1(+) granule neurons and Zbtb20(+)/KA1(+) pyramidal neurons, both of which were electrically functional with network formation. Thus, we have developed an in vitro model that recapitulates human hippocampus development, allowing the generation of functional hippocampal granule- and pyramidal-like neurons.
Asunto(s)
Plexo Coroideo/embriología , Hipocampo/embriología , Células Madre Embrionarias Humanas/citología , Células Piramidales/citología , Telencéfalo/embriología , Proteínas Morfogenéticas Óseas/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Plexo Coroideo/efectos de los fármacos , Hipocampo/efectos de los fármacos , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Inmunohistoquímica , Técnicas In Vitro , Neuronas/citología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Telencéfalo/efectos de los fármacos , Proteínas Wnt/farmacología , Vía de Señalización WntRESUMEN
Human embryonic stem cells (hESCs) can survive and proliferate for an extended period of time in culture, but unlike that of tumor-derived cells, this form of cellular immortality does not depend on genomic aberrations. In this study, we sought to elucidate the molecular basis of this long-term growth property of hESCs. We found that the survival of hESCs depends on the small GTPase Rho and its activator AKAP-Lbc. We show that AKAP-Lbc/Rho signaling sustains the nuclear function of the transcriptional cofactors YAP and TAZ by modulating actin microfilament organization. By inducing reprogramming and differentiation, we found that dependency on this Rho signaling pathway is associated with the pluripotent state. Thus, our findings show that the capacity of hESCs to undergo long-term expansion in vitro is intrinsically coupled to their cellular identity through interconnected molecular circuits that link cell survival to pluripotency.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Citoesqueleto de Actina , Aciltransferasas , Proteínas de Ciclo Celular , Proliferación Celular , Autorrenovación de las Células , Supervivencia Celular , Células Cultivadas , Células Madre Embrionarias Humanas/citología , HumanosRESUMEN
Embryonic stem (ES) cells have a remarkable capacity to self-organize complex, multi-layered optic cups in vitro via a culture technique called SFEBq. During both SFEBq and in vivo optic cup development, Rax (Rx) expressing neural retina epithelial (NRE) tissues utilize Fgf and Wnt/ß-catenin signalling pathways to differentiate into neural retina (NR) and retinal-pigmented epithelial (RPE) tissues, respectively. How these signaling pathways affect gene expression during optic tissue formation has remained largely unknown, especially at the transcriptome scale. Here, we address this question using RNA-Seq. We generated Rx+ optic tissue using SFEBq, exposed these tissues to either Fgf or Wnt/ß-catenin stimulation, and assayed their gene expression across multiple time points using RNA-Seq. This comparative dataset will help elucidate how Fgf and Wnt/ß-catenin signaling affect gene expression during optic tissue differentiation and will help inform future efforts to optimize in vitro optic tissue culture technology.
Asunto(s)
Diferenciación Celular , Retina , Epitelio Pigmentado de la Retina , Transcriptoma , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Retina/citología , Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Vía de Señalización WntRESUMEN
During cerebellar development, the main portion of the cerebellar plate neuroepithelium gives birth to Purkinje cells and interneurons, whereas the rhombic lip, the germinal zone at its dorsal edge, generates granule cells and cerebellar nuclei neurons. However, it remains elusive how these components cooperate to form the intricate cerebellar structure. Here, we found that a polarized cerebellar structure self-organizes in 3D human embryonic stem cell (ESC) culture. The self-organized neuroepithelium differentiates into electrophysiologically functional Purkinje cells. The addition of fibroblast growth factor 19 (FGF19) promotes spontaneous generation of dorsoventrally polarized neural-tube-like structures at the level of the cerebellum. Furthermore, addition of SDF1 and FGF19 promotes the generation of a continuous cerebellar plate neuroepithelium with rhombic-lip-like structure at one end and a three-layer cytoarchitecture similar to the embryonic cerebellum. Thus, human-ESC-derived cerebellar progenitors exhibit substantial self-organizing potential for generating a polarized structure reminiscent of the early human cerebellum at the first trimester.
Asunto(s)
Cerebelo/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Técnicas de Cultivo de Célula , Células Cultivadas , Quimiocina CXCL12/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Neuronas/citología , Células de Purkinje/citologíaRESUMEN
Hypothalamic neurons orchestrate many essential physiological and behavioral processes via secreted neuropeptides, and are relevant to human diseases such as obesity, narcolepsy and infertility. We report the differentiation of human pluripotent stem cells into many of the major types of neuropeptidergic hypothalamic neurons, including those producing pro-opiolemelanocortin, agouti-related peptide, hypocretin/orexin, melanin-concentrating hormone, oxytocin, arginine vasopressin, corticotropin-releasing hormone (CRH) or thyrotropin-releasing hormone. Hypothalamic neurons can be generated using a 'self-patterning' strategy that yields a broad array of cell types, or via a more reproducible directed differentiation approach. Stem cell-derived human hypothalamic neurons share characteristic morphological properties and gene expression patterns with their counterparts in vivo, and are able to integrate into the mouse brain. These neurons could form the basis of cellular models, chemical screens or cellular therapies to study and treat common human diseases.