RESUMEN
Organoid-based models of murine and human innate lymphoid cell precursor (ILCP) maturation are presented. First, murine intestinal and pulmonary organoids are harnessed to demonstrate that the epithelial niche is sufficient to drive tissue-specific maturation of all innate lymphoid cell (ILC) groups in parallel, without requiring subset-specific cytokine supplementation. Then, more complex human induced pluripotent stem cell (hiPSC)-based gut and lung organoid models are used to demonstrate that human epithelial cells recapitulate maturation of ILC from a stringent systemic human ILCP population, but only when the organoid-associated stromal cells are depleted. These systems offer versatile and reductionist models to dissect the impact of environmental and mucosal niche cues on ILC maturation. In the future, these could provide insight into how ILC activity and development might become dysregulated in chronic inflammatory diseases.
Asunto(s)
Células Madre Pluripotentes Inducidas , Organoides , Animales , Diferenciación Celular , Humanos , Inmunidad Innata , Inmunoterapia , Linfocitos , RatonesRESUMEN
The Wnt-pathway is part of a signalling network that regulates many aspects of cell biology. Recently, we discovered crosstalk between AMPA/Kainate-type ionotropic glutamate receptors (iGluRs) and the Wnt-pathway during the initial Wnt3a-interaction at the cytonemes of mouse embryonic stem cells (ESCs). Here, we demonstrate that this crosstalk persists throughout the Wnt3a-response in ESCs. Both AMPA and Kainate receptors regulate early Wnt3a-recruitment, dynamics on the cell membrane, and orientation of the spindle towards a Wnt3a-source at mitosis. AMPA receptors specifically are required for segregating cell fate components during Wnt3a-mediated asymmetric cell division (ACD). Using Wnt-pathway component knockout lines, we determine that Wnt co-receptor Lrp6 has particular functionality over Lrp5 in cytoneme formation, and in facilitating ACD. Both Lrp5 and 6, alongside pathway effector ß-catenin act in concert to mediate the positioning of the dynamic interaction with, and spindle orientation to, a localised Wnt3a-source. Wnt-iGluR crosstalk may prove pervasive throughout embryonic and adult stem cell signalling.
Asunto(s)
División Celular , Células Madre Embrionarias de Ratones/metabolismo , Receptores AMPA/metabolismo , Receptores de Ácido Kaínico/metabolismo , Vía de Señalización Wnt , Proteína Wnt3A/metabolismo , Animales , Diferenciación Celular , Línea Celular , Linaje de la Célula , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Microscopía Confocal , Microscopía Fluorescente , Microscopía por Video , Receptor Cross-Talk , Receptores AMPA/genética , Receptores de Ácido Kaínico/genética , Factores de Tiempo , Proteína Wnt3A/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Asymmetric histone inheritance can regulate cell-fate determination in Drosophila male germline stem cells. However, it remains elusive how this mechanism may be used in mammalian system. Recently, we show mouse embryonic stem cells (mESCs) with Wnt3a beads display non-overlapping H3/H4 patterns. Here, we present a detailed protocol for tracking histone inheritance in asymmetrically dividing mESCs at single-cell resolution. This protocol will establish a new system to study histone inheritance in cultured mammalian cells and could be applied to other parallel systems. For complete details on the use and execution of this protocol, please refer to Tran et al. (2012), Habib et al. (2013), Lowndes et al. (2017), and Ma et al. (2020).
Asunto(s)
Histonas/metabolismo , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Análisis de la Célula Individual/métodos , Poliposis Adenomatosa del Colon/metabolismo , Animales , División Celular Asimétrica , Biomarcadores/metabolismo , Ciclo Celular , Células Cultivadas , Expresión Génica , Ratones , Ratones Transgénicos , Mitosis , Plásmidos/metabolismo , Transgenes , Proteína Wnt3A/metabolismoRESUMEN
Wnt3a-coated beads can induce asymmetric divisions of mouse embryonic stem cells (mESCs), resulting in one self-renewed mESC and one differentiating epiblast stem cell. This provides an opportunity for studying histone inheritance pattern at a single-cell resolution in cell culture. Here, we report that mESCs with Wnt3a-bead induction display nonoverlapping preexisting (old) versus newly synthesized (new) histone H3 patterns, but mESCs without Wnt3a beads have largely overlapping patterns. Furthermore, H4K20me2/3, an old histone-enriched modification, displays a higher instance of asymmetric distribution on chromatin fibers from Wnt3a-induced mESCs than those from non-induced mESCs. These locally distinct distributions between old and new histones have both cellular specificity in Wnt3a-induced mESCs and molecular specificity for histones H3 and H4. Given that post-translational modifications at H3 and H4 carry the major histone modifications, our findings provide a mammalian cell culture system to study histone inheritance for maintaining stem cell fate and for resetting it during differentiation.
Asunto(s)
Histonas/metabolismo , Células Madre Embrionarias de Ratones , Proteína Wnt3A/farmacología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Desarrollo Embrionario , Estratos Germinativos/efectos de los fármacos , Estratos Germinativos/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Procesamiento Proteico-Postraduccional , Proteína Wnt3A/metabolismoRESUMEN
Spatial cellular organization is fundamental for embryogenesis. Remarkably, coculturing embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) recapitulates this process, forming embryo-like structures. However, mechanisms driving ESC-TSC interaction remain elusive. We describe specialized ESC-generated cytonemes that react to TSC-secreted Wnts. Cytoneme formation and length are controlled by actin, intracellular calcium stores, and components of the Wnt pathway. ESC cytonemes select self-renewal-promoting Wnts via crosstalk between Wnt receptors, activation of ionotropic glutamate receptors (iGluRs), and localized calcium transients. This crosstalk orchestrates Wnt signaling, ESC polarization, ESC-TSC pairing, and consequently synthetic embryogenesis. Our results uncover ESC-TSC contact-mediated signaling, reminiscent of the glutamatergic neuronal synapse, inducing spatial self-organization and embryonic cell specification.