RESUMEN
The human lung differs substantially from its mouse counterpart, resulting in a distinct distal airway architecture affected by disease pathology in chronic obstructive pulmonary disease. In humans, the distal branches of the airway interweave with the alveolar gas-exchange niche, forming an anatomical structure known as the respiratory bronchioles. Owing to the lack of a counterpart in mouse, the cellular and molecular mechanisms that govern respiratory bronchioles in the human lung remain uncharacterized. Here we show that human respiratory bronchioles contain a unique secretory cell population that is distinct from cells in larger proximal airways. Organoid modelling reveals that these respiratory airway secretory (RAS) cells act as unidirectional progenitors for alveolar type 2 cells, which are essential for maintaining and regenerating the alveolar niche. RAS cell lineage differentiation into alveolar type 2 cells is regulated by Notch and Wnt signalling. In chronic obstructive pulmonary disease, RAS cells are altered transcriptionally, corresponding to abnormal alveolar type 2 cell states, which are associated with smoking exposure in both humans and ferrets. These data identify a distinct progenitor in a region of the human lung that is not found in mouse that has a critical role in maintaining the gas-exchange compartment and is altered in chronic lung disease.
Asunto(s)
Bronquiolos , Hurones , Células Madre Multipotentes , Alveolos Pulmonares , Animales , Bronquiolos/citología , Linaje de la Célula , Humanos , Pulmón/patología , Ratones , Células Madre Multipotentes/citología , Alveolos Pulmonares/citología , Enfermedad Pulmonar Obstructiva CrónicaRESUMEN
Zebrafish transgenesis is increasingly popular owing to the optical transparency and external development of embryos, which provide a scalable vertebrate model for in vivo experimentation. The ability to express transgenes in a tightly controlled spatio-temporal pattern is an important prerequisite for exploitation of zebrafish in a wide range of biomedical applications. However, conventional transgenesis methods are plagued by position effects: the regulatory environment of genomic integration sites leads to variation of expression patterns of transgenes driven by engineered cis-regulatory modules. This limitation represents a bottleneck when studying the precise function of cis-regulatory modules and their subtle variants or when various effector proteins are to be expressed for labelling and manipulation of defined sets of cells. Here, we provide evidence for the efficient elimination of variability of position effects by developing a PhiC31 integrase-based targeting method. To detect targeted integration events, a simple phenotype scoring of colour change in the lens of larvae is used. We compared PhiC31-based integration and Tol2 transgenesis in the analysis of the activity of a novel conserved enhancer from the developmentally regulated neural-specific esrrga gene. Reporter expression was highly variable among independent lines generated with Tol2, whereas all lines generated with PhiC31 into a single integration site displayed nearly identical, enhancer-specific reporter expression in brain nuclei. Moreover, we demonstrate that a modified integrase system can also be used for the detection of enhancer activity in transient transgenesis. These results demonstrate the power of the PhiC31-based transgene integration for the annotation and fine analysis of transcriptional regulatory elements and it promises to be a generally desirable tool for a range of applications, which rely on highly reproducible patterns of transgene activity in zebrafish.
Asunto(s)
Efectos de la Posición Cromosómica/genética , Marcación de Gen , Mutagénesis Insercional/genética , Transgenes/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Encéfalo/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Genes Reporteros/genética , Sitios Genéticos/genética , Genoma/genética , Integrasas/metabolismo , Cristalino/metabolismo , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Xenopus laevis/genéticaRESUMEN
The tight junction protein claudin 6 (CLDN6) is differentially expressed on cancer cells with almost no expression in healthy tissue. However, achieving therapeutic MAb specificity for this 4 transmembrane protein is challenging because it is nearly identical to the widely expressed CLDN9, with only 3 extracellular amino acids different. Most other CLDN6 MAbs, including those in clinical development are cross-reactive with CLDN9, and several trials have now been stopped. Here we isolated rare MAbs that bind CLDN6 with up to picomolar affinity and display minimal cross-reactivity with CLDN9, 22 other CLDN family members, or across the human membrane proteome. Amino acid-level epitope mapping distinguished the binding sites of our MAbs from existing clinical-stage MAbs. Atomic-level epitope mapping identified the structural mechanism by which our MAbs differentiate CLDN6 and CLDN9 through steric hindrance at a single molecular contact point, the γ carbon on CLDN6 residue Q156.
RESUMEN
Dyskeratosis congenita (DC) is a rare genetic disorder characterized by deficiencies in telomere maintenance leading to very short telomeres and the premature onset of certain age-related diseases, including pulmonary fibrosis (PF). PF is thought to derive from epithelial failure, particularly that of type II alveolar epithelial (AT2) cells, which are highly dependent on Wnt signaling during development and adult regeneration. We use human induced pluripotent stem cell-derived AT2 (iAT2) cells to model how short telomeres affect AT2 cells. Cultured DC mutant iAT2 cells accumulate shortened, uncapped telomeres and manifest defects in the growth of alveolospheres, hallmarks of senescence, and apparent defects in Wnt signaling. The GSK3 inhibitor, CHIR99021, which mimics the output of canonical Wnt signaling, enhances telomerase activity and rescues the defects. These findings support further investigation of Wnt agonists as potential therapies for DC-related pathologies.
Asunto(s)
Disqueratosis Congénita , Células Madre Pluripotentes Inducidas , Telomerasa , Células Epiteliales Alveolares/metabolismo , Disqueratosis Congénita/genética , Disqueratosis Congénita/patología , Glucógeno Sintasa Quinasa 3 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Telomerasa/genética , Telomerasa/metabolismo , Telómero/metabolismoRESUMEN
Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway.
Asunto(s)
Epitelio/metabolismo , Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Pulmón/citología , Análisis de la Célula Individual , Animales , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula , Plasticidad de la Célula , Epitelio/ultraestructura , Genes Reporteros , Humanos , Células Madre Pluripotentes Inducidas/citología , Cinética , Ratones , Secretoglobinas/metabolismo , Análisis de Secuencia de ARN , Solubilidad , Esferoides Celulares/citología , Esferoides Celulares/metabolismo , Factores de Tiempo , Transcriptoma/genética , Vía de Señalización WntRESUMEN
Although many factors contribute to cellular differentiation, the role of mitochondria Ca2+ dynamics during development remains unexplored. Because mammalian embryonic epiblasts reside in a hypoxic environment, we intended to understand whether mCa2+ and its transport machineries are regulated during hypoxia. Tissues from multiple organs of developing mouse embryo evidenced a suppression of MICU1 expression with nominal changes on other MCU complex components. As surrogate models, we here utilized human embryonic stem cells (hESCs)/induced pluripotent stem cells (hiPSCs) and primary neonatal myocytes to delineate the mechanisms that control mCa2+ and bioenergetics during development. Analysis of MICU1 expression in hESCs/hiPSCs showed low abundance of MICU1 due to its direct repression by Foxd1. Experimentally, restoration of MICU1 established the periodic cCa2+ oscillations and promoted cellular differentiation and maturation. These findings establish a role of mCa2+ dynamics in regulation of cellular differentiation and reveal a molecular mechanism underlying this contribution through differential regulation of MICU1.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Factores de Transcripción Forkhead/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factores de Transcripción Forkhead/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Interferencia de ARNRESUMEN
Abetalipoproteinemia (ABL) is an inherited disorder of lipoprotein metabolism resulting from mutations in microsomal triglyceride transfer protein (MTTP). In addition to expression in the liver and intestine, MTTP is expressed in cardiomyocytes, and cardiomyopathy has been reported in several ABL cases. Using induced pluripotent stem cells (iPSCs) generated from an ABL patient homozygous for a missense mutation (MTTPR46G), we show that human hepatocytes and cardiomyocytes exhibit defects associated with ABL disease, including loss of apolipoprotein B (apoB) secretion and intracellular accumulation of lipids. MTTPR46G iPSC-derived cardiomyocytes failed to secrete apoB, accumulated intracellular lipids, and displayed increased cell death, suggesting intrinsic defects in lipid metabolism due to loss of MTTP function. Importantly, these phenotypes were reversed after the correction of the MTTPR46G mutation by CRISPR/Cas9 gene editing. Together, these data reveal clear cellular defects in iPSC-derived hepatocytes and cardiomyocytes lacking MTTP activity, including a cardiomyocyte-specific regulated stress response to elevated lipids.
Asunto(s)
Apolipoproteínas B/metabolismo , Proteínas Portadoras/metabolismo , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Estrés Fisiológico , Abetalipoproteinemia/metabolismo , Edición Génica , Humanos , FenotipoRESUMEN
Genome-wide association studies have struggled to identify functional genes and variants underlying complex phenotypes. We recruited a multi-ethnic cohort of healthy volunteers (n = 91) and used their tissue to generate induced pluripotent stem cells (iPSCs) and hepatocyte-like cells (HLCs) for genome-wide mapping of expression quantitative trait loci (eQTLs) and allele-specific expression (ASE). We identified many eQTL genes (eGenes) not observed in the comparably sized Genotype-Tissue Expression project's human liver cohort (n = 96). Focusing on blood lipid-associated loci, we performed massively parallel reporter assays to screen candidate functional variants and used genome-edited stem cells, CRISPR interference, and mouse modeling to establish rs2277862-CPNE1, rs10889356-DOCK7, rs10889356-ANGPTL3, and rs10872142-FRK as functional SNP-gene sets. We demonstrated HLC eGenes CPNE1, VKORC1, UBE2L3, and ANGPTL3 and HLC ASE gene ACAA2 to be lipid-functional genes in mouse models. These findings endorse an iPSC-based experimental framework to discover functional variants and genes contributing to complex human traits.
Asunto(s)
Sitios Genéticos , Variación Genética , Hepatocitos/citología , Células Madre Pluripotentes Inducidas/citología , Lípidos/sangre , Animales , Secuencia de Bases , Estudios de Cohortes , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Ratones , Especificidad de Órganos/genética , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genéticaRESUMEN
Although significant advancement has been made in the induced pluripotent stem cell (iPSC) field, current methods for iPSC derivation are labor intensive and costly. These methods involve manual selection, expansion, and characterization of multiple clones for each reprogrammed cell sample and therefore significantly hampers the feasibility of studies where a large number of iPSCs need to be derived. To develop higher throughput iPSC reprogramming methods, we generated iPSCs as a pooled culture using rigorous cell surface pluripotent marker selection with TRA-1-60 or SSEA4 antibodies followed by Magnetic Activated Cell Sorting (MACS). We observed that pool-selected cells are similar or identical to clonally derived iPSC lines from the same donor by all criteria examined, including stable expression of endogenous pluripotency genes, normal karyotype, loss of exogenous reprogramming factors, and in vitro spontaneous and lineage directed differentiation potential. This strategy can be generalized for iPSC generation using both integrating and non-integrating reprogramming methods. Our studies provide an attractive alternative to clonal derivation of iPSCs using rigorously selected cell pools and is amenable to automation.