Severe influenza disease strikes otherwise healthy children and remains unexplained. We report compound heterozygous null mutations in IRF7, which encodes the transcription factor interferon ...regulatory factor 7, in an otherwise healthy child who suffered life-threatening influenza during primary infection. In response to influenza virus, the patient's leukocytes and plasmacytoid dendritic cells produced very little type I and III interferons (IFNs). Moreover, the patient's dermal fibroblasts and induced pluripotent stem cell (iPSC)–derived pulmonary epithelial cells produced reduced amounts of type I IFN and displayed increased influenza virus replication. These findings suggest that IRF7-dependent amplification of type I and III IFNs is required for protection against primary infection by influenza virus in humans. They also show that severe influenza may result from single-gene inborn errors of immunity.
Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human ...lung development. Here we describe a strategy for directed differentiation of hPSCs into developmental lung progenitors, and their subsequent differentiation into predominantly distal lung epithelial cells. The protocol entails four stages that recapitulate lung development, and it takes ∼50 d. First, definitive endoderm (DE) is induced in the presence of high concentrations of activin A. Subsequently, lung-biased anterior foregut endoderm (AFE) is specified by sequential inhibition of bone morphogenetic protein (BMP), transforming growth factor-β (TGF-β) and Wnt signaling. AFE is then ventralized by applying Wnt, BMP, fibroblast growth factor (FGF) and retinoic acid (RA) signaling to obtain lung and airway progenitors. Finally, these are further differentiated into more mature epithelial cells types using Wnt, FGF, cAMP and glucocorticoid agonism. This protocol is conducted in defined conditions, it does not involve genetic manipulation of the cells and it results in cultures in which the majority of the cells express markers of various lung and airway epithelial cells, with a predominance of cells identifiable as functional type II alveolar epithelial cells.
The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening and ...studies of human lung development. We have established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation of hPSCs in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. The type II alveolar epithelial cells were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing retinoic acid, Wnt and BMP-agonists to signaling pathways critical for early lung development in the mouse-recapitulated defects in corresponding genetic mouse knockouts. As this protocol generates most cell types of the respiratory system, it may be useful for deriving patient-specific therapeutic cells.
The entire lung epithelium arises from SRY box 9 (SOX9)-expressing progenitors that form the respiratory tree and differentiate into airway and alveolar cells. Despite progress in understanding their ...initial specification within the embryonic foregut, how these progenitors are subsequently maintained is less clear. Using inducible, progenitor-specific genetic mosaic mouse models, we showed that β-catenin (CTNNB1) maintains lung progenitors by promoting a hierarchical lung progenitor gene signature, suppressing gastrointestinal (GI) genes, and regulating NK2 homeobox 1 (NKX2.1) and SRY box 2 (SOX2) in a developmental stage-dependent manner. At the early, but not later, stage post-lung specification, CTNNB1 cell-autonomously maintained normal NKX2.1 expression levels and suppressed ectopic SOX2 expression. Genetic epistasis analyses revealed that CTNNB1 is required for fibroblast growth factor (Fgf)/Kirsten rat sarcoma viral oncogene homolog (
)-mediated promotion of the progenitors.
screening of Eurexpress and translating ribosome affinity purification (TRAP)-RNAseq identified a progenitor gene signature, a subset of which depends on CTNNB1. Wnt signaling also maintained NKX2.1 expression and suppressed GI genes in cultured human lung progenitors derived from embryonic stem cells.
The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed ...differentiation of human iPSCs into airway basal cells (“iBCs”), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato), we track and purify these cells as they first emerge as developmentally immature NKX2-1GFP+ lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1GFP+/TP63tdTomato+ cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.
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•Directed differentiation of human iPSCs generates airway basal cells (“iBCs”)•iBCs self-renew and display multipotent differentiation in vitro and in vivo•By single-cell RNA-seq, iBCs are highly similar to adult primary airway basal cells•iBCs enable modeling of acquired and genetic airway diseases
Hawkins and colleagues report a directed differentiation protocol enabling the derivation of airway basal cells (“iBCs”) from human iPSCs. iBCs recapitulate hallmark stem cell properties of primary basal cells, including self-renewal and multi-lineage differentiation, thus enabling modeling of airway diseases in vitro and repopulation of tracheal xenografts in vivo.
Autosomal recessive IRF7 and IRF9 deficiencies impair type I and III IFN immunity and underlie severe influenza pneumonitis. We report three unrelated children with influenza A virus (IAV) infection ...manifesting as acute respiratory distress syndrome (IAV-ARDS), heterozygous for rare
variants (P554S in two patients and P680L in the third) causing autosomal dominant (AD) TLR3 deficiency. AD TLR3 deficiency can underlie herpes simplex virus-1 (HSV-1) encephalitis (HSE) by impairing cortical neuron-intrinsic type I IFN immunity to HSV-1. TLR3-mutated leukocytes produce normal levels of IFNs in response to IAV. In contrast, TLR3-mutated fibroblasts produce lower levels of IFN-β and -λ, and display enhanced viral susceptibility, upon IAV infection. Moreover, the patients' iPSC-derived pulmonary epithelial cells (PECs) are susceptible to IAV. Treatment with IFN-α2b or IFN-λ1 rescues this phenotype. AD TLR3 deficiency may thus underlie IAV-ARDS by impairing TLR3-dependent, type I and/or III IFN-mediated, PEC-intrinsic immunity. Its clinical penetrance is incomplete for both IAV-ARDS and HSE, consistent with their typically sporadic nature.
It has been postulated that during human fetal development, all cells of the lung epithelium derive from embryonic, endodermal, NK2 homeobox 1-expressing (NKX2-1+) precursor cells. However, this ...hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity, these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support, this NKX2-1+ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively, when recombined with fetal mouse lung mesenchyme, the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved, stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47hiCD26lo cell surface phenotype that allows their prospective isolation from untargeted, patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine.
In living tissues, mechanical stiffness and biological function are intrinsically linked. Alterations in the stiffness of tissues can induce pathological interactions that affect cellular activity ...and tissue function. Underlying connections between tissue stiffness and disease highlights the importance of accurate quantitative characterizations of soft tissue mechanics, which can improve our understanding of disease and inform therapeutic development. In particular, accurate measurement of lung mechanical properties has been especially challenging due to the anatomical and mechanobiological complexities of the lung. Discrepancies between measured mechanical properties of dissected lung tissue samples and intact lung tissues in vivo has limited the ability to accurately characterize integral lung mechanics. Here, we report a non-destructive vacuum-assisted method to evaluate mechanical properties of soft biomaterials, including intact tissues and hydrogels. Using this approach, we measured elastic moduli of rat lung tissue that varied depending on stress-strain distribution throughout the lung. We also observed that the elastic moduli of enzymatically disrupted lung parenchyma increased by at least 64%. The reported methodology enables assessment of the nonlinear viscoelastic characteristics of intact lungs under normal and abnormal (i.e., injured, diseased) conditions and allows measurement of mechanical properties of tissue-mimetic biomaterials for use in therapeutics or in vitro models.
Accurate quantification of tissue stiffness is critical for understanding mechanisms of disease and developing effective therapeutics. Current modalities to measure tissue stiffness are destructive and preclude accurate assessment of lung mechanical properties, as lung mechanics are determined by complex features of the intact lung. To address the need for alternative methods to assess lung mechanics, we report a non-destructive vacuum-based approach to quantify tissue stiffness. We applied this method to correlate lung tissue mechanics with tissue disruption, and to assess the stiffness of biomaterials. This method can be used to inform the development of tissue-mimetic materials for use in therapeutics and disease models, and could potentially be applied for in-situ evaluation of tissue stiffness as a diagnostic or prognostic tool.
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The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), ...we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSC–derived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3a–regulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.
Arsenic is a well-established human carcinogen that is chronically consumed in drinking water by millions of people worldwide. Recent evidence has suggested that arsenic is a genotoxic carcinogen. ...Furthermore, we have shown that mitochondria mediate the mutagenic effects of arsenic in mammalian cells, as arsenic did not induce nuclear mutations in mitochondrial DNA (mtDNA)-depleted cells. Using the human-hamster hybrid A(L) cells, we show here that arsenic alters mitochondrial function by decreasing cytochrome c oxidase function and oxygen consumption but increasing citrate synthase function. These alterations correlated with depletion in mtDNA copy number and increase in large heteroplasmic mtDNA deletions. In addition, mtDNA isolated periodically from cultures treated continuously with arsenic did not consistently display the same deletion pattern, indicating that the mitochondrial genome was subjected to repeated and continuous damage. These data support the theory that the mitochondria, and particularly mtDNA, are important targets of the mutagenic effects of arsenic in mammalian cells.
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