Effective derivation of functional airway organoids from induced pluripotent stem cells (iPSCs) would provide valuable models of lung disease and facilitate precision therapies for airway disorders ...such as cystic fibrosis. However, limited understanding of human airway patterning has made this goal challenging. Here, we show that cyclical modulation of the canonical Wnt signaling pathway enables rapid directed differentiation of human iPSCs via an NKX2-1+ progenitor intermediate into functional proximal airway organoids. We find that human NKX2-1+ progenitors have high levels of Wnt activation but respond intrinsically to decreases in Wnt signaling by rapidly patterning into proximal airway lineages at the expense of distal fates. Using this directed approach, we were able to generate cystic fibrosis patient-specific iPSC-derived airway organoids with a defect in forskolin-induced swelling that is rescued by gene editing to correct the disease mutation. Our approach has many potential applications in modeling and drug screening for airway diseases.
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•Wnt signaling regulates lung differentiation of human pluripotent stem cells•Withdrawal of Wnt signaling from lung progenitors prompts rapid proximal patterning•Purified lung progenitors differentiate to airway organoids in low Wnt conditions•Derived organoids exhibit CFTR-dependent swelling in response to forskolin
Kotton and colleagues show that carefully timed regulation of Wnt signaling can direct human pluripotent cells to differentiate rapidly into functional airway epithelial organoids with many potential applications in disease modeling, drug screening, and precision medicine, and for diseases such as cystic fibrosis.
Recent studies have shown that the respiratory system has an extensive ability to respond to injury and regenerate lost or damaged cells. The unperturbed adult lung is remarkably quiescent, but after ...insult or injury progenitor populations can be activated or remaining cells can re-enter the cell cycle. Techniques including cell-lineage tracing and transcriptome analysis have provided novel and exciting insights into how the lungs and trachea regenerate in response to injury and have allowed the identification of pathways important in lung development and regeneration. These studies are now informing approaches for modulating the pathways that may promote endogenous regeneration as well as the generation of exogenous lung cell lineages from pluripotent stem cells. The emerging advances, highlighted in this Review, are providing new techniques and assays for basic mechanistic studies as well as generating new model systems for human disease and strategies for cell replacement.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The mammalian lung epithelium is composed of a wide array of specialized cells that have adapted to survive environmental exposure and perform the tasks necessary for respiration. Although the ...majority of these cells are remarkably quiescent during adult lung homeostasis, a growing body of literature has demonstrated the capacity of these epithelial lineages to proliferate in response to injury and regenerate lost or damaged cells. In this review, we focus on the regionally distinct lung epithelial cell types that contribute to repair after injury, and we address current controversies regarding whether elite stem cells or frequent facultative progenitors are the predominant participants. We also shed light on the newly emerging approaches for exogenously generating similar lung epithelial lineages from pluripotent stem cells.
Alveolar epithelial type II cells (AEC2s) are the facultative progenitors of lung alveoli and serve as the surfactant-producing cells of air-breathing organisms. Although primary human AEC2s are ...difficult to maintain stably in cell cultures, recent advances have facilitated the derivation of AEC2-like cells from human pluripotent stem cells (hPSCs) in vitro. Here, we provide a detailed protocol for the directed differentiation of hPSCs into self-renewing AEC2-like cells that can be maintained for up to 1 year in culture as epithelial-only spheres without the need for supporting mesenchymal feeder cells. The month-long protocol requires recapitulation of the sequence of milestones associated with in vivo development of the distal lung, beginning with differentiation of cells into anterior foregut endoderm, which is followed by their lineage specification into NKX2-1
lung progenitors and then distal/alveolar differentiation to produce progeny that express transcripts and possess functional properties associated with AEC2s.
A hallmark of severe COVID-19 pneumonia is SARS-CoV-2 infection of the facultative progenitors of lung alveoli, the alveolar epithelial type 2 cells (AT2s). However, inability to access these cells ...from patients, particularly at early stages of disease, limits an understanding of disease inception. Here, we present an in vitro human model that simulates the initial apical infection of alveolar epithelium with SARS-CoV-2 by using induced pluripotent stem cell-derived AT2s that have been adapted to air-liquid interface culture. We find a rapid transcriptomic change in infected cells, characterized by a shift to an inflammatory phenotype with upregulation of NF-κB signaling and loss of the mature alveolar program. Drug testing confirms the efficacy of remdesivir as well as TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in alveolar cells. Our model system reveals cell-intrinsic responses of a key lung target cell to SARS-CoV-2 infection and should facilitate drug development.
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•iPSC-derived alveolar type 2 cells (iAT2s) are permissive to SARS-CoV-2 infection•SARS-CoV-2 induces an iAT2-intrinsic cytotoxicity and inflammatory response•iAT2s effectively model antiviral drug response and may be used for further drug development
Huang et al. show that human iPSC-derived alveolar type 2 cells (iAT2s) can be used to model COVID-19. They find that iAT2s in air-liquid interface culture are permissive to SARS-CoV-2 infection and show that SARS-CoV-2 induces a rapid inflammatory phenotype predominated by NF-κB signaling.
Differentiation of functional thyroid epithelia from pluripotent stem cells (PSCs) holds the potential for application in regenerative medicine. However, progress toward this goal is hampered by ...incomplete understanding of the signaling pathways needed for directed differentiation without forced overexpression of exogenous transgenes. Here we use mouse PSCs to identify key conserved roles for BMP and FGF signaling in regulating thyroid lineage specification from foregut endoderm in mouse and Xenopus. Thyroid progenitors derived from mouse PSCs can be matured into thyroid follicular organoids that provide functional secretion of thyroid hormones in vivo and rescue hypothyroid mice after transplantation. Moreover, by stimulating the same pathways, we were also able to derive human thyroid progenitors from normal and disease-specific iPSCs generated from patients with hypothyroidism resulting from NKX2-1 haploinsufficiency. Our studies have therefore uncovered the regulatory mechanisms that underlie early thyroid organogenesis and provide a significant step toward cell-based regenerative therapy for hypothyroidism.
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•BMP4 and FGF2 are necessary and sufficient to specify the thyroid lineage•Pathways regulating thyroid specification are conserved across species•Transplantation of ESC-derived thyroid cells regenerates in vivo function•Patient-specific thyroid progenitors can be differentiated from human iPSCs
The molecular pathways governing thyroid differentiation are poorly understood. Kurmann et al. show that BMP4 and FGF2 activate key pathways that drive thyroid specification in vivo and in vitro, enabling differentiation of mouse and human pluripotent stem cells into thyroid follicular cells that produce thyroid hormones and rescue mouse hypothyroidism.
Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and ...protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2-infected Calu-3 and A549
lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in
knockout A549
cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in
knockout A549
cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.
Claudin 18 (CLDN18) is a tight junction protein that is highly expressed in the lung. While mice lacking CLDN18 exhibit the expected loss of epithelial integrity in the lung, these animals also have ...unexpectedly large lungs. In this issue of the JCI, Zhou, Flodby, and colleagues reveal that the increased lung size of Cldn18-/- mice is the result of increased type 2 alveolar epithelial (AT2) cell proliferation. This increase in proliferation was shown to be driven by translocation of the transcriptional regulator Yes-associated protein (YAP) to the nucleus and subsequent induction of proliferative pathways. CLDN18-deficent mice also had increased frequency of lung adenocarcinomas. Together, the results of this study advance our understanding of the mechanisms that likely regulate homeostasis of the normal lung as well as promote the proliferative state of malignant cells found in lung adenocarcinomas thought to originate from AT2 cells.
The respiratory system, which includes the trachea, airways, and distal alveoli, is a complex multi-cellular organ that intimately links with the cardiovascular system to accomplish gas exchange. In ...this review and as members of the NIH/NHLBI-supported Progenitor Cell Translational Consortium, we discuss key aspects of lung repair and regeneration. We focus on the cellular compositions within functional niches, cell-cell signaling in homeostatic health, the responses to injury, and new methods to study lung repair and regeneration. We also provide future directions for an improved understanding of the cell biology of the respiratory system, as well as new therapeutic avenues.
Two populations of Nkx2-1+ progenitors in the developing foregut endoderm give rise to the entire postnatal lung and thyroid epithelium, but little is known about these cells because they are ...difficult to isolate in a pure form. We demonstrate here the purification and directed differentiation of primordial lung and thyroid progenitors derived from mouse embryonic stem cells (ESCs). Inhibition of TGFβ and BMP signaling, followed by combinatorial stimulation of BMP and FGF signaling, can specify these cells efficiently from definitive endodermal precursors. When derived using Nkx2-1GFP knockin reporter ESCs, these progenitors can be purified for expansion in culture and have a transcriptome that overlaps with developing lung epithelium. Upon induction, they can express a broad repertoire of markers indicative of lung and thyroid lineages and can recellularize a 3D lung tissue scaffold. Thus, we have derived a pure population of progenitors able to recapitulate the developmental milestones of lung/thyroid development.
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► ESCs differentiate to lung and thyroid lineages via Nkx2-1+ endodermal progenitors ► Combined BMP and FGF signaling are required to specify these Nkx2.1+ lineages ► Nkx2-1+ progenitors can be purified and expanded in culture ► The resulting cells can recellularize 3D lung scaffolds
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