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1.
Nature ; 604(7904): 120-126, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35355013

RESUMO

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.


Assuntos
Bronquíolos , Furões , Células-Tronco Multipotentes , Alvéolos Pulmonares , Animais , Bronquíolos/citologia , Linhagem da Célula , Humanos , Pulmão/patologia , Camundongos , Células-Tronco Multipotentes/citologia , Alvéolos Pulmonares/citologia , Doença Pulmonar Obstrutiva Crônica
2.
Development ; 144(21): 3879-3893, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28947536

RESUMO

The in vitro-directed differentiation of pluripotent stem cells (PSCs) through stimulation of developmental signaling pathways can generate mature somatic cell types for basic laboratory studies or regenerative therapies. However, there has been significant uncertainty regarding a method to separately derive lung versus thyroid epithelial lineages, as these two cell types each originate from Nkx2-1+ foregut progenitors and the minimal pathways claimed to regulate their distinct lineage specification in vivo or in vitro have varied in previous reports. Here, we employ PSCs to identify the key minimal signaling pathways (Wnt+BMP versus BMP+FGF) that regulate distinct lung- versus thyroid-lineage specification, respectively, from foregut endoderm. In contrast to most previous reports, these minimal pathways appear to be evolutionarily conserved between mice and humans, and FGF signaling, although required for thyroid specification, unexpectedly appears to be dispensable for lung specification. Once specified, distinct Nkx2-1+ lung or thyroid progenitor pools can now be independently derived for functional 3D culture maturation, basic developmental studies or future regenerative therapies.


Assuntos
Padronização Corporal , Diferenciação Celular , Pulmão/citologia , Pulmão/embriologia , Células-Tronco Pluripotentes/citologia , Transdução de Sinais , Glândula Tireoide/citologia , Animais , Biomarcadores/metabolismo , Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem da Célula , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Endoderma/citologia , Endoderma/metabolismo , Células Epiteliais/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Reprodutibilidade dos Testes , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Glândula Tireoide/embriologia , Transcriptoma/genética , Proteínas Wnt/metabolismo
3.
Cell Syst ; 15(4): 307-321.e10, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38508187

RESUMO

Receptor-mediated signaling plays a central role in tissue regeneration, and it is dysregulated in disease. Here, we build a signaling-response map for a model regenerative human tissue: the airway epithelium. We analyzed the effect of 17 receptor-mediated signaling pathways on organotypic cultures to determine changes in abundance and phenotype of epithelial cell types. This map recapitulates the gamut of known airway epithelial signaling responses to these pathways. It defines convergent states induced by multiple ligands and diverse, ligand-specific responses in basal cell and secretory cell metaplasia. We show that loss of canonical differentiation induced by multiple pathways is associated with cell-cycle arrest, but that arrest is not sufficient to block differentiation. Using the signaling-response map, we show that a TGFB1-mediated response underlies specific aberrant cells found in multiple lung diseases and identify interferon responses in COVID-19 patient samples. Thus, we offer a framework enabling systematic evaluation of tissue signaling responses. A record of this paper's transparent peer review process is included in the supplemental information.


Assuntos
Células Epiteliais , Pulmão , Humanos , Epitélio , Células Epiteliais/metabolismo , Pulmão/metabolismo , Diferenciação Celular/genética , Transdução de Sinais/genética
4.
bioRxiv ; 2022 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-36597531

RESUMO

Receptor-mediated signaling plays a central role in tissue regeneration, and it is dysregulated in disease. Here, we build a signaling-response map for a model regenerative human tissue: the airway epithelium. We analyzed the effect of 17 receptor-mediated signaling pathways on organotypic cultures to determine changes in abundance and phenotype of all epithelial cell types. This map recapitulates the gamut of known airway epithelial signaling responses to these pathways. It defines convergent states induced by multiple ligands and diverse, ligand-specific responses in basal-cell and secretory-cell metaplasia. We show that loss of canonical differentiation induced by multiple pathways is associated with cell cycle arrest, but that arrest is not sufficient to block differentiation. Using the signaling-response map, we show that a TGFB1-mediated response underlies specific aberrant cells found in multiple lung diseases and identify interferon responses in COVID-19 patient samples. Thus, we offer a framework enabling systematic evaluation of tissue signaling responses.

5.
Curr Protoc Stem Cell Biol ; 45(1): e51, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-30040246

RESUMO

New protocols to efficiently generate functional airway epithelial organoids from human pluripotent stem cells (PSCs) would represent a major advance towards effective disease modeling, drug screening and cell based therapies for lung disorders. This unit describes an approach using stage-specific signaling pathway manipulation to differentiate cells to proximal airway epithelium via key developmental intermediates. Cells are directed via definitive endoderm (DE) to anterior foregut, and then specified to NKX2-1+ lung epithelial progenitors. These lung progenitors are purified using cell surface marker sorting and replated in defined culture conditions to form three-dimensional, epithelial-only airway organoids. This directed differentiation approach using serum-free, defined media also includes protocols for evaluation of DE induction, intracellular FACS analysis of NKX2-1 specification efficiency and enrichment, and approaches for characterization and expansion of airway organoids. Taken together, this represents an efficient and reproducible approach to generate expandable airway organoids from human PSCs for use in numerous downstream applications. © 2018 by John Wiley & Sons, Inc.


Assuntos
Células Epiteliais/citologia , Pulmão/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Engenharia Tecidual/métodos , Antígenos CD/metabolismo , Diferenciação Celular , Separação Celular , Sistema Digestório/citologia , Endoderma/citologia , Fluorescência , Humanos
6.
Stem Cell Reports ; 10(5): 1579-1595, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29657097

RESUMO

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.


Assuntos
Epitélio/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Pulmão/citologia , Análise de Célula Única , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula , Plasticidade Celular , Epitélio/ultraestrutura , Genes Reporter , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Cinética , Camundongos , Secretoglobinas/metabolismo , Análise de Sequência de RNA , Solubilidade , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Fatores de Tempo , Transcriptoma/genética , Via de Sinalização Wnt
7.
Cell Stem Cell ; 20(6): 844-857.e6, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28366587

RESUMO

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.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/metabolismo , Mucosa Respiratória/metabolismo , Fator Nuclear 1 de Tireoide/metabolismo , Via de Sinalização Wnt , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Mucosa Respiratória/citologia
8.
J Clin Invest ; 127(6): 2277-2294, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28463226

RESUMO

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.


Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Separação Celular , Células Cultivadas , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Fator Nuclear 1 de Tireoide , Transcriptoma
9.
Cell Stem Cell ; 21(4): 472-488.e10, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28965766

RESUMO

Lung alveoli, which are unique to air-breathing organisms, have been challenging to generate from pluripotent stem cells (PSCs) in part because there are limited model systems available to provide the necessary developmental roadmaps for in vitro differentiation. Here we report the generation of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, from human PSCs. Using multicolored fluorescent reporter lines, we track and purify human SFTPC+ alveolar progenitors as they emerge from endodermal precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells form monolayered epithelial "alveolospheres" in 3D cultures without the need for mesenchymal support, exhibit self-renewal capacity, and display additional AEC2 functional capacities. Footprint-free CRISPR-based gene correction of PSCs derived from patients carrying a homozygous surfactant mutation (SFTPB121ins2) restores surfactant processing in AEC2s. Thus, PSC-derived AEC2s provide a platform for disease modeling and future functional regeneration of the distal lung.


Assuntos
Diferenciação Celular , Células Epiteliais/citologia , Células-Tronco Pluripotentes/citologia , Alvéolos Pulmonares/citologia , Sequência de Bases , Linhagem Celular , Proliferação de Células , Autorrenovação Celular , Separação Celular , Células Epiteliais/ultraestrutura , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Pneumopatias/patologia , Modelos Biológicos , Alvéolos Pulmonares/ultraestrutura , Surfactantes Pulmonares/metabolismo , Fator Nuclear 1 de Tireoide/metabolismo , Fatores de Tempo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt
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