Prospective isolation of NKX2-1–expressing human lung progenitors derived from pluripotent stem cells

Hawkins, Finn; Krämer, Philipp; Jacob, Anjali; Driver, Ian; Thomas, Dylan C.; McCauley, Katherine B.; Skvir, Nicholas; Crane, Ana M.; Kurmann, Anita; Hollenberg, Anthony N.; Nguyen, Sinead; Wong, Brandon G.; Khalil, Ahmad S.; Huang, Sarah X.L.; Guttentag, Susan H.; Rock, Jason R.; Shannon, John M.; Davis, Brian R.; Kotton, Darrell N.

doi:10.1172/jci89950

Cited by 184 publications

(333 citation statements)

References 61 publications

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“…To extend this approach to PSCs that do not contain an NKX2-1 knockin reporter, we next developed a strategy to identify AEC2-competent NKX2-1+ progenitors in the RUES2 cell line, which has a single SFTPC tdTomato reporter. We have previously shown that CD47 hi /CD26 lo day 15 cells are highly enriched in NKX2-1+ lung progenitors (Hawkins et al, 2017). In keeping with these findings, we observed that sorting for RUES2 CD47 hi /CD26 lo cells on day 15 identified the entirety of the SFTPC-competent population, enriching the SFTPC+ yield in the day 21 population approximately 4-fold over unsorted cells (Figure S2C).…”

Section: Resultsmentioning

confidence: 99%

“…BU3 and C17 iPSC lines carrying the NKX2-1 GFP reporter were obtained from our prior studies (Hawkins et al, 2017). These lines were derived from a normal donor (BU3) (Kurmann et al, 2015) and an individual with cystic fibrosis (C17) carrying a published compound heterozygous CFTR genotype (Crane et al, 2015), respectively.…”

Section: Star+methodsmentioning

confidence: 99%

“…The TALENs and donor vector plasmids were co-transfected into the following PSC lines: RUES2, C17 NKX2-1 GFP , and BU3 NKX2-1 GFP (Hawkins et al, 2017) using a lipofectamine based transfection protocol. Each line was plated onto a mitomycin C-inactivated DR4 mouse embryonic fibroblast (MEF) feeder layer and cultured in human iPSC media (WiCell) in a 6 well plate.…”

Section: Star+methodsmentioning

confidence: 99%

“…Several groups have reported differentiation protocols that result in lung progenitors expressing the transcriptional regulator NKX2-1 as well as more differentiated populations containing cells expressing alveolar marker genes such as SFTPC (Huang et al, 2014; Gotoh et al, 2014; Dye et al, 2015; Hawkins et al, 2017). Next logical steps for the field include comparison of iPSC-derived putative AEC2s (iAEC2s) to primary controls, assessment of their maturation relative to the developing human lung, and evaluation of their ability to model human alveolar disease in vitro.…”

Section: Introductionmentioning

confidence: 99%

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Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells

et al. 2017

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SUMMARY 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.

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Section: Resultsmentioning

confidence: 99%

Section: Star+methodsmentioning

confidence: 99%

Section: Star+methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells

et al. 2017

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“…However, we note that, in general, these methods generate a very low percentage of NKX2-1 + lung progenitors and very few specific bronchiolar, or alveolar, lineage cells (see ). Moreover, there is extensive variability between hPSC cell lines in terms of their differentiation competence (Hawkins et al, 2017). …”

Section: Studies Of Human Lung Development Using Pluripotent Stem Cellsmentioning

confidence: 99%

Human lung development: recent progress and new challenges

2018

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Recent studies have revealed biologically significant differences between human and mouse lung development, and have reported new in vitro systems that allow experimental manipulation of human lung models. At the same time, emerging clinical data suggest that the origins of some adult lung diseases are found in embryonic development and childhood. The convergence of these research themes has fuelled a resurgence of interest in human lung developmental biology. In this Review, we discuss our current understanding of human lung development, which has been profoundly influenced by studies in mice and, more recently, by experiments using in vitro human lung developmental models and RNA sequencing of human foetal lung tissue. Together, these approaches are helping to shed light on the mechanisms underlying human lung development and disease, and may help pave the way for new therapies.

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Advanced In Vitro Lung Models for Drug and Toxicity Screening: The Promising Role of Induced Pluripotent Stem Cells

Moreira¹,

Müller

Costa³

et al. 2021

Advanced Biology

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Respiratory diseases are among the global leading causes of morbidity and mortality. Acute conditions arising from infection, such as pneumonia and tuberculosis, affect millions of people worldwide, the latter being the most common lethal infectious disease with 1.4 million annual deaths. [1] Upon infection, the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), which caused a worldwide pandemic, leads to the clinical picture of the coronavirus disease-2019 (COVID-19) with possibly severe and life-threatening progression. Lung cancer is the most frequently diagnosed malignancy and the main cause of cancer-related death, [2] with markedly low survival rates especially when the diagnosis is performed at an advanced state of the disease. [3] Moreover, chronic respiratory diseases (CRDs), including chronic obstructive pulmonary disease (COPD), asthma, and interstitial lung disease (ILD), have consistently received less attention in comparison to other non-communicable diseases, continuing to exert a considerable socioeconomic impact. [4,5] Risk factors for the development of respiratory diseases include, for example, tobacco use and second-hand smoke, as well as exposure to air-pollutants, which has been accentuated with the widespread use of fossil fuels. [4] It is clear that a great number of these risks can be mitigated by lifestyle changes, promoted by anti-tobacco campaigns already in place at a global scale and by the use of renewable, clean energy sources, and two recent studies indicate that the age-standardized incidence and prevalence of CRDs has decreased from 1990 to 2017. [4,5] However, the risk of developing CRDs, particularly COPD, appears to increase steeply with age; [4,5] most strikingly, these diseases were the third leading cause of death in 2017 [4] and potentially in 2020. [6] In addition to microbial, environmental, and lifestyle-related factors, a large number of lung diseases have a genetic origin. [7] Cystic fibrosis (CF), for example, is an incurable hereditary disorder known to originate from different mutations in the gene coding for the CF transmembrane conductance regulator (CFTR), an ion transporter, resulting in severe alterations in pulmonary physiology that culminate in impaired lung function, respiratory distress and, ultimately, death. [8,9]

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Prospective isolation of NKX2-1–expressing human lung progenitors derived from pluripotent stem cells

Cited by 184 publications

References 61 publications

Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells

Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells

Human lung development: recent progress and new challenges

Advanced In Vitro Lung Models for Drug and Toxicity Screening: The Promising Role of Induced Pluripotent Stem Cells

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