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Global gene expression kinetics of early human lung development modeled by directed differentiation of human PSCs using an NKX2-1GFP iPSC reporter

ABSTRACT: It has been postulated that during human fetal development all cells of the lung epithelium derive from an embryonic endodermal NKX2-1+ precursor, however, this hypothesis has not been formally tested due to an inability to purify or track this theorized cell for detailed characterization. Here we engineer and developmentally differentiate NKX2-1GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate a human primordial lung progenitor that expresses NKX2-1 but is initially devoid of markers of differentiated lung lineages. As these progenitors move through the earliest moments of lung lineage specification from definitive endoderm they can be imaged in real time or isolated for time-series global transcriptomic profiling. We performed microarray analysis of 5 timepoints of human iPSC to lung directed differentiation compared to week 21 human fetal lung and Neural NKX2-1+ cell controls. These profiles indicate that evolutionarily conserved, stage-dependent developmental gene signatures are expressed in primordial human lung progenitors. Using a TALEN-targeted fluorescent reporter to purify iPSC-derived lung progenitors (C17 NKX2-1GFP) we analyzed cells at major developmental time points in vitro (undifferentiated iPSC, definitive endoderm, anterior foregut endoderm and sorted NKX2-1GFP+ and NKX2-1GFP- cells on day 15 and day 28 of the protocol). We also differentiated NXK2-1GFP iPSC in a neural protocol and isolated neural NKX2-1GFP+ cells. Approximately 90% pure human fetal lung epithelial cells from week 21 embryos were used as controls.

ORGANISM(S): Homo sapiens

PROVIDER: GSE83310 | GEO | 2017/05/02

SECONDARY ACCESSION(S): PRJNA325535

REPOSITORIES: GEO

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Project description:The derivation of self-renewing tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would shorten the time needed to engineer mature cell types in vitro and would have broad reaching implications for the field of regenerative medicine. Here we report the directed differentiation of human iPSCs into putative airway basal cells (“iBCs”), a population resembling the epithelial stem cell of lung airways. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato) we track and purify these cells over time, as they first emerge from iPSC-derived foregut endoderm as developmentally immature NKX2-1GFP+ lung progenitors which then augment a TP63 program during subsequent proximal airway epithelial patterning. These cells clonally proliferate, initially as NKX2-1GFP+/ TP63tdTomato+ immature airway progenitors that lack expression of the adult basal cell surface marker, NGFR. However, in response to primary basal cell media, NKX2-1GFP+/ TP63tdTomato+ cells upregulate NGFR and display the molecular and functional phenotype of airway basal stem cells, including the capacity to clonally self-renew or undergo multilineage ciliated and secretory epithelial differentiation in air-liquid interface cultures. iBCs and their differentiated progeny recapitulate several fundamental physiologic features of normal primary airway epithelial cells and model perturbations that characterize acquired and genetic airway diseases. In an asthma model of mucus metaplasia, the inflammatory cytokine IL-13 induced an increase in MUC5AC+ cells similar to primary cells. CFTR-dependent chloride flux in airway epithelium generated from cystic fibrosis iBCs or their syngeneic CFTR-corrected controls exhibited a pattern consistent with the flux measured in primary diseased and normal human airway epithelium, respectively. Finally, multiciliated cells generated from an individual with primary ciliary dyskinesia recapitulated the ciliary beat and ultrastructural defects observed in the donor. Thus, we demonstrate the successful de novo generation of a tissue-resident stem cell-like population in vitro from iPSCs, an approach which should facilitate disease modeling and future regenerative therapies for a variety of diseases affecting the lung airways.Single-cell RNA-Sequencing profiling of human iPSC-derived basal cells, airway epithelium compared to primary human basal cells and airway epithelium.

Project description:Lung adenocarcinoma is responsible for significant global mortality with limited effective treatments. Although some studies suggest that these tumors arise from alveolar epithelial type 2 cells (AEC2s), there is scant information regarding the early events that might occur in human AEC2s at the inception of oncogenesis. This limitation, is partially due to a lack of human model systems that recapitulate the initiation of oncogenesis in AEC2s. Unfortunately, primary AEC2s from patients are difficult to access in vivo or stably maintain in cell cultures. Hence, we sought to develop an in vitro system to model the early stages of oncogenesis utilizing human induced AEC2s (iAEC2s) generated through the directed differentiation of induced pluripotent stem cells (iPSCs). To this end, we selected a normal human iPSC line we have previously engineered to carry fluorochrome reporters targeted to lung epithelial-specific loci, NKX2-1GFP and SFTPCtdTomato that enable monitoring and purification of alveolar lung epithelial cells. To test the effects of adenocarcinoma oncogene induction in these cells, we targeted a third locus, AAVS1 using gene editing to engineer a doxycycline-inducible cassette encoding mutant KRASG12D, the most commonly found oncogene in lung adenocarcinomas. Successful induction of KRASG12D with doxycycline was demonstrated in both the targeted undifferentiated iPSCs as well as in the iAEC2s derived from these cells. We profiled the downstream effects of KRASG12D induction in iAEC2s, comparing dox vs vehicle exposed cells by cell counting, FACS for NKX2-1GFP/SFTPCtdTomato, RT-qPCR, deep proteomic and phosphoproteomic analyses, and scRNA-sequencing. Through this characterization, we found that induction of KRASG12D robustly activates MAPK signaling resulting in a shift of iAEC2s away from their mature alveolar program towards a distal lung epithelial progenitor phenotype. Successful modeling of lung adenocarcinoma with this model system has a variety of future applications, including testing unknown mechanisms for oncogenesis, discovery of novel biomarkers of disease, or development of new effective treatment methods through drug screening.

Project description:Lung adenocarcinoma is responsible for significant global mortality with limited effective treatments. Although some studies suggest that these tumors arise from alveolar epithelial type 2 cells (AEC2s), there is scant information regarding the early events that might occur in human AEC2s at the inception of oncogenesis. This limitation, is partially due to a lack of human model systems that recapitulate the initiation of oncogenesis in AEC2s. Unfortunately, primary AEC2s from patients are difficult to access in vivo or stably maintain in cell cultures. Hence, we sought to develop an in vitro system to model the early stages of oncogenesis utilizing human induced AEC2s (iAEC2s) generated through the directed differentiation of induced pluripotent stem cells (iPSCs). To this end, we selected a normal human iPSC line we have previously engineered to carry fluorochrome reporters targeted to lung epithelial-specific loci, NKX2-1GFP and SFTPCtdTomato that enable monitoring and purification of alveolar lung epithelial cells. To test the effects of adenocarcinoma oncogene induction in these cells, we targeted a third locus, AAVS1 using gene editing to engineer a doxycycline-inducible cassette encoding mutant KRASG12D, the most commonly found oncogene in lung adenocarcinomas. Successful induction of KRASG12D with doxycycline was demonstrated in both the targeted undifferentiated iPSCs as well as in the iAEC2s derived from these cells. We profiled the downstream effects of KRASG12D induction in iAEC2s, comparing dox vs vehicle exposed cells by cell counting, FACS for NKX2-1GFP/SFTPCtdTomato, RT-qPCR, deep proteomic and phosphoproteomic analyses, and scRNA-sequencing. Through this characterization, we found that induction of KRASG12D robustly activates MAPK signaling resulting in a shift of iAEC2s away from their mature alveolar program towards a distal lung epithelial progenitor phenotype, indicated by the upregulation of lung progenitor and proliferation markers (e.g. SOX9, ETV4, LEF1, TM4SF1, MKI67, and TOP2A) while maintaining NKX2-1 expression, at the expense of mature alveolar markers (e.g. SFTPC, SFTPB, NAPSA, and LPCAT1). Successful modeling of lung adenocarcinoma with this model system has a variety of future applications, including testing unknown mechanisms for oncogenesis, discovery of novel biomarkers of disease, or development of new effective treatment methods through drug screening.

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Global gene expression kinetics of early human lung development modeled by directed differentiation of human PSCs using an NKX2-1GFP iPSC reporter

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