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Single cell RNA-sequencing of day 15 cells derived from the directed differentiation of human iPSCs towards lung epithelium

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. Progress has been made in deriving lung epithelial cells from human induced pluripotent stem cells (iPSCs). However, little is known about the heterogeneity or genetic programs of the cells generated using these lung differentiation protocols. We engineered and differentiated NKX2-1GFP reporter iPSCs in vitro, recapitulating the major developmental milestones of lung development, to generate and isolate human primordial lung progenitors (day 15 of differentiation) that expresses NKX2-1 but are initially devoid of markers of differentiated lung lineages. To further characterize the cells generated in the lung directed differentiation protocol we performed single cell RNA-seq analysis of cells on day 15 of lung directed differentiation. We analyzed sorted NKX2-1GFP+ cells for the iPSC line C17 and cells from the iPSC line BU3.

ORGANISM(S): Homo sapiens

PROVIDER: GSE96106 | GEO | 2017/05/02

SECONDARY ACCESSION(S): PRJNA378848

REPOSITORIES: GEO

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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: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, 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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Single cell RNA-sequencing of day 15 cells derived from the directed differentiation of human iPSCs towards lung epithelium

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