Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA325535 | ENA |
REPOSITORIES: ENA
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Project description: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.
2017-05-02 | GSE83310 | GEO
Project description: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.
2017-05-02 | GSE96106 | GEO
Project description: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. We used microarrays to detail the global transcriptomes of mouse embryonic stem cells differentiated in vitro into putative thyroid vs lung epithelial lineages using serum-free media supplemented with either BMP4+FGF2 (thyroid media) or BMP4+Wnt3a (lung media.) Differentiated cells carried an Nkx2-1mCherry knock-in reporter to allow sorting of mCherry + vs - cells in each condition on day 14 of differentiation for global transcriptomic profiling.
2017-10-31 | GSE92916 | GEO
Project description:We perform transcriptomic profiling of a directed differentiation protocol for generating distal lung epithelial cells and alveolar type II epithelial cells (AEC2s) from pluripotent stem cells (PSCs) using bi-fluorescently targeted iPSC cell line BU3-NGST. On day 33 of distal lung differentiation, we sorted SFTPCtdTomato/NKX2-1GFP double positive cells and NKX2-1GFP single positive cells for Fluidigm single cell RNA sequencing analysis. We find canonical AEC2s genes expressed in both sorted populations but at a higher level in the SFTPCtdTomato/NKX2-1GFP double positive population. We also find genes associated with cellular proliferation to be enriched in the NKX2-1GFP single positive population, suggestive of a less mature but more proliferative subset of PSC-derived AEC2s.
2021-06-01 | GSE152654 | GEO
Project description:Global gene expression of human iPSC directed differentation towards smooth muscle cells using an Acta2eGFP human iPSC reporter line
| PRJNA548124 | ENA
Project description:We perform time-series global transcriptomic profiling of a directed differentiation protocol for generating alveolar epithelial type II cells (AEC2s) from pluripotent stem cells (PSCs). We analyzed 3 different timepoints in the RUES2 differentiation: 1) Day 0 undifferentiated cells, 2) Day 15 lung progenitors highly enriched in NKX2-1+ cells by CD47hi/CD26lo sorting, and 3) the outgrowth of these purified progenitors in 3D culture sorted again on Day 35 based on SFTPCtdTomato+ and SFTPCtdTomato- gating. For comparison to primary cells, we simultaneously sequenced RNA from purified primary fetal (21 week gestation) distal alveolar epithelial progenitors and purified adult human (HTII-280 sorted) AEC2s. In order to evaluate the effect of cell culture on primary fetal alveolar cells, parallel aliquots of the fetal cells were also exposed to 4 days of culture in DCI media. We find that AEC2 maturation involves downregulation of Wnt signaling activity, and that the highest differentially expressed transcripts in iPSC-derived AEC2s encode genes associated with lamellar body and surfactant biogenesis.
2019-09-19 | GSE131768 | GEO
Project description:We perform time-series global transcriptomic profiling of a directed differentiation protocol for generating alveolar epithelial type II cells (AEC2s) from pluripotent stem cells (PSCs). We analyzed 3 different timepoints in the RUES2 differentiation: 1) Day 0 undifferentiated cells, 2) Day 15 lung progenitors highly enriched in NKX2-1+ cells by CD47hi/CD26lo sorting, and 3) the outgrowth of these purified progenitors in 3D culture sorted again on Day 35 based on SFTPCtdTomato+ and SFTPCtdTomato- gating. For comparison to primary cells, we simultaneously sequenced RNA from purified primary fetal (21 week gestation) distal alveolar epithelial progenitors and purified adult human (HTII-280 sorted) AEC2s. In order to evaluate the effect of cell culture on primary fetal alveolar cells, parallel aliquots of the fetal cells were also exposed to 4 days of culture in DCI media. We find that AEC2 maturation involves downregulation of Wnt signaling activity, and that the highest differentially expressed transcripts in iPSC-derived AEC2s encode genes associated with lamellar body and surfactant biogenesis.
2019-09-24 | GSE137811 | GEO
Project description:We perform time-series global transcriptomic profiling of a directed differentiation protocol for generating alveolar epithelial type II cells (AEC2s) from pluripotent stem cells (PSCs). We analyzed 3 different timepoints in the RUES2 differentiation: 1) Day 0 undifferentiated cells, 2) Day 15 lung progenitors highly enriched in NKX2-1+ cells by CD47hi/CD26lo sorting, and 3) the outgrowth of these purified progenitors in 3D culture sorted again on Day 35 based on SFTPCtdTomato+ and SFTPCtdTomato- gating. For comparison to primary cells, we simultaneously sequenced RNA from purified primary fetal (21 week gestation) distal alveolar epithelial progenitors and purified adult human (HTII-280 sorted) AEC2s. In order to evaluate the effect of cell culture on primary fetal alveolar cells, parallel aliquots of the fetal cells were also exposed to 4 days of culture in DCI media. We find that AEC2 maturation involves downregulation of Wnt signaling activity, and that the highest differentially expressed transcripts in iPSC-derived AEC2s encode genes associated with lamellar body and surfactant biogenesis.
2017-10-03 | GSE96642 | GEO
Project description:Anterior foregut endoderm (AFE) gives rise to many tissue types of interest for therapeutic research including the esophagus, salivary glands, lung, thymus, parathyroid and thyroid. Despite its importance, only few reports describe the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation. Here, we describe a novel protocol to derive a subdomain of AFE, identified by expression of Pax9, from PSCs using small molecules and chemically defined conditions. Generation of a reporter PSC line allows isolation and characterization of Pax9+ AFE cells. When transplanted in vivo, Pax9+ AFE can form several distinct types of complex anterior foregut epithelia including mucosal glands and stratified squamous epithelium. Finally, we show that the directed differentiation protocol can be used to generate AFE from DiGeorge Syndrome patient-specific human induced PSCs, thus creating a platform to produce anterior foregut derivatives for therapy and to enable the study of disorders of the AFE. Total RNA obtained from FACS purified from in vitro dervied mouse definitive endoderm, anterior foregut and ES cells. AFE cells were derived from a 129X1/SvJ background, DE cells from 129X1/SvJ x 129S1/SV-+p+Tyr- cKitlSl-J/+ (R1 ES cells) and non reporter ES cells from a 129P2/OlaHsd background.
2013-04-15 | E-GEOD-42139 | biostudies-arrayexpress
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.
2022-02-15 | PXD019240 | Pride