Gene induction during differentiation of human pulmonary type II cells in vitro
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE3306 | GEO | 2005/09/21
SECONDARY ACCESSION(S): PRJNA92821
REPOSITORIES: GEO
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Project description:Studies of human fetal lung in explant culture and in isolated epithelial cells have demonstrated that both glucocorticoids and cyclic AMP promote differentiated alveolar type II cell phenotype as assessed by ultrastructural morphology and surfactant production. This project profiles changes in gene expression associated with hormone induced differentiation. Undifferentiated human fetal lung (13-20 wk) epithelial cells were cultured in serum-free medium (control) or with dexamethasone/8-Bromo cyclic AMP/isobutylmethylxanthine (DCI) to promote type II cell differentiation. RNA from five sets of experiments (10 samples) was evaluated using the U133A Affymetrix GeneChip set. Experiment Overall Design: Cells were isolated from 13 individual lungs and the cells from each were cultured in the absence (control) and presence of DCI dexamethasone (10 nM)/8-Br-cyclic AMP (0.1 mM)/isobutylxanthine for 72 h. RNA was isolated from the control and treated cells of each prep. For the 5 sets of microarray experiments (10 total chips, 2 for each control and treated pair), RNA from 2 of the cell preps was analysed as individual microarray comparisons. RNA from 11 cell preps was pooled for the other 3 experiments (4, 4, and 3 cell preps) to provide control and DCI-treated RNA pools.
2007-11-22 | E-GEOD-3306 | biostudies-arrayexpress
Project description:Normal lung function relies on mature function of alveolar type II cels, which have numerous functions including to regulate ion and fluid flux, produce immune molecules, and synthesize and secrete surfactant to stabilize air spaces. Differentiation of type II cells from precursor epithelial cells is accelerated by exposure of cultured cells to glucocorticoid and cAMP. In these studies we used DNA microarray analysis to identify genes of both fetal and adult type II cells that are regulated by glucocorticoid plus cAMP. In the first series of experiments we isolated epithelial cells from fetal human lung and cultured cells for 5 days with or without hormone treatment (dexamethasone plus 8-Br-cAMP plus isobutylmethylxanthine, DCI) and then performed DNA microarray analysis. In the second series of experiments, we isolated type II cells from adult human postmortem lung and cultured cells for 5 days with or without DCI and performed DNA microarray analysis.
2009-12-30 | E-GEOD-19699 | biostudies-arrayexpress
Project description:Alveolar epithelial type II (ATII)-like cells can be generated from murine embryonic stem cells (ESCs), although to date, no robust protocols applying specific differentiation factors are established. We hypothesized that the keratinocyte growth factor (KGF), an important mediator of lung organogenesis and primary ATII cell maturation and proliferation, together with dexamethasone, 8-bromoadenosine-cAMP, and isobutylmethylxanthine (DCI), which induce maturation of primary fetal ATII cells, also support the alveolar differentiation of murine ESCs. Here we demonstrate that the above stimuli synergistically potentiate the alveolar differentiation of ESCs as indicated by increased expression of the surfactant proteins (SP-) C and SP-B. This effect is most profound if KGF is supplied not only in the late stage, but at least also during the intermediate stage of differentiation. Our results indicate that KGF most likely does not enhance the generation of (mes)endodermal or NK2 homeobox 1 (Nkx2.1) expressing progenitor cells but rather, supported by DCI, accelerates further differentiation/maturation of respiratory progeny in the intermediate phase and maturation/proliferation of emerging ATII cells in the late stage of differentiation. Ultrastructural analyses confirmed the presence of ATII-like cells with intracellular composite and lamellar bodies. Finally, induced pluripotent stem cells (iPSCs) were generated from transgenic mice with ATII cell-specific lacZ reporter expression. Again, KGF and DCI synergistically increased SP-C and SP-B expression in iPSC cultures, and lacZ expressing ATII-like cells developed. In conclusion, ATII cell-specific reporter expression enabled the first reliable proof for the generation of murine iPSC-derived ATII cells. In addition, we have shown KGF and DCI to synergistically support the generation of ATII-like cells from ESCs and iPSCs. Combined application of these factors will facilitate more efficient generation of stem cell-derived ATII cells for future basic research and potential therapeutic application. 10 samples in total. mESCs at d8 of differentiation (Control) mESCs at d8 of differentiation with KGF treatment mESCs at d17 of differentiation (Control) mESCs at d17 of differentiation with KGF treatment mESCs at d17 of differentiation with DCI treatment mESCs at d17 of differentiation with KGF and DCI treatment mESCs at d24 of differentiation (Control) mESCs at d24 of differentiation with KGF treatment mESCs at d24 of differentiation with DCI treatment mESCs at d24 of differentiation with KGF and DCI treatment
2013-01-01 | E-GEOD-33308 | biostudies-arrayexpress
Project description:Alveolar epithelial type II (ATII)-like cells can be generated from murine embryonic stem cells (ESCs), although to date, no robust protocols applying specific differentiation factors are established. We hypothesized that the keratinocyte growth factor (KGF), an important mediator of lung organogenesis and primary ATII cell maturation and proliferation, together with dexamethasone, 8-bromoadenosine-cAMP, and isobutylmethylxanthine (DCI), which induce maturation of primary fetal ATII cells, also support the alveolar differentiation of murine ESCs. Here we demonstrate that the above stimuli synergistically potentiate the alveolar differentiation of ESCs as indicated by increased expression of the surfactant proteins (SP-) C and SP-B. This effect is most profound if KGF is supplied not only in the late stage, but at least also during the intermediate stage of differentiation. Our results indicate that KGF most likely does not enhance the generation of (mes)endodermal or NK2 homeobox 1 (Nkx2.1) expressing progenitor cells but rather, supported by DCI, accelerates further differentiation/maturation of respiratory progeny in the intermediate phase and maturation/proliferation of emerging ATII cells in the late stage of differentiation. Ultrastructural analyses confirmed the presence of ATII-like cells with intracellular composite and lamellar bodies. Finally, induced pluripotent stem cells (iPSCs) were generated from transgenic mice with ATII cell-specific lacZ reporter expression. Again, KGF and DCI synergistically increased SP-C and SP-B expression in iPSC cultures, and lacZ expressing ATII-like cells developed. In conclusion, ATII cell-specific reporter expression enabled the first reliable proof for the generation of murine iPSC-derived ATII cells. In addition, we have shown KGF and DCI to synergistically support the generation of ATII-like cells from ESCs and iPSCs. Combined application of these factors will facilitate more efficient generation of stem cell-derived ATII cells for future basic research and potential therapeutic application.
2013-01-01 | GSE33308 | 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.
2019-09-19 | 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.
2017-10-03 | GSE96642 | 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:ADCY9 encodes AC9, which is 1 of 9 transmembrane AC isoforms (AC1-9) that catalyze the synthesis of cyclic AMP from ATP and are regulated primarily by heterotrimeric G-protein-coupled receptors. We ectopically expressed AC9 in 393P cells and found that, relative to empty vector controls, the AC9 transfectants had higher cyclic AMP levels and were more migratory and invasive in Boyden chambers. We examined the prognostic value of an AC9-regulated gene expression signature derived from 393P_AC9 cells and 393P_vector cells. We probed a compendium of 1,586 clinically annotated human lung adenocarcinomas for the manifestation of the AC9 gene signature and found that the manifestation of the signature was correlated with a significantly shorter duration of survival.
2018-09-18 | GSE102168 | GEO
Project description:The cAMP response element binding protein (Creb) is a member of a leucine zipper transcription factor family that regulates gene expression primarily in response to the intracellular cAMP signalling pathway. Previous studies have shown Creb1-null mice suffer respiratory failure with lung atelectasis and a large reduction in Sftpd mRNA. Using a new line of Creb1-null mice we have further investigated Creb function in the developing mouse lung, focussing on differentiation of the airway epithelium. The lungs of Creb1-null fetal mice showed normal respiratory development until E17.5 when proximal and distal airways fail to inflate. Subsequent ultrastructural analysis of the lungs of E17.5 Creb1-null fetal mice revealed a defect in AEC differentiation with a reduction in proportions of type-II AECs and in particular, a very large reduction in type-I AECs. Furthermore, immunostaining for the proximal epithelial cell markers Scgb1a1 (also known as CC10) (Clara cells), Foxj1 (Ciliated cells), and CGRP (Neuroendocrine cells) showed delayed or defective proximal epithelial differentiation in Creb1-null fetal lungs. Quantitative real time PCR (qRT-PCR) analysis at E17.5 in Creb1-null fetal lungs showed differential expression of mRNAs for Creb/Atf1 subfamily members, surfactant-associated proteins, type-I AEC markers and proximal epithelial markers. Furthermore, whole-genome microarray analysis at E17.5 in Creb1-null fetal lungs has provided novel genes which will prove useful to further investigate Creb-mediated signalling in lung development. Together these results demonstrate that Creb plays a key role in determining cell lineages and differentiation of the developing lung epithelium.
2009-04-30 | E-MEXP-1295 | biostudies-arrayexpress
Project description:We had previously discovered that the transcription factor OVO-like 1 (OVOL1) was highly induced during trophoblast differentiation. In this study, we used an lentiviral shRNA strategy to decrease OVOL1 expression in BeWo trophoblast cells. Control cells were transduced with shRNAs targeting no known mammalian transcript (shCont). Following stimulation of differentiation (48h exposure to 8-bromo-cyclic adenosine monophosphate), a RNA-seq approach was used to determine global transcript differences in OVOL1-knockdown cells compared to control cells. Trophoblast cells transduced with control shRNAs were used as controls. Cells transduced with shRNAs targeting OVOL1 were used as treatment. All cells received 250 uM 8-bromo-cyclic adenosine monophosphate to stimulate differentiation. Three independent replicates of control and treatment groups were analyzed.
2015-08-01 | E-GEOD-66962 | biostudies-arrayexpress