A novel human fetal lung-derived alveolar organoid model reveals mechanisms of surfactant protein C maturation relevant to interstitial lung disease
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ABSTRACT:
PROVIDER: PRJNA994820 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25276734_1.fastq.gz | Fastqsanger.gz | |||
| SRR25276734_2.fastq.gz | Fastqsanger.gz | |||
| SRR25276735_1.fastq.gz | Fastqsanger.gz | |||
| SRR25276735_2.fastq.gz | Fastqsanger.gz | |||
| SRR25276736_1.fastq.gz | Fastqsanger.gz |
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Project description:During human lung development the distal tip epithelial cells act as multipotent progenitors. From ~15 pcw (post conception weeks) the human tip epithelium retains progenitor marker expression, but also upregulates markers of the AT2 lineage. Immature AT2 cells appear in the tissue from 17 pcw. We have recently shown that 16-22 pcw epithelial tip cells can be expanded as organoids and differentiated to AT2 cells. However, the differentiated AT2 cells were not proliferative, limiting their use for functional studies and genetic manipulation. We have now developed a highly robust, efficient, and scalable culture condition (AT2 medium) that induces the differentiation of 16-22 pcw fetal lung tip cells into mature AT2 cells which grow as expandable 3D organoids. In this study, we characterise the fetal-derived AT2 cells, showing that they are stable over long-term passaging, efficiently process and secrete surfactant, and can differentiate into AT1 cells in vitro and in mouse lung transplantation assays. Forward genetic screen identifies candidate effectors of SFTPC trafficking which we validate using CRISPR interference (CRISPRi) in the AT2 organoids. We show that the trafficking of SFTPC requires ubiquitination by HECT domain E3 ligases, particularly Itch, and that their depletion phenocopies the pathological redistribution seen for the SFTPCI73T variant.
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Project description:Organoid models have been one of the most exciting advancements in stem cell research of the past decade. Here we describe a strategy for directed differentiation of human pluripotent stem cells into distal lung organoids that can be used to model interstitial lung disease, viral infection and human endoderm and lung development. This protocol entails five stages that recapitulate lung development. hPSCs are sequentially specified to definitive endoderm, anterior foregut endoderm, ventral anterior foregut endoderm, lung bud organoids, and finally branching lung organoids that can be maintained, while progressively maturing up to the a stage consistent with the second trimester of human gestation, for more than 180 days. This protocol is conducted in defined, serum-free conditions and does not require lineage-specific reporters or cell purification. We also provide a protocol for the generation of single cell suspensions for single cell RNAseq, and for clearing and 3D light sheet fluorescence imaging.
2023-10-17 | GSE215825 | GEO
Project description:This study uses microarray analyses to examine gene expression profiles for Mycobacterium tuberculosis (Mtb) induced by exposure in vitro to bovine lung surfactant preparations that vary in apoprotein content: (i) whole lung surfactant (WLS) containing the complete mix of endogenous lipids and surfactant proteins (SP)-A, -B, -C, and -D; (ii) extracted lung surfactant (CLSE) containing lipids plus SP-B and -C; (iii) column-purified surfactant lipids (PPL) containing no apoproteins, and (iv) purified human SP-A. Exposure to WLS evoked a multitude of transcriptional responses in Mtb, with 52 genes up-regulated and 23 genes down-regulated at 30 min exposure, plus 146 genes up-regulated and 27 genes down-regulated at 2 h. Notably, WLS rapidly induced several membrane-associated lipases that presumptively act on surfactant lipids as substrates, and a large number of genes involved in the synthesis of phthiocerol dimycocerosate (PDIM), a cell wall component known to be important in macrophage interactions and Mtb virulence. Exposure of Mtb to CLSE, PPL, or purified SP-A caused a substantially weaker transcriptional response (≤ 20 genes were induced) suggesting that interactions among multiple lipid-protein components of WLS may contribute to its effects on Mtb transcription.
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2011-04-19 | E-GEOD-21411 | biostudies-arrayexpress
Project description:This study uses microarray analyses to examine gene expression profiles for Mycobacterium tuberculosis (Mtb) induced by exposure in vitro to bovine lung surfactant preparations that vary in apoprotein content: (i) whole lung surfactant (WLS) containing the complete mix of endogenous lipids and surfactant proteins (SP)-A, -B, -C, and -D; (ii) extracted lung surfactant (CLSE) containing lipids plus SP-B and -C; (iii) column-purified surfactant lipids (PPL) containing no apoproteins, and (iv) purified human SP-A. Exposure to WLS evoked a multitude of transcriptional responses in Mtb, with 52 genes up-regulated and 23 genes down-regulated at 30 min exposure, plus 146 genes up-regulated and 27 genes down-regulated at 2 h. Notably, WLS rapidly induced several membrane-associated lipases that presumptively act on surfactant lipids as substrates, and a large number of genes involved in the synthesis of phthiocerol dimycocerosate (PDIM), a cell wall component known to be important in macrophage interactions and Mtb virulence. Exposure of Mtb to CLSE, PPL, or purified SP-A caused a substantially weaker transcriptional response (⤠20 genes were induced) suggesting that interactions among multiple lipid-protein components of WLS may contribute to its effects on Mtb transcription. Whole lung surfactant (WLS) was obtained by centrifugation of lavage fluid (0.15M NaCl) from the intact lungs of freshly killed calves (ONY, Inc, Amherst, NY). Centrifugation was done at 12,000 x g at room temperature to pellet the active large aggregate surfactant fraction. CLSE was prepared by extraction of WLS into chloroform-methanol by the method of Bligh and Dyer (1959), as described in our prior work (Notter et al. 2002). CLSE is conceptually identical to the substance of the clinical surfactant Infasurf®. The composition of CLSE by weight was approximately 94% phospholipid, 4.5% cholesterol, and 1.5% protein (a mixture of SP-B and SP-C) as defined previously (Notter et al. 2002). Mixed lung surfactant phospholipids (PPL) were isolated from CLSE by column chromatography with an elution solvent of 1:1 (v/v) chloroform-methanol plus 5% 0.1 N HCl. Two passes through a Sephadex LH-20 column (Pharmacia-LKB Biotechnology, Piscataway, NJ) separated phospholipids away from hydrophobic surfactant apoproteins and neutral lipids. Acid remaining in PPL fractions was removed by a second chloroform-methanol extraction. Final PPL had a protein content below the limits of detection of both the Folin-phenol assay of Lowry et al. (1951) and the amido black assay of Kaplin and Pedersen (1989) modified by the addition of 15% sodium dodecyl sulfate (SDS) to allow accurate quantitation in the presence of lipid. Human SP-A was a gift from Dr. J. R. Wright at Duke University, and was isolated from bronchoalveolar lavage from patients with alveolar proteinosis as described previously (Wright et al. 1987). For exposure studies with Mtb, amounts of WLS, CLSE, and PPL used per experiment were 3.3 mg in a total volume of 4 ml. In preliminary studies, this concentration was shown to induce a transcriptional response in Mtb without affecting viability after a 2 h treatment as determined by serial dilutions compared to untreated Mtb. SP-A was studied at a level of 0.165 mg based on its putative concentration of approximately 5% in endogenous surfactant. To determine the effects of lung surfactant on the transcriptional response of Mtb, preparations of WLS, CLSE, or PPL (in 650 µl PBS) were initially coated onto the bottom surface of a 25 cm2 cell culture flask for 30 min at 37oC to mimic a surfactant layer. A minimum of two independent biological replicates were studied for each surfactant preparation. Purified human SP-A was also examined for its effects on Mtb transcription, and phosphate buffered saline (PBS) was used as a âno treatmentâ control. In experiments with SP-A (in 650 µl PBS), the pure apoprotein was either pre-coated on the flask as done with the surfactant preparations, or was directly added and incubated with Mtb during study. Mtb was added to the flask in 3.35 ml of DMEM (supplemented with 2% FCS and 5 mM HEPES) and exposed to coated surfactant/SP-A or PBS control for 30 min or 2h at 37oC, followed by RNA isolation as detailed by Rohde et al. (2008). GTC lysis/stabilization buffer was added, and pelleted Mtb were washed and then digested with lysozyme. TRIzol (Invitrogen) was added, and organisms were disrupted using glass beads in a Mini-Bead Beater (BioSpec Products, Inc. Bartlesville, OK). After extraction of Mtb lysates with chloroform, ethanol was added to the aqueous phase before loading samples onto RNeasy mini columns (Qiagen, Valencia, CA). Subsequent steps were then performed according to the manufacturerâs instructions. Following elution with RNase-free water, the RNA samples were treated with DNase (Turbo DNAfree, Applied Biosystems, Foster City, CA), and stored at -70oC until analysis. The MessageAmpTMII-Bacteria system (Applied Biosystems) was used to amplify mycobacterial RNA for all samples [16]. Total RNA was reverse transcribed to yield double-stranded amino-allyl modified aRNA, which was labeled with Alexa Fluor 555 and Alexa Fluor 647 (Invitrogen). Excess reactive dyes were removed with a Mega Clear purification kit (Applied Biosystems). Next, Alexa-labeled aRNA from paired samples was dried using a Speedvac, and re-suspended in hybridization buffer (5x SSC, 25% formamide, 0.15% SDS, and 25 μg salmon sperm DNA). The mixed samples were denatured by being heated to 95oC for and then cooled to 50oC prior to loading them onto microarrays. The arrays were hybridized for approximately 15 h at 45oC, washed and then dried by centrifugation. Fluorescence intensity data from each array were collected with a GenePix 4000B scanner (Axon Instruments, Union City, CA) using GenePixPro3.0 software (Axon Instruments). Image analysis, i.e. spot identification, background measurements, quality assessment and flagging were conducted with Imagene software (Version 6.0, BioDiscovery, Inc., El Segundo, CA). Poor quality spots were filtered and removed from the analysis. Subsequent normalization, statistical analysis (i.e. t-test), and visualization of array data was conducted using Genespring (Version 7, Agilent Technologies, Palo Alto, CA).
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