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Global transcriptomic analyses of pluripotent stem cell-derived type II alveolar epithelial-like cells identified by ABCA3:GFP and SFTPCtdTomato bi-fluorescent reporters

ABSTRACT: Global transcriptomic analyses of pluripotent stem cell-derived type II alveolar epithelial-like cells identified by ABCA3:GFP and SFTPCtdTomato bi-fluorescent reporters

PROVIDER: PRJNA639975 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12032850_1.fastq.gz	Fastqsanger.gz
	SRR12032850_2.fastq.gz	Fastqsanger.gz
	SRR12032851_1.fastq.gz	Fastqsanger.gz
	SRR12032851_2.fastq.gz	Fastqsanger.gz
	SRR12032852_1.fastq.gz	Fastqsanger.gz

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Global transcriptomic analyses of pluripotent stem cell-derived type II alveolar epithelial-like cells identified by ABCA3:GFP and SFTPCtdTomato bi-fluorescent reporters

Project description:We performed transcriptomic profiling of cells derived from human pluripotent stem cells (PSCs) using distal lung directed differentiation protocol previously described to generate alveolar type II epithelial-like cells (iAEC2s). We used the RUES2-STAG human embryonic stem cell line containing bi-fluorescently targeted ABCA3:GFP knock-in fusion reporter and SFTPCtdTomato knock-in reporter to potentially identify heterogenous iAEC2 populations and determine their gene expression profiles. On day 36 of differentiation, we sorted SFTPCtdTomato/ABCA3:GFP double positive (DP), ABCA3:GFP single positive (SP) and reporter double negative (DN) cells for transcriptomic analysis by bulk RNA sequencing. We found DN cells to be enriched in non-lung endodermal cells. Comparison between the reporter positive iAEC2 populations revealed high transcriptomic similarity with expression of canonical AEC2 transcripts in both the DP and SP cells. Analyses of differentially expressed transcript between the DP and SP cells revealed higher levels of surfactant and lamellar body-related transcripts in the DP cells. Moreover, we find enrichment of proliferative markers associated with cellular proliferation indicating the ABCA3:GFP single positive iAEC2s to potentially be a more proliferative subset of iAEC2s.

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Bulk RNA sequencing of wildtype and ABCA3 mutant human iPSC-derived mutant containing the ABCA3:GFP fusion reporter

Project description:We performed transcriptomic profiling of induced pluripotent stem cell (iPSC)-derived iAEC2s containing wildtype, E690K homozygous ABCA3 mutantion, and W308R homozygous ABCA3 mutation. We first performed CRISPR-Cas9 gene editing on a wildtype human iPSC line (BU3) to engineer a GFP reporter bi-allelically targeted at the stop codon of the endigenous ABCA3 locus, thus generating an ABCA3:GFP fusion reporter line (as previous published iin Sun. et al, 2021). Next we utilized CRISPR-Cas9 gene-mutagenesis to introduce two homozygous ABCA3 mutations (E690K and W308R), resulting in two, new ABCA3 mutant iPSC lines containg the ABCA3:GFP fusion reporter, totally a total of 3 ABCA3:GFP fusion iPSC lines (wildtype, E690K-AG, W308R-AG). Following triplicate distal lung directed differentiations of all 3 ABCA3:GFP fusion iPSC lines we sorted the ABCA3:GFP positive iAEC2s on day 43 for transcriptomic analyses.

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Generation of ABCA3:GFP expressing type II alveolar epithelial-like cells from human pluripotent stem cells

Project description:We perform transcriptomic profiling of cells derived from human induced pluripotent stem cells (iPSCs) using distal lung directed differentiation protocol previously described to generate alveolar type II epithelial-like cells (iAEC2s). We use the BU3 ABCA3:GFP iPSC line containing a GFP knock-in fluorescent reporter for the canonical AEC2 gene, ABCA3, to sort out pure populations of GFP positive and negative cells on day 31 of distal lung differentiation for bulk RNA sequencing analysis. We find canonical AEC2s transcripts as well as lamellar body-associated transcripts to be enriched in the ABCA3:GFP positive population suggestive of enrichment of AEC2 program. In contrast, we find non-lung endodermal lineages to be enriched in the ABCA3:GFP negative cells. Combined, these findings indicate the reliability of the ABCA3:GFP reporter in enriching iAEC2s in our in vitro distal lung directed differentiation protocol.

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Heterogeneity in human iPSC-derived alveolar epithelial type II cells revealed with ABCA3/SFTPC reporters

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mCherry contains a fluorescent protein isoform that interferes with its reporter function

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