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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE152500 | GEO | 2021/06/01

REPOSITORIES: GEO

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Project description:We performed transcriptomic profiling of freshly isolated primary adult human alveolar epithelial type 2 cells (AEC2s), their isogenic cultured progeny, and human induced pluripotent stem cell-derived AEC2s (iAEC2s). Distal lung preparations from adult donor lung explants from two individuals were cryopreserved using methods we have previously described. Primary AEC2s were purified using fluorescence activated cell sorting (FACS) to isolate cells co-expressing EPCAM and HTII-280. To establish cultures of primary AEC2s, these cells were combined with MRC5 fibroblasts at an 1:10 ratio on cell culture inserts in CK+DCI medium. iAEC2s were derived using our previously published lung directed differentiation protocol. We used the SPC2 human iPSC line and specifically the SPC2-ST-B2 (SFTPC tdT/WT ) clone containing a SFTPC tdTomato knock-in reporter. SFTPC tdTomato+ cells were sorted on day 41 of differentiation. iAEC2s were single-cell passaged in self-renewing feeder-free 3D cultures approximately every 2 weeks. We used the 10X Chromium System to generate single-cell transcriptomic data from 5 samples prepared in parallel and sequenced on the same day. We profiled (a) preculture primary AEC2s, (b) cultured primary AEC2s, (c) 3D feeder-free iAEC2s, (d) feeder-free iAEC2 cultured on cell culture inserts, and (e) iAEC2s cultured on cell inserts with MRC5 fibroblasts. The cultured primary AEC2 samples and the iAEC2 sample co-cultured with MRC5s were sorted for live EPCAM+ cells prior to encapsulation. All other samples were sorted for live cells. We find that although each population (fresh, primary cultured, and iPSC-derived AEC2s) occupies a distinct transcriptomic space, both cultured primary AEC2s and cultured feeder-free iAEC2s closely resemble their primary pre-cultured counterparts. We also find a continuum of progressively more proliferative states (from fresh primary to cultured primary to cultured iAEC2s) inversely associated with progressively less mature AEC2 states.

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

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