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

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. Overall design: Global transcriptomic analysis of day 31 PSC-derived cells comparing ABCA3:GFP reporter positive vs ABCA3:GFP reporter negative cells in triplicate differentiations (n=3)

我们采用既往报道的远端肺定向分化方案，对人诱导多能干细胞（human induced pluripotent stem cells, iPSCs）来源的细胞开展转录组分析，该方案可诱导生成II型肺泡上皮样细胞（iAEC2s）。本研究使用携带有针对经典AEC2基因ABCA3的GFP敲入荧光报告基因的BU3 ABCA3:GFP诱导多能干细胞系，在远端肺分化第31天时分选GFP阳性与阴性的纯细胞群，用于批量RNA测序（bulk RNA sequencing）分析。我们发现，ABCA3:GFP阳性细胞群中富集了经典AEC2的转录本以及板层小体（lamellar body）相关转录本，提示该细胞群的AEC2程序得到有效富集。与之相反，ABCA3:GFP阴性细胞群中则富集了非肺内胚层谱系相关的转录本。综合上述实验结果，证实了ABCA3:GFP报告系统在本研究的体外远端肺定向分化方案中，可有效富集iAEC2s。整体实验设计：对诱导多能干细胞分化第31天的细胞开展全局转录组分析，比较ABCA3:GFP报告阳性与报告阴性细胞群，设置3次生物学重复（n=3）