GRAMD2+ alveolar type I cell plasticity facilitates cell state transitions in organoid culture. GRAMD2+ alveolar type I cell plasticity facilitates cell state transitions in organoid culture

Alveolar epithelial regeneration is critical for normal lung function and becomes dysregulated in disease. While alveolar type 2 (AT2) and club cells are known distal lung epithelial progenitors, determining if alveolar epithelial type 1 (AT1) cells also contribute to alveolar regeneration has been hampered by lack of highly specific mouse models labeling AT1 cells. To address this, the Gramd2CreERT2 transgenic strain was generated and crossed to ROSAmTmG mice. Extensive cellular characterization, including distal lung immunofluorescence and cytospin staining, confirmed that GRAMD2+ AT1 cells are highly enriched for green fluoresecent protein (GFP). Interestingly, Gramd2CreERT2 GFP+ cells were able to form colonies in organoid co-culture with Mlg fibroblasts. Temporal scRNAseq revealed that Gramd2+ AT1 cells transition through numerous intermediate lung epithelial cell states including basal, secretory and AT2 cell in organoids while acquiring proliferative capacity. Our results indicate that Gramd2+ AT1 cells are highly plastic suggesting they may contribute to alveolar regeneration. Overall design: Primary isolation of FACS sorted GFP+ cells were defined as day 0. On days 8, 13, and 20 of 3D culture, to obtain single cells, colonies were dissociated with dispase (#354235, BD Gibco) and TrypLE express (#12604013, Gibco). Biotinylated EpCAM Ab (#130-096-419, Miltenyi Biotec, San Diego, California), anti-biotin microbeads (#130-900-485, Miltenyi Biotec,) and MS columns (#130-042-201, Miltenyi Biotec) were used to enrich for epithelial cells. For each time point, cells were counted, and viability was measured using an automated cell counter (Countess 3, Thermo Fisher Scientific). Single cell suspensions were processed using Chromium Single Cell 3’ Reagent Kits v3.1 (10x Genomics, PN-1000128, PN-1000127 and PN-1000213). Briefly, cells resuspended in the reaction mix were loaded into the Chromium Single Cell Chip G, together with the 10x Barcoded Gel Beads and partitioning oil. The Chip G was placed into the 10x Chromium controller for cell partitioning, targeting a final number of 10,000 cells per sample. After partitioning, the GEMs (Gel Bead-In EMulsions) were transferred to the C1000 Touch Thermal Cycler for the first phase of reverse transcription followed by library preparation following the manufacturer’s instructions. Libraries were deep sequenced using the Illumina NovaSeq platform.

肺泡上皮再生对维持肺部正常生理功能至关重要，且在疾病状态下会发生失调。已知肺泡Ⅱ型（alveolar type 2, AT2）细胞与棒状细胞是远端肺上皮祖细胞，但此前因缺乏可特异性标记肺泡Ⅰ型（alveolar epithelial type 1, AT1）细胞的小鼠模型，学界对AT1细胞是否参与肺泡再生的研究始终受阻。

为解决这一科学问题，研究人员构建了Gramd2CreERT2转基因小鼠品系，并将其与ROSAmTmG小鼠杂交。通过远端肺免疫荧光染色、细胞离心涂片染色等多维度细胞表征实验，证实GRAMD2阳性AT1细胞可高效富集绿色荧光蛋白（green fluorescent protein, GFP）。

值得注意的是，在与Mlg成纤维细胞共培养的类器官体系中，Gramd2CreERT2 GFP阳性细胞可形成细胞克隆集落。时序性单细胞RNA测序（single-cell RNA sequencing, scRNAseq）结果显示，在类器官培养过程中，Gramd2阳性AT1细胞会经历多种肺上皮细胞中间状态，包括基底细胞、分泌细胞及AT2细胞，并在此过程中获得增殖能力。

本研究结果表明，Gramd2阳性AT1细胞具有高度可塑性，提示其可能参与肺泡再生过程。

整体实验设计：以流式细胞术分选得到的GFP阳性原代细胞设为第0天样本。分别在3D培养的第8、13和20天，使用dispase（货号#354235, BD Gibco）与TrypLE Express（货号#12604013, Gibco）消化细胞集落以获取单细胞悬液。采用生物素化EpCAM抗体（货号#130-096-419, Miltenyi Biotec, 美国加利福尼亚州圣地亚哥）、抗生物素微珠（货号#130-900-485, Miltenyi Biotec）及MS分选柱（货号#130-042-201, Miltenyi Biotec）富集上皮细胞。对每个时间点的细胞进行计数，并通过自动细胞计数仪（Countess 3, 赛默飞世尔科技）检测细胞活力。

单细胞悬液采用Chromium单细胞3’转录组试剂盒v3.1（10x Genomics，货号PN-1000128、PN-1000127及PN-1000213）进行建库。具体流程如下：将重悬于反应体系的细胞与10x条形码凝胶微珠、分区油共同加载至Chromium单细胞芯片G中，随后将芯片G置于10x Chromium控制器中进行细胞分区，目标每样本最终获取10000个细胞。分区完成后，将凝胶微珠乳液（Gel Bead-In EMulsions，以下简称GEMs）转移至C1000 Touch热循环仪中进行第一轮反转录，随后按照制造商说明书完成文库制备。文库采用Illumina NovaSeq测序平台进行深度测序。