Identification of a multipotent lung progenitor for lung regeneration

We recently showed that a suspension of mouse or human fetal or adult lung cells infused intravenously following appropriate conditioning of recipient mice leads to marked lung chimerism within alveolar and bronchiolar lineages, in distinct 'patches'. A large proportion of these donor-derived 'patches' contain both epithelial and endothelial cells. We show here, using R26R-Confetti mice as donors, that these multi-lineage patches are derived from a single lung progenitor. Fluorescence activated cell sorting of adult mouse lung cells revealed that the putative patch-forming progenitors co-express the endothelial marker CD31 (PECAM-1) and the epithelial marker CD326 (EPCAM). Transgenic Cre/lox mice expressing GFP under different promoters support this duality by demonstrating expression of additional epithelial and endothelial markers in this double-positive lung subpopulation. These findings could pave the way not only for effective lung regeneration but also for better understanding of lung vasculature and airways development and maintenance in health and disease. Long term chimerism in recipient lungs after transplantation of td-tomato labeled cells: In our previous studies we followed by immuno-histology (IH) lung chimerism up to 4 months. We have now performed 6-8 months follow up of chimerism using and combining results of immunohistology with single cell RNA transcriptome analysis , showing donor derived epithelial (ciliated cells, club cells, AT1 and AT2 cells,) and endothelial clusters (lymphatics, endothelial progenitors, as well as the newly described gCap -“general”, and aCap -“aerocytes” capillary cells). The resulting clusters were defined based on marker genes and gene set analysis results, using publicaly available LGEA (Lung Gene Expression Analysis) Web Portal. For definition of each cluster we used well definied hallmark gene sets. We conducted single cell transcriptome analysis of the chimeric lung, using FACS purified donor- and recepient-derived lung cells. Three chimeric lungs were first verified to exhibit significant chimerism by fluorescent microscopy, pooled, enzymatically dissociated and FACS separated after gating on CD45-, single, live cells into donor and recipient compartments based on Td-Tomato expression.

我们此前的研究证实，对受体小鼠进行适当预处理后，经静脉输注小鼠或人胎儿/成人肺细胞悬液，可在肺泡和细支气管谱系中形成显著的肺嵌合现象，且呈现清晰的"斑块"结构。
此类供体来源的"斑块"中，有较大比例同时包含上皮细胞与内皮细胞。
本研究通过以R26R-Confetti小鼠作为供体，证实这些多谱系斑块源自单个肺祖细胞。
对成年小鼠肺细胞进行荧光激活细胞分选（Fluorescence Activated Cell Sorting，FACS）后发现，潜在的斑块形成祖细胞共表达内皮标志物CD31（PECAM-1）与上皮标志物CD326（EPCAM）。
在不同启动子下表达绿色荧光蛋白（GFP）的转基因Cre/lox小鼠模型证实，这种双阳性肺细胞亚群同时表达更多上皮与内皮标志物，从而支持了上述双重表型的结论。
本研究结果不仅可为有效的肺再生研究奠定基础，还有助于更深入阐明肺血管系统与气道在健康及疾病状态下的发育与维持机制。
td-Tomato标记细胞移植后受体肺的长期嵌合现象：
在既往研究中，我们通过免疫组织化学（IH）方法追踪肺嵌合现象长达4个月。
本研究现已通过免疫组织化学与单细胞RNA转录组分析相结合的方法，将嵌合现象追踪时长延长至6-8个月，结果显示供体来源的上皮细胞群（纤毛细胞、棒状细胞、AT1及AT2细胞）与内皮细胞群（淋巴管、内皮祖细胞，以及最新报道的gCap——"通用型"、aCap——"气细胞型"毛细血管细胞）均存在嵌合。
上述细胞群的分类基于标志物基因与基因集分析结果，通过公开可用的LGEA（Lung Gene Expression Analysis）网络门户完成。
本研究使用经过充分验证的特征性基因集完成各细胞群的定义。
本研究通过荧光激活细胞分选纯化供体与受体来源的肺细胞，对嵌合肺组织开展单细胞转录组分析。
首先通过荧光显微镜确认3个嵌合肺组织存在显著嵌合现象，随后将其混合并进行酶解消化；基于CD45阴性、单细胞、活细胞设门，再根据td-Tomato的表达情况，通过荧光激活细胞分选将细胞分为供体群与受体群。