Laboratory for Lung Development and Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.
Department of Drug Modality Development, Osaka Research Center for Drug Discovery, Otsuka Pharmaceutical Co., Ltd., Minoh, Japan.
Stem Cells. 2023 Aug 16;41(8):809-820. doi: 10.1093/stmcls/sxad044.
Single-cell RNA sequencing is a valuable tool for dissecting cellular heterogeneity in complex systems. However, it is still challenging to estimate the proliferation and differentiation potentials of subpopulations within dormant tissue stem cells. Here, we established a new single-cell analysis method for profiling the organoid-forming capacity and differentiation potential of tissue stem cells to disclose stem cell subpopulations by integrating single-cell morphometrics, organoid-forming assay, and RNA sequencing, a method named scMORN. To explore lung epithelial stem cells, we initially developed feeder-free culture system, which could expand all major lung stem cells, including basal, club, and alveolar type 2 (AT2) cells, and found that club cells contained a subpopulation, which showed better survival rate and high proliferation capacity and could differentiate into alveolar cells. Using the scMORN method, we discovered a club cell subpopulation named Muc5b+ and large club (ML-club) cells that efficiently formed organoids than other club or AT2 cells in our feeder-free organoid culture and differentiated into alveolar cells in vitro. Single-cell transcriptome profiling and immunohistochemical analysis revealed that ML-club cells localized at the intrapulmonary proximal airway and distinct from known subpopulations of club cells such as BASCs. Furthermore, we identified CD14 as a cell surface antigen of ML-club cells and showed that purified CD14+ club cells engrafted into injured mouse lungs had better engraftment rate and expansion than other major lung stem cells, reflecting the observations in organoid culture systems. The scMORN method could be adapted to different stem cell tissues to discover useful stem-cell subpopulations.
单细胞 RNA 测序是剖析复杂系统中细胞异质性的一种有效工具。然而，对于休眠组织干细胞内亚群的增殖和分化潜力的评估仍然具有挑战性。在这里，我们建立了一种新的单细胞分析方法，用于分析类器官形成能力和组织干细胞的分化潜能，通过整合单细胞形态计量学、类器官形成测定和 RNA 测序来揭示干细胞亚群，该方法称为 scMORN。为了探索肺上皮干细胞，我们最初开发了无饲养层培养系统，该系统可以扩增所有主要的肺干细胞，包括基底细胞、Club 细胞和肺泡 II 型（AT2）细胞，并发现 Club 细胞包含一个亚群，该亚群表现出更好的存活率、高增殖能力，并能分化为肺泡细胞。使用 scMORN 方法，我们发现了一个 Club 细胞亚群，命名为 Muc5b+和大 Club（ML-club）细胞，它们在我们的无饲养层类器官培养中比其他 Club 或 AT2 细胞更有效地形成类器官，并在体外分化为肺泡细胞。单细胞转录组谱分析和免疫组织化学分析表明，ML-club 细胞定位于肺内近端气道，与已知的 Club 细胞亚群（如 BASCs）不同。此外，我们确定 CD14 是 ML-club 细胞的细胞表面抗原，并表明纯化的 CD14+ Club 细胞植入损伤的小鼠肺中比其他主要肺干细胞具有更好的植入率和扩增率，这反映了在类器官培养系统中的观察结果。scMORN 方法可以适应不同的干细胞组织，以发现有用的干细胞亚群。
