To the Editor, Upon entry into the lungs, SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) receptor to facilitate viral entry into the epithelial cells that cover the airways and the alveolar gas-exchanging space, leading to extensive epithelial injury, which contributes to local inflammatory storm and a series of respiratory syndromes.1 A number of drugs, based on their modes of action, have been suggested as therapeutic candidates for COVID-19, and some of them are being evaluated in accelerated clinical trials.1 The antimalarial drugs chloroquine and hydroxychloroquine and the anti-influenza drug umifenovir inhibit endocytosis of the virus. The HIV protease inhibitors lopinavir/ritonavir and broad-spectrum antiviral agents ribavirin and favipiravir affect the ability of the virus to replicate in host cells. However, the efficacy and safety of these drugs are still controversial in the treatment of COVID-19 patients. The human lung harbours epithelial stem/progenitor cells that are activated to bring about the repair of airways and the respiratory epithelium after lung injury.2 Among them are airway club cells and alveolar type 2 (AT2) cells. The identity and distribution of club and AT2 cells are very similar in the lungs of mice and humans, thereby enabling successful evaluation of the functionality of human club and AT2 cells in mouse models.3 Club cells proliferate and differentiate into ciliated cells and goblet cells. At the terminal end of the respiratory tree, AT2 cells self-renew and generate AT1 gas-exchanging cells. Surprisingly, both club and AT2 cells are targeted by the SARS-CoV-2 because of abundant expression of ACE2 on the cell surface.4 Damage to the club and AT2 cells compromises the epithelial regenerative capacity in the lung. Insufficient epithelial repair could lead to progression of lung fibrosis, which is thought to occur in COVID-19 patients.5 Stem/progenitor cell–derived three-dimensional organoids serve not only as a model for evaluating stem/progenitor cell function in vitro, but also as a powerful platform for drug screening and safety assays. Much effort has been invested in the discovery of promising drug candidates and their testing in stringent clinical trials. However, care should be taken to select the right drug candidate that would not sacrifice the regenerative potential of the lung epithelium for better prognosis in COVID-19 treatment. Mouse club and AT2 cells were sorted and cultured in a 3D organoid-based system as previously described (Figure 1A and B).6 Chloroquine was shown to inhibit the production of SARS-CoV-2 (EC50 = 1.13 μmol/L). 7 Chloroquine did not affect colony-forming efficiency (CFE) or the growth of organoids derived from club or AT2 cells (Figure 1C-D and G-H). The differentiation potential of club cells into ciliated cells (Foxj1) and goblet cells (Foxa3), and AT2 differentiation into AT1 cells (T1α) was also not affected (Figure 1E-F and I). The EC50 value of remdesivir (GS-5734) against SARS-CoV-2 was estimated at 0.77 μmol/L.7 Remdesivir (at 1 and 10 μmol/L) decreased the CFE of club cells, but promoted the growth of club organoids with negligible effect on ciliated and goblet cell differentiation (Figure 1C-F), the CFE of AT2 cells and the size of AT2 cell–derived organoids (Figure 1G-H). Remdesivir enhanced AT1 cell differentiation at both doses, but reduced the CFE of AT2 cells at high doses (Figure 1G and I). Lopinavir and ritonavir were confirmed to inhibit SARS-CoV-2 (EC50 values of lopinavir and ritonavir were 10.40 μmol/L and 8.63 μmol/L, respectively).8,9 Lopinavir at 10 μmol/L decreased CFEs of both club and AT2 cells (Figure 1C and G). The growth of club celland AT2 cell–derived organoids was inhibited in the presence of lopinavir at 50 μΜ (Figure 1D and H). Lopinavir abrogated club cell differentiation but not AT2 cell differentiation (Figure 1E, F and I). Ritonavir reduced the CFE and organoid growth of both club and AT2 cells (Figure 1C-D and G-H). Ritonavir also suppressed ciliated cell differentiation, but goblet cell differentiation and AT1 differentiation remained unaffected (Figure 1E, F and I). Similar to lopinavir, umifenovir (EC50 value against SARS-CoV-2 was 3.32 μmol/L) decreased the CFE and organoid growth of both club and AT2 cells in a dose-dependent fashion (Figure 1C-D and G-H).8 Umifenovir at 10 μmol/L affected the differentiation potential of both club cells and AT2 cells was also not affected. Ribavirin was shown to inhibit SARS-CoV-2 with EC50 at 109.5 μmol/L. 7 Ribavirin at the concentration of 100 μmol/L decreased CFEs of club and AT2 cells, and limited AT2 organoid growth (Figure 1C, G and H). Club organoid growth, ciliated cell differentiation and AT1 cell differentiation were affected by ribavirin at a higher dose (500 μmol/L) (Figure 1D-F and I). Favipiravir exhibited antiviral activity against SARS-CoV-2 with an EC50 value of 61.88 μmol/L. 7 Favipiravir (up to 500 μmol/L) had little effect on the proliferation and differentiation of club cells (Figure 1C-F). Favipiravir inhibited the CFE of AT2 cells without affecting their organoid growth and AT1 differentiation (Figure 1G-I).

中文翻译：

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类器官技术证明了 COVID-19 潜在药物对肺再生的影响

致编辑，SARS-CoV-2进入肺部后，利用血管紧张素转换酶2（ACE2）受体促进病毒进入覆盖气道和肺泡气体交换空间的上皮细胞，导致广泛的上皮细胞损伤，这会导致局部炎症风暴和一系列呼吸综合征。 1 根据其作用方式，许多药物已被建议作为 COVID-19 的治疗候选药物，其中一些正在加速临床试验中进行评估.1 抗疟药氯喹和羟氯喹以及抗流感药乌米诺韦抑制病毒的内吞作用。HIV蛋白酶抑制剂洛匹那韦/利托那韦和广谱抗病毒药物利巴韦林和法匹拉韦影响病毒在宿主细胞中复制的能力。然而，这些药物在治疗 COVID-19 患者中的有效性和安全性仍存在争议。人肺含有上皮干/祖细胞，在肺损伤后，这些干细胞/祖细胞被激活以修复气道和呼吸道上皮。2 其中包括气道俱乐部细胞和肺泡 2 (AT2) 细胞。在小鼠和人类的肺中，club 和 AT2 细胞的身份和分布非常相似，从而能够成功评估小鼠模型中人类 club 和 AT2 细胞的功能。3 Club 细胞增殖并分化为纤毛细胞和杯状细胞。在呼吸树的末端，AT2 细胞自我更新并产生 AT1 气体交换细胞。令人惊讶的是，由于 ACE2 在细胞表面大量表达，俱乐部和 AT2 细胞都被 SARS-CoV-2 靶向。4 对俱乐部和 AT2 细胞的损伤会损害肺中的上皮再生能力。上皮修复不足可能导致肺纤维化进展，这被认为发生在 COVID-19 患者中。5 干/祖细胞衍生的三维类器官不仅可作为体外评估干/祖细胞功能的模型，而且也是药物筛选和安全性分析的强大平台。在发现有前景的候选药物及其在严格的临床试验中进行测试方面投入了大量精力。但是，应注意选择正确的候选药物，该候选药物不会牺牲肺上皮的再生潜力，以便在 COVID-19 治疗中获得更好的预后。如前所述，在基于 3D 类器官的系统中对小鼠俱乐部和 AT2 细胞进行分类和培养（图 1A 和 B）。6 氯喹显示出抑制 SARS-CoV-2 的产生（EC50 = 1.13 μmol/L）。7 氯喹不影响集落形成效率 (CFE) 或源自棍棒或 AT2 细胞的类器官的生长（图 1C-D 和 GH）。俱乐部细胞向纤毛细胞 (Foxj1) 和杯状细胞 (Foxa3) 的分化潜能，以及 AT2 向 AT1 细胞 (T1α) 的分化潜能也不受影响（图 1E-F 和 I）。瑞德西韦 (GS-5734) 对 SARS-CoV-2 的 EC50 值估计为 0.77 μmol/L。7 Remdesivir（1 和 10 μmol/L）降低了俱乐部细胞的 CFE，但促进了俱乐部类器官的生长对纤毛和杯状细胞分化的影响可以忽略不计（图 1C-F）、AT2 细胞的 CFE 和 AT2 细胞衍生的类器官的大小（图 1G-H）。瑞德西韦在两种剂量下都增强了 AT1 细胞分化，但在高剂量下降低了 AT2 细胞的 CFE（图 1G 和 I）。洛匹那韦和利托那韦被证实可抑制 SARS-CoV-2（洛匹那韦和利托那韦的 EC50 值分别为 10.40 μmol/L 和 8.63 μmol/L）。8,9 10 μmol/L 的洛匹那韦降低了 club 和 AT2 细胞的 CFE （图 1C 和 G）。在 50 μM 的洛匹那韦存在下，棒状细胞和 AT2 细胞衍生的类器官的生长受到抑制（图 1D 和 H）。洛匹那韦消除了俱乐部细胞分化，但不消除 AT2 细胞分化（图 1E、F 和 I）。利托那韦降低了俱乐部和 AT2 细胞的 CFE 和类器官生长（图 1C-D 和 GH）。利托那韦还抑制纤毛细胞分化，但杯状细胞分化和 AT1 分化不受影响（图 1E、F 和 I）。与洛匹那韦类似，乌米诺韦（针对 SARS-CoV-2 的 EC50 值为 3。32 μmol/L) 以剂量依赖性方式降低俱乐部细胞和 AT2 细胞的 CFE 和类器官生长（图 1C-D 和 GH）。8 10 μmol/L 的 Umifenovir 影响俱乐部细胞和 AT2 细胞的分化潜能也没有受到影响。利巴韦林以 109.5 μmol/L 的 EC50 抑制 SARS-CoV-2。7 浓度为 100 μmol/L 的利巴韦林降低了 club 和 AT2 细胞的 CFE，并限制了 AT2 类器官的生长（图 1C、G 和 H）。俱乐部类器官生长、纤毛细胞分化和 AT1 细胞分化受较高剂量 (500 μmol/L) 的利巴韦林影响（图 1D-F 和 I）。法匹拉韦对 SARS-CoV-2 具有抗病毒活性，EC50 值为 61.88 μmol/L。7 Favipiravir（高达 500 μmol/L）对俱乐部细胞的增殖和分化几乎没有影响（图 1C-F）。