Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, Massachusetts.
The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts.
Am J Respir Cell Mol Biol. 2021 Oct;65(4):442-460. doi: 10.1165/rcmb.2020-0259OC.
Alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, are typically identified through the use of the canonical markers, SFTPC and ABCA3. Self-renewing AEC2-like cells have been generated from human induced pluripotent stem cells (iPSCs) through the use of knock-in SFTPC fluorochrome reporters. However, developmentally, SFTPC expression onset begins in the fetal distal lung bud tip and thus is not specific to mature AEC2s. Furthermore, SFTPC reporters appear to identify only those iPSC-derived AEC2s (iAEC2s) expressing the highest SFTPC levels. Here, we generate an ABCA3 knock-in GFP fusion reporter (ABCA3:GFP) that enables the purification of iAEC2s while allowing visualization of lamellar bodies, organelles associated with AEC2 maturation. Using an SFTPC and ABCA3:GFP bifluorescent line for distal lung-directed differentiation, we observe later onset of ABCA3:GFP expression and broader identification of the subsequently emerging iAEC2 population based on ABCA3:GFP expression compared with SFTPC expression. Comparing ABCA3:GFP/SFTPC double-positive with ABCA3:GFP single-positive (SP) cells by RNA sequencing and functional studies reveals iAEC2 cellular heterogeneity with both populations functionally processing surfactant proteins but the SP cells exhibiting faster growth kinetics, increased clonogenicity, increased expression of progenitor markers, lower levels of SFTPC expression, and lower levels of AEC2 maturation markers. Over time, we observe that each population (double-positive and SP) gives rise to the other and each can serve as the parents of indefinitely self-renewing iAEC2 progeny. Our results indicate that iAEC2s are a heterogeneous population of cells with differing proliferation versus maturation properties, the majority of which can be tracked and purified using the ABCA3:GFP reporter or surrogate cell surface proteins, such as SLC34A2 and CPM.
肺泡上皮细胞 2 型（AEC2）是肺肺泡的可选祖细胞，通常通过使用经典标志物 SFTPC 和 ABCA3 来识别。通过使用带有 knock-in SFTPC 荧光报告基因的人诱导多能干细胞（iPSC）已经生成了自我更新的 AEC2 样细胞。然而，从发育上讲，SFTPC 的表达起始于胎儿远端肺芽尖端，因此特异性不强。此外，SFTPC 报告基因似乎仅识别那些表达最高 SFTPC 水平的 iPSC 衍生 AEC2（iAEC2）。在这里，我们生成了一种 ABCA3 knock-in GFP 融合报告基因（ABCA3：GFP），它可以纯化 iAEC2，同时允许观察板层小体，这是与 AEC2 成熟相关的细胞器。使用 SFTPC 和 ABCA3：GFP 双荧光线进行远端肺定向分化，我们观察到 ABCA3：GFP 的表达出现较晚，并且与 SFTPC 表达相比，基于 ABCA3：GFP 表达更广泛地识别随后出现的 iAEC2 群体。通过 RNA 测序和功能研究比较 ABCA3：GFP/SFTPC 双阳性与 ABCA3：GFP 单阳性（SP）细胞，发现 iAEC2 细胞具有异质性，两个群体都能处理表面活性剂蛋白，但 SP 细胞表现出更快的生长动力学、更高的克隆形成能力、更高的祖细胞标志物表达水平、更低的 SFTPC 表达水平和更低的 AEC2 成熟标志物水平。随着时间的推移，我们观察到每个群体（双阳性和 SP）都能产生另一个群体，并且每个群体都可以作为无限自我更新的 iAEC2 后代的亲本。我们的结果表明，iAEC2 是一个具有不同增殖与成熟特性的异质细胞群体，其中大多数可以使用 ABCA3：GFP 报告基因或替代细胞表面蛋白（如 SLC34A2 和 CPM）进行跟踪和纯化。
