Center for Regenerative Medicine, Boston University and Boston Medical Center; The Pulmonary Center and Department of Medicine, Boston University School of Medicine; QIMR Berghofer Medical Research Institute.
Center for Regenerative Medicine, Boston University and Boston Medical Center; The Pulmonary Center and Department of Medicine, Boston University School of Medicine.
J Vis Exp. 2022 Apr 15(182). doi: 10.3791/63875.
In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as "matrix"), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 10 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPC expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.
在肺部，肺泡上皮细胞是一种免受环境刺激的物理屏障，在维持体内平衡和疾病中发挥着重要作用。II 型肺泡上皮细胞（AT2）是远端肺上皮的兼性祖细胞。AT2 的功能障碍和损伤可能来自于各种肺部疾病，并促进这些疾病的发展。因此，更好地了解 AT2 的生物学特性对于理解肺部生物学和疾病至关重要；然而，原代人 AT2 通常难以分离，供应有限。为了克服这些限制，可以通过一种定向分化方案来生成人诱导多能干细胞（iPSC）衍生的 II 型肺泡上皮细胞（iAT2），该方案可重现体内肺发育过程。iAT2 在无饲养层的条件下生长，与人类成人原代 AT2 具有相似的转录组程序，并执行 AT2 的关键功能，如表面活性剂的产生、包装和分泌。本方案详细介绍了通过在三维（3D）培养中连续传代或使 iAT2 适应气液界面（ALI）培养来维持自我更新的 iAT2 的方法。在 3D 可溶性基底膜基质（以下简称“基质”）中进行平板培养之前，先制备 iAT2 的单细胞悬液，在其中，它们自组装成单层上皮球体。可以将 3D 培养中的 iAT2 连续解离成单细胞悬液进行传代或在 2D ALI 培养中平板培养。在 ALI 培养中，iAT2 形成一个具有极性的单层，其顶表面暴露在空气中，使这个平台易于进行环境暴露。因此，本方案生成了大量的 iAT2，每个输入细胞可产生超过 1 x 10 的细胞，在 15 次传代中保持 SFTPC 表达的 AT2 程序。所得细胞代表了一个可重复且相关的平台，可用于研究基因突变、模拟环境暴露或筛选药物。
