背景:干细胞特性使基底细胞成为气道疾病离体建模的理想选择。然而,到目前为止,允许它们在体外广泛连续扩增和保持真正干细胞特性的方法仍在等待建立。本研究旨在开发小鼠气道基底干细胞(ABSCs)的无饲养层培养系统,在体外维持其干细胞潜能,为进一步深入研究和机理探索提供实验依据。
方法:我们使用含有ROCK抑制剂Y-27632的3T3-CM,MEF-CM,和RbEF-CM以确定可以维持小鼠ABSCs体外干细胞形态的合适的无饲养层培养系统。免疫细胞荧光用于鉴定获得的细胞的基底细胞标志物。进行连续繁殖以观察在该培养系统中是否可以保留干细胞形态和基底细胞标记。接下来,我们通过评估种群倍增时间和集落形成效率来检查体外扩增和自我更新能力。此外,通过体外分化培养和3D气管试验检测分化潜能.
结果:当使用含有ROCK抑制剂Y-27632的3T3-CM与Matrigel包被的培养皿组合培养小鼠ABSC时,它们可以稳定地扩增和维持干细胞样克隆。我们证实获得的克隆包含p63/Krt5双阳性ABSC。在不断通过和维护文化中,我们发现它可以在体外传代到至少15代,稳定维持其干细胞形态,基底细胞标记,和体外扩增和自我更新能力。同时,通过体外分化培养和三维气管球培养,我们发现除了保持自我更新,小鼠ABSC可以分化为其他气道上皮细胞,例如乙酰化微管蛋白(Act-Tub)纤毛和MUC5AC粘液分泌细胞。然而,它们未能分化成肺泡上皮细胞,包括肺泡Ⅰ型和肺泡Ⅱ型。
结论:我们建立了一种体外无饲养层培养系统,使小鼠ABSCs保持其干细胞特性,包括自我更新和气道上皮分化潜能,同时保持体外扩增的稳定性。
BACKGROUND: The stem cell characteristic makes basal cells desirable for ex vivo modeling of airway diseases. However, to date, approaches allowing them extensively in vitro serial expansion and maintaining bona fide stem cell property are still awaiting to be established. This study aims to develop a feeder-free culture system of mouse airway basal stem cells (ABSCs) that sustain their stem cell potential in vitro, providing an experimental basis for further in-depth research and mechanism exploration.
METHODS: We used ROCK inhibitor Y-27632-containing 3T3-CM, MEF-CM, and RbEF-CM to determine the proper feeder-free culture system that could maintain in vitro stem cell morphology of mouse ABSCs. Immunocytofluorescence was used to identify the basal cell markers of obtained cells. Serial propagation was carried out to observe whether the stem cell morphology and basal cell markers could be preserved in this cultivation system. Next, we examined the in vitro expansion and self-renewal ability by evaluating population doubling time and colony-forming efficiency. Moreover, the differentiation potential was detected by an in vitro differentiation culture and a 3D tracheosphere assay.
RESULTS: When the mouse ABSCs were cultured using 3T3-CM containing ROCK inhibitor Y-27632 in combination with Matrigel-coated culture dishes, they could stably expand and maintain stem cell-like clones. We confirmed that the obtained clones comprised p63/Krt5 double-positive ABSCs. In continuous passage and maintenance culture, we found that it could be subculture to at least 15 passages in vitro, stably maintaining its stem cell morphology, basal cell markers, and in vitro expansion and self-renewal capabilities. Meanwhile, through in vitro differentiation culture and 3D tracheosphere culture, we found that in addition to maintaining self-renewal, mouse ABSCs could differentiate into other airway epithelial cells such as acetylated tubulin (Act-Tub) + ciliated and MUC5AC + mucus-secreting cells. However, they failed to differentiate into alveoli epithelial cells, including alveolar type I and alveolar type II.
CONCLUSIONS: We established an in vitro feeder-free culture system that allows mouse ABSCs to maintain their stem cell characteristics, including self-renewal and airway epithelium differentiation potential, while keeping up in vitro expansion stability.