Hayashi Masamichi, Yamamoto Naoki, Hiramatsu Noriko, Isogai Sumito, Gotoh Yusuke, Goto Yasuhiro, Kondo Masashi, Imaizumi Kazuyoshi
Department of Respiratory Medicine, School of Medicine, Fujita Health University, Toyoake, Aichi, 470-1192, Japan.
Regenerative Medicine Support Promotion Facility, Center for Research Promotion and Support, Fujita Health University, Toyoake, Aichi, 470-1192, Japan.
In Vitro Cell Dev Biol Anim. 2018 Oct;54(9):648-657. doi: 10.1007/s11626-018-0287-x. Epub 2018 Aug 25.
In recent research on regenerative medicine, three-dimensional (3D) tissue reconstruction using the induced pluripotent stem cell (iPS cell) differentiated cells has attracted attention. In this study, mouse lungs at 1.5, 10, and 20 d old were subjected to enzyme treatment, and aggregates formed in serum-free suspension culture (3D-culture) were observed. The number of aggregates formed was the highest in 1.5 d. The cell aggregates in which the interior of the aggregate is filled and form small vacuoles and the organoid-like aggregates having a relatively large vacuole inside and forming the alveolar-like structure were observed. At 1.5 d, the formation ratio of the organoid-like aggregates was the highest and aggregate size was small at 20 d. For the cell aggregates derived from 1.5 d, positive cells of SSEA-1, CD29, CD90, CD105, alveolar epithelial stem cell marker of SP-C, and Sca-1 were observed in the center. In the cell aggregates derived from 10 d, the expression level of 1.5 d each protein markers and OCT4 gene of transcription factor was decreased, and furthermore, markers were hardly observed in the organoid-like aggregates derived from 10 d. In addition, cells surrounding the vacuole of organoid-like aggregate obtained over 10 d differentiated into periodic acid-Schiff (PAS), podoplanin-positive cells. When the formed cell aggregates were dispersed, cell aggregates and organoid-like aggregates were reformed. Comparing 3D-culture and adhesion culture (2D-culture), SP-C expression of 10 d of cells was maintained. Expression of markers of undifferentiated markers and alveolar tissue stem cells decreased when cell aggregates were cultured with the addition of fetal bovine serum.
在再生医学的最新研究中，利用诱导多能干细胞（iPS细胞）分化细胞进行三维（3D）组织重建受到了关注。在本研究中，对1.5、10和20日龄的小鼠肺进行酶处理，并观察在无血清悬浮培养（3D培养）中形成的聚集体。形成的聚集体数量在1.5日龄时最高。观察到聚集体内部充满并形成小液泡的细胞聚集体以及内部有相对较大液泡并形成肺泡样结构的类器官聚集体。在1.5日龄时，类器官聚集体的形成率最高，而在20日龄时聚集体尺寸较小。对于源自1.5日龄的细胞聚集体，在中心观察到SSEA-1、CD29、CD90、CD105、肺泡上皮干细胞标志物SP-C和Sca-1的阳性细胞。在源自10日龄的细胞聚集体中，每种蛋白质标志物和转录因子OCT4基因在1.5日龄时的表达水平降低，此外，在源自10日龄的类器官聚集体中几乎观察不到这些标志物。此外，10日龄以上获得的类器官聚集体液泡周围的细胞分化为过碘酸希夫（PAS）、血小板内皮细胞黏附分子阳性细胞。当形成的细胞聚集体分散时，细胞聚集体和类器官聚集体会重新形成。比较3D培养和贴壁培养（2D培养），10日龄细胞的SP-C表达得以维持。当细胞聚集体在添加胎牛血清的情况下培养时，未分化标志物和肺泡组织干细胞的标志物表达降低。
