Workman Michael J, Gleeson John P, Troisi Elissa J, Estrada Hannah Q, Kerns S Jordan, Hinojosa Christopher D, Hamilton Geraldine A, Targan Stephan R, Svendsen Clive N, Barrett Robert J
Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California.
Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California.
Cell Mol Gastroenterol Hepatol. 2017 Dec 29;5(4):669-677.e2. doi: 10.1016/j.jcmgh.2017.12.008. eCollection 2018.
Human intestinal organoids derived from induced pluripotent stem cells have tremendous potential to elucidate the intestinal epithelium's role in health and disease, but it is difficult to directly assay these complex structures. This study sought to make this technology more amenable for study by obtaining epithelial cells from induced pluripotent stem cell-derived human intestinal organoids and incorporating them into small microengineered Chips. We then investigated if these cells within the Chip were polarized, had the 4 major intestinal epithelial subtypes, and were biologically responsive to exogenous stimuli.
Epithelial cells were positively selected from human intestinal organoids and were incorporated into the Chip. The effect of continuous media flow was examined. Immunocytochemistry and hybridization were used to demonstrate that the epithelial cells were polarized and possessed the major intestinal epithelial subtypes. To assess if the incorporated cells were biologically responsive, Western blot analysis and quantitative polymerase chain reaction were used to assess the effects of interferon (IFN)-γ, and fluorescein isothiocyanate-dextran 4 kDa permeation was used to assess the effects of IFN-γ and tumor necrosis factor-α on barrier function.
The optimal cell seeding density and flow rate were established. The continuous administration of flow resulted in the formation of polarized intestinal folds that contained Paneth cells, goblet cells, enterocytes, and enteroendocrine cells along with transit-amplifying and stem cells. Administration of IFN-γ for 1 hour resulted in the phosphorylation of STAT1, whereas exposure for 3 days resulted in a significant upregulation of IFN-γ related genes. Administration of IFN-γ and tumor necrosis factor-α for 3 days resulted in an increase in intestinal permeability.
We demonstrate that the Intestine-Chip is polarized, contains all the intestinal epithelial subtypes, and is biologically responsive to exogenous stimuli. This represents a more amenable platform to use organoid technology and will be highly applicable to personalized medicine and a wide range of gastrointestinal conditions.
源自诱导多能干细胞的人肠道类器官在阐明肠道上皮在健康和疾病中的作用方面具有巨大潜力，但直接检测这些复杂结构却很困难。本研究旨在通过从诱导多能干细胞衍生的人肠道类器官中获取上皮细胞并将其整合到小型微工程芯片中，使这项技术更易于研究。然后，我们研究了芯片内的这些细胞是否极化、是否具有4种主要的肠道上皮亚型以及对外源刺激是否具有生物学反应。
从人肠道类器官中阳性选择上皮细胞并整合到芯片中。检测了连续培养基流动的影响。采用免疫细胞化学和杂交技术证明上皮细胞极化并拥有主要的肠道上皮亚型。为了评估整合的细胞是否具有生物学反应，使用蛋白质印迹分析和定量聚合酶链反应评估干扰素（IFN）-γ的作用，使用异硫氰酸荧光素-葡聚糖4 kDa渗透评估IFN-γ和肿瘤坏死因子-α对屏障功能的影响。
确定了最佳的细胞接种密度和流速。持续给予流动导致形成极化的肠褶，其中包含潘氏细胞、杯状细胞、肠上皮细胞和肠内分泌细胞以及过渡增殖细胞和干细胞。给予IFN-γ 1小时导致STAT1磷酸化，而暴露3天导致IFN-γ相关基因显著上调。给予IFN-γ和肿瘤坏死因子-α 3天导致肠道通透性增加。
我们证明肠道芯片是极化的，包含所有肠道上皮亚型，并且对外源刺激具有生物学反应。这代表了一个更易于使用类器官技术的平台，将高度适用于个性化医学和广泛的胃肠道疾病。
