Department of Pediatric Surgery, McGovern Medical School at UTHealth, Houston, Texas.
Department of Medicine Section of Gastroenterology and Hepatology, Baylor College of Medicine, Houston, Texas.
J Surg Res. 2021 Mar;259:500-508. doi: 10.1016/j.jss.2020.10.002. Epub 2020 Nov 7.
Short bowel syndrome is a potentially fatal condition with inadequate management options. Tissue-engineered small intestine (TESI) is a promising solution, but confirmation of TESI function will be crucial before human application. We sought to define intestinal epithelial barrier function in human intestinal organoid (HIO)-derived TESI.
HIOs were generated in vitro from human embryonic stem cells. After 1 mo, HIOs were collected for analysis or transplanted into the kidney capsule of immunocompromised mice. Transplanted HIOs (tHIOs) were harvested for analysis at 4 or 8 wk. Reverse transcription quantitative polymerase chain reaction and immunofluorescent staining were performed for tight junction components: claudin 3 (CLDN3), claudin 15 (CLDN15), occludin (OCLN), and zonula occludens-1, or tight junction protein-1 (TJP1/ZO-1).
Four-week-old tHIOs demonstrated significantly (P < 0.05) higher levels of CLDN15 (6x), OCLN (4x), and TJP1/ZO-1 (3x) normalized to GAPDH than in vitro HIOs. Eight-week-old tHIOs demonstrated significantly (P < 0.05) higher expression levels of CLDN3 (26x), CLDN15 (29x), OCLN (4x), and TJP1/ZO-1 (5x) than in vitro HIOs. There was no significant difference in expression of these tight junction components between 4- and 8-week-old tHIOs. Immunofluorescent staining revealed the presence of claudin 3, claudin 15, occludin, and zonula occludens-1 in both in vitro HIOs and tHIOs; however, the morphology appeared more mature in tHIOs.
In vitro HIOs have lower levels of tight junction mRNA, and tight junction proteins appear morphologically immature. Transplantation facilitates maturation of the HIOs and enhances select tight junction gene expression.
短肠综合征是一种潜在致命的疾病，目前治疗选择有限。组织工程小肠（TESI）是一种很有前途的解决方案，但在人体应用之前，确认 TESI 的功能将是至关重要的。我们试图确定源自人肠类器官（HIO）的 TESI 的肠上皮屏障功能。
从人胚胎干细胞体外生成 HIO。1 个月后，收集 HIO 进行分析或移植到免疫缺陷小鼠的肾包膜下。4 或 8 周后收获移植的 HIO（tHIO）进行分析。进行逆转录定量聚合酶链反应和免疫荧光染色以检测紧密连接成分： Claudin 3（CLDN3）、Claudin 15（CLDN15）、Occludin（OCLN）和紧密连接蛋白-1（TJP1/ZO-1）。
4 周龄的 tHIO 与体外 HIO 相比，CLDN15（6 倍）、OCLN（4 倍）和 TJP1/ZO-1（3 倍）的表达水平显著升高（P < 0.05）。8 周龄的 tHIO 与体外 HIO 相比，CLDN3（26 倍）、CLDN15（29 倍）、OCLN（4 倍）和 TJP1/ZO-1（5 倍）的表达水平显著升高（P < 0.05）。4 周龄和 8 周龄 tHIO 之间这些紧密连接成分的表达水平没有差异。免疫荧光染色显示，体外 HIO 和 tHIO 均存在 Claudin 3、Claudin 15、Occludin 和紧密连接蛋白-1；然而，tHIO 中的形态看起来更成熟。
体外 HIO 的紧密连接 mRNA 水平较低，紧密连接蛋白在形态上显得不成熟。移植有助于 HIO 的成熟，并增强了某些紧密连接基因的表达。
