Mariaelena A Boyle1, David J Sequeira1, Eoin P McNeill1, Zachary K Criss2, Noah F Shroyer2, Allison L Speer3. 1. Department of Pediatric Surgery, McGovern Medical School at UTHealth, Houston, Texas. 2. Department of Medicine Section of Gastroenterology and Hepatology, Baylor College of Medicine, Houston, Texas. 3. Department of Pediatric Surgery, McGovern Medical School at UTHealth, Houston, Texas. Electronic address: Allison.L.Speer@uth.tmc.edu.
Abstract
BACKGROUND: 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. MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: 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. MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.
Authors: Jennifer L Holmes; Christina M Van Itallie; Julia E Rasmussen; James M Anderson Journal: Gene Expr Patterns Date: 2006-02-02 Impact factor: 1.224
Authors: Michael Meir; Sven Flemming; Natalie Burkard; Johanna Wagner; Christoph-Thomas Germer; Nicolas Schlegel Journal: Am J Physiol Gastrointest Liver Physiol Date: 2016-05-05 Impact factor: 4.052
Authors: Tracy C Grikscheit; Aleem Siddique; Erin R Ochoa; Ashok Srinivasan; Eben Alsberg; Richard A Hodin; Joseph P Vacanti Journal: Ann Surg Date: 2004-11 Impact factor: 12.969
Authors: Frédéric G Sala; Jamil A Matthews; Allison L Speer; Yasuhiro Torashima; Erik R Barthel; Tracy C Grikscheit Journal: Tissue Eng Part A Date: 2011-05-04 Impact factor: 3.845
Authors: Michael J Workman; Maxime M Mahe; Stephen Trisno; Holly M Poling; Carey L Watson; Nambirajan Sundaram; Ching-Fang Chang; Jacqueline Schiesser; Philippe Aubert; Edouard G Stanley; Andrew G Elefanty; Yuichiro Miyaoka; Mohammad A Mandegar; Bruce R Conklin; Michel Neunlist; Samantha A Brugmann; Michael A Helmrath; James M Wells Journal: Nat Med Date: 2016-11-21 Impact factor: 87.241
Authors: Alexander R Cortez; Holly M Poling; Nicole E Brown; Akaljot Singh; Maxime M Mahe; Michael A Helmrath Journal: Surgery Date: 2018-07-30 Impact factor: 4.348
Authors: Stacy R Finkbeiner; Jennifer J Freeman; Minna M Wieck; Wael El-Nachef; Christopher H Altheim; Yu-Hwai Tsai; Sha Huang; Rachel Dyal; Eric S White; Tracy C Grikscheit; Daniel H Teitelbaum; Jason R Spence Journal: Biol Open Date: 2015-10-12 Impact factor: 2.643
Authors: Shane M Hickey; Ben Ung; Christie Bader; Robert Brooks; Joanna Lazniewska; Ian R D Johnson; Alexandra Sorvina; Jessica Logan; Carmela Martini; Courtney R Moore; Litsa Karageorgos; Martin J Sweetman; Douglas A Brooks Journal: Cells Date: 2021-12-23 Impact factor: 6.600