Advertisement

Pediatric Surgery International

, Volume 35, Issue 1, pp 3–7 | Cite as

Neonatal intestinal organoids as an ex vivo approach to study early intestinal epithelial disorders

  • Bo Li
  • Carol Lee
  • Marissa Cadete
  • Hiromu Miyake
  • Dorothy Lee
  • Agostino PierroEmail author
Original Article

Abstract

Background

Adult intestinal organoids have been used to study ex vivo intestinal injury in adulthood. However, the neonatal intestinal epithelium has many unique features that are different from adult mature intestine. Establishing a neonatal ex vivo organoid model is essential to study the epithelial physiology in early postnatal development and to investigate derangements associated with disease processes during the neonatal period like necrotizing enterocolitis (NEC).

Methods

Fresh and frozen terminal ileum was harvested from mice pups on postnatal day 9. Crypts were isolated and organoids were cultured. Organoids were exposed to hypoxia and lipopolysaccharide (LPS) for 48 h to induce epithelial injury. Inflammatory cytokines and tight junction proteins were evaluated.

Results

Robust intestinal organoids can be formed from both fresh and frozen intestinal tissue of neonatal mice pups. Hypoxia and LPS administration induced intestinal inflammation and disrupted tight junctions in these neonatal intestinal organoids.

Conclusions

We have established a novel method to grow organoids from neonatal intestine. We demonstrated that these organoids respond to the injury occurring during neonatal intestinal diseases such as NEC by increasing the organoid inflammation and by disrupting the organoid barrier function. Organoids provide an ex vivo platform to study intestinal physiology and pathology during the neonatal period.

Keywords

Neonatal intestinal injury Intestinal organoids Necrotizing enterocolitis Stress factors 

Notes

Acknowledgements

This work is supported by a Canadian Institutes of Health Research (CIHR) Foundation Grant 353857. AP is the Robert M. Filler Chair of Surgery, The Hospital for Sick Children (HSC). BL is the recipient of HSC Restracomp Fellowship.

Author contributions

BL, CL, and AP designed experiments; CL, MC, HM, DL, and RW performed experiments; BL, CL, and MC wrote the manuscript; AP provided advice and supervision; all the authors reviewed and revised the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interest.

References

  1. 1.
    Walton KD, Freddo AM, Wang S, Gumucio DL (2016) Generation of intestinal surface: an absorbing tale. Development 143(13):2261–2272.  https://doi.org/10.1242/dev.135400 CrossRefGoogle Scholar
  2. 2.
    Ley RE, Peterson DA, Gordon JI (2006) Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124(4):837–848.  https://doi.org/10.1016/j.cell.2006.02.017 CrossRefGoogle Scholar
  3. 3.
    Cheng H, Leblond CP (1974) Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types. Am J Anat 141(4):537–561.  https://doi.org/10.1002/aja.1001410407 CrossRefGoogle Scholar
  4. 4.
    Sato T, Clevers H (2013) Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340(6137):1190–1194.  https://doi.org/10.1126/science.1234852 CrossRefGoogle Scholar
  5. 5.
    Yui S, Nakamura T, Sato T, Nemoto Y, Mizutani T, Zheng X, Ichinose S, Nagaishi T, Okamoto R, Tsuchiya K, Clevers H, Watanabe M (2012) Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5(+) stem cell. Nat Med 18(4):618–623.  https://doi.org/10.1038/nm.2695 CrossRefGoogle Scholar
  6. 6.
    Clevers H (2016) Modeling development and disease with organoids. Cell 165(7):1586–1597.  https://doi.org/10.1016/j.cell.2016.05.082 CrossRefGoogle Scholar
  7. 7.
    Schweiger PJ, Jensen KB (2016) Modeling human disease using organotypic cultures. Curr Opin Cell Biol 43:22–29.  https://doi.org/10.1016/j.ceb.2016.07.003 CrossRefGoogle Scholar
  8. 8.
    Dutta D, Heo I, Clevers H (2017) Disease modeling in stem cell-derived 3D organoid systems. Trends Mol Med 23(5):393–410.  https://doi.org/10.1016/j.molmed.2017.02.007 CrossRefGoogle Scholar
  9. 9.
    Stenson WF (2013) Postnatal growth in the intestine. Curr Opin Gastroenterol 29(2):107–111.  https://doi.org/10.1097/MOG.0b013e32835d9ec3 CrossRefGoogle Scholar
  10. 10.
    Herbst JJ, Sunshine P (1969) Postnatal development of the small intestine of the rat. Changes in mucosal morphology at weaning. Pediatr Res 3(1):27–33CrossRefGoogle Scholar
  11. 11.
    Paran TS, Rolle U, Puri P (2006) Postnatal development of the mucosal plexus in the porcine small and large intestine. Pediatr Surg Int 22(12):997–1001.  https://doi.org/10.1007/s00383-006-1786-5 CrossRefGoogle Scholar
  12. 12.
    Unthank JL, Lash JM, Bohlen HG (1990) Maturation of the rat intestinal microvasculature from juvenile to early adult life. Am J Physiol 259(2 Pt 1):G282–G289.  https://doi.org/10.1152/ajpgi.1990.259.2.G282 Google Scholar
  13. 13.
    Trahair JF (1989) Remodeling of the rat small intestinal mucosa during the suckling period. J Pediatr Gastroenterol Nutr 9(2):232–237CrossRefGoogle Scholar
  14. 14.
    Dehmer JJ, Garrison AP, Speck KE, Dekaney CM, Van Landeghem L, Sun X, Henning SJ, Helmrath MA (2011) Expansion of intestinal epithelial stem cells during murine development. PLoS One 6(11):e27070.  https://doi.org/10.1371/journal.pone.0027070 CrossRefGoogle Scholar
  15. 15.
    Rouwet EV, Heineman E, Buurman WA, ter Riet G, Ramsay G, Blanco CE (2002) Intestinal permeability and carrier-mediated monosaccharide absorption in preterm neonates during the early postnatal period. Pediatr Res 51(1):64–70.  https://doi.org/10.1203/00006450-200201000-00012 CrossRefGoogle Scholar
  16. 16.
    Baumler AJ, Sperandio V (2016) Interactions between the microbiota and pathogenic bacteria in the gut. Nature 535(7610):85–93.  https://doi.org/10.1038/nature18849 CrossRefGoogle Scholar
  17. 17.
    Cummins AG, Thompson FM (1997) Postnatal changes in mucosal immune response: a physiological perspective of breast feeding and weaning. Immunol Cell Biol 75(5):419–429.  https://doi.org/10.1038/icb.1997.67 CrossRefGoogle Scholar
  18. 18.
    Lee C, Minich A, Li B, Miyake H, Seo S, Pierro A (2018) Influence of stress factors on intestinal epithelial injury and regeneration. Pediatr Surg Int 34(2):155–160.  https://doi.org/10.1007/s00383-017-4183-3 CrossRefGoogle Scholar
  19. 19.
    Dekkers JF, Wiegerinck CL, de Jonge HR, Bronsveld I, Janssens HM, de Winter-de Groot KM, Brandsma AM, de Jong NW, Bijvelds MJ, Scholte BJ, Nieuwenhuis EE, van den Brink S, Clevers H, van der Ent CK, Middendorp S, Beekman JM (2013) A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med 19(7):939–945.  https://doi.org/10.1038/nm.3201 CrossRefGoogle Scholar
  20. 20.
    Li B, Lee C, Filler T, Hock A, Wu RY, Li Q, Chen S, Koike Y, Ip W, Chi L, Zani-Ruttenstock E, Maattanen P, Gonska T, Delgado-Olguin P, Zani A, Sherman PM, Pierro A (2017) Inhibition of corticotropin-releasing hormone receptor 1 and activation of receptor 2 protect against colonic injury and promote epithelium repair. Sci Rep 7:46616.  https://doi.org/10.1038/srep46616 CrossRefGoogle Scholar
  21. 21.
    Sodhi CP, Neal MD, Siggers R, Sho S, Ma C, Branca MF, Prindle T Jr, Russo AM, Afrazi A, Good M, Brower-Sinning R, Firek B, Morowitz MJ, Ozolek JA, Gittes GK, Billiar TR, Hackam DJ (2012) Intestinal epithelial Toll-like receptor 4 regulates goblet cell development and is required for necrotizing enterocolitis in mice. Gastroenterology 143(3):708–718 e705.  https://doi.org/10.1053/j.gastro.2012.05.053 CrossRefGoogle Scholar
  22. 22.
    McElroy SJ, Castle SL, Bernard JK, Almohazey D, Hunter CJ, Bell BA, Al Alam D, Wang L, Ford HR, Frey MR (2014) The ErbB4 ligand neuregulin-4 protects against experimental necrotizing enterocolitis. Am J Pathol 184(10):2768–2778.  https://doi.org/10.1016/j.ajpath.2014.06.015 CrossRefGoogle Scholar
  23. 23.
    Zani A, Cananzi M, Fascetti-Leon F, Lauriti G, Smith VV, Bollini S, Ghionzoli M, D’Arrigo A, Pozzobon M, Piccoli M, Hicks A, Wells J, Siow B, Sebire NJ, Bishop C, Leon A, Atala A, Lythgoe MF, Pierro A, Eaton S, De Coppi P (2014) Amniotic fluid stem cells improve survival and enhance repair of damaged intestine in necrotising enterocolitis via a COX-2 dependent mechanism. Gut 63(2):300–309.  https://doi.org/10.1136/gutjnl-2012-303735 CrossRefGoogle Scholar
  24. 24.
    Ling X, Linglong P, Weixia D, Hong W (2016) Protective effects of bifidobacterium on intestinal barrier function in LPS-induced enterocyte barrier injury of Caco-2 monolayers and in a rat NEC model. PLoS One 11(8):e0161635.  https://doi.org/10.1371/journal.pone.0161635 CrossRefGoogle Scholar
  25. 25.
    Khailova L, Dvorak K, Arganbright KM, Halpern MD, Kinouchi T, Yajima M, Dvorak B (2009) Bifidobacterium bifidum improves intestinal integrity in a rat model of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 297(5):G940–G949.  https://doi.org/10.1152/ajpgi.00141.2009 CrossRefGoogle Scholar
  26. 26.
    Senger S, Ingano L, Freire R, Anselmo A, Zhu W, Sadreyev R, Walker WA, Fasano A (2018) Human fetal-derived enterospheres provide insights on intestinal development and a novel model to study necrotizing enterocolitis (NEC). Cell Mol Gastroenterol Hepatol 5(4):549–568.  https://doi.org/10.1016/j.jcmgh.2018.01.014 CrossRefGoogle Scholar
  27. 27.
    Walsh AJ, Cook RS, Sanders ME, Arteaga CL, Skala MC (2016) Drug response in organoids generated from frozen primary tumor tissues. Sci Rep 6:18889.  https://doi.org/10.1038/srep18889 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Bo Li
    • 1
  • Carol Lee
    • 1
  • Marissa Cadete
    • 1
  • Hiromu Miyake
    • 1
  • Dorothy Lee
    • 1
  • Agostino Pierro
    • 1
    Email author
  1. 1.Division of General and Thoracic Surgery, Translational Medicine ProgramThe Hospital for Sick ChildrenTorontoCanada

Personalised recommendations