Eur J Pediatr Surg 2020; 30(01): 079-084
DOI: 10.1055/s-0039-1693726
Original Article
Georg Thieme Verlag KG Stuttgart · New York

Liver Organoids Generated from Mice with Necrotizing Enterocolitis Have Reduced Regenerative Capacity

Hiromu Miyake
1   Division of General and Thoracic Surgery, Hospital for Sick Children, Toronto, Ontario, Canada
2   Department of Pediatric Surgery, Shizuoka Children's Hospital, Shizuoka, Japan
,
Carol Lee
1   Division of General and Thoracic Surgery, Hospital for Sick Children, Toronto, Ontario, Canada
,
Shogo Seo
1   Division of General and Thoracic Surgery, Hospital for Sick Children, Toronto, Ontario, Canada
,
Bo Li
1   Division of General and Thoracic Surgery, Hospital for Sick Children, Toronto, Ontario, Canada
,
1   Division of General and Thoracic Surgery, Hospital for Sick Children, Toronto, Ontario, Canada
› Author Affiliations
Further Information

Publication History

15 May 2019

15 June 2019

Publication Date:
31 July 2019 (online)

Abstract

Introduction Necrotizing enterocolitis (NEC) is one of the most severe gastrointestinal diseases in infancy. NEC can cause metabolic derangements, multi-organ injury including severe liver damage. The mechanism leading to hepatic damage in NEC remains unclear. The aim of this study is to establish and characterize liver organoids from NEC mice.

Materials and Methods Following ethical approval (#44032), we induced experimental NEC from postnatal day 5 (P5) to P9 using C57BL/6 mice pups. NEC was induced by gavage formula feeding, gavage lipopolysaccharide (LPS) administration, and hypoxia. Breastfed pups were used as control. On P9, NEC and control pups were sacrificed and liver tissue was harvested and organoids were generated. Organoid size was recorded daily (day 2–4) by measuring their surface area and organoid growth was calculated. RNA was extracted on day 4 after liver organoid generation.

Results Organoid growth rate was significantly lower in NEC liver organoids compared to control liver organoids. mRNA expression of liver progenitor cells markers of LGR5 and SOX9 was lower in NEC liver organoids compared to control liver organoids. Similarly, expression of proliferation markers of Ki67 and PCNA was lower in NEC liver organoids.

Conclusion We report a novel technique to generate liver organoids during NEC. These organoids are characterized by reduced progenitor cells, reduced proliferation, and overall impaired regenerative capacity. Liver progenitor cells are important targets to prevent liver damage in NEC and promote recovery.

 
  • References

  • 1 Neu J, Walker WA. Necrotizing enterocolitis. N Engl J Med 2011; 364 (03) 255-264
  • 2 Hall NJ, Eaton S, Pierro A. Royal Australasia of Surgeons Guest Lecture. Necrotizing enterocolitis: prevention, treatment, and outcome. J Pediatr Surg 2013; 48 (12) 2359-2367
  • 3 Hull MA, Fisher JG, Gutierrez IM. , et al. Mortality and management of surgical necrotizing enterocolitis in very low birth weight neonates: a prospective cohort study. J Am Coll Surg 2014; 218 (06) 1148-1155
  • 4 Sonntag J, Wagner MH, Waldschmidt J, Wit J, Obladen M. Multisystem organ failure and capillary leak syndrome in severe necrotizing enterocolitis of very low birth weight infants. J Pediatr Surg 1998; 33 (03) 481-484
  • 5 Morecroft JA, Spitz L, Hamilton PA, Holmes SJ. Necrotizing enterocolitis--multisystem organ failure of the newborn?. Acta Paediatr Suppl 1994; 396: 21-23
  • 6 Halpern MD, Holubec H, Dominguez JA. , et al. Hepatic inflammatory mediators contribute to intestinal damage in necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 2003; 284 (04) G695-G702
  • 7 Halpern MD, Holubec H, Clark JA. , et al. Epidermal growth factor reduces hepatic sequelae in experimental necrotizing enterocolitis. Biol Neonate 2006; 89 (04) 227-235
  • 8 Huch M, Boj SF, Clevers H. Lgr5(+) liver stem cells, hepatic organoids and regenerative medicine. Regen Med 2013; 8 (04) 385-387
  • 9 Forbes SJ, Newsome PN. Liver regeneration - mechanisms and models to clinical application. Nat Rev Gastroenterol Hepatol 2016; 13 (08) 473-485
  • 10 Khan Z, Orr A, Michalopoulos GK, Ranganathan S. Immunohistochemical analysis of the stem cell marker LGR5 in pediatric liver disease. Pediatr Dev Pathol 2017; 20 (01) 16-27
  • 11 Huch M, Dorrell C, Boj SF. , et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 2013; 494 (7436): 247-250
  • 12 Miyake H, Li B, Lee C. , et al. Liver damage, proliferation, and progenitor cell markers in experimental necrotizing enterocolitis. J Pediatr Surg 2018; 53 (05) 909-913
  • 13 Huch M, Gehart H, van Boxtel R. , et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 2015; 160 (1-2): 299-312
  • 14 Broutier L, Andersson-Rolf A, Hindley CJ. , et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 2016; 11 (09) 1724-1743
  • 15 Broutier L, Mastrogiovanni G, Verstegen MMA. , et al. Human primary liver cancer-derived organoid cultures for disease modeling and drug screening. Nat Med 2017; 23 (12) 1424-1435
  • 16 Barlow B, Santulli TV, Heird WC, Pitt J, Blanc WA, Schullinger JN. An experimental study of acute neonatal enterocolitis--the importance of breast milk. J Pediatr Surg 1974; 9 (05) 587-595
  • 17 Zani A, Cordischi L, Cananzi M. , et al. Assessment of a neonatal rat model of necrotizing enterocolitis. Eur J Pediatr Surg 2008; 18 (06) 423-426
  • 18 Katoonizadeh A, Nevens F, Verslype C, Pirenne J, Roskams T. Liver regeneration in acute severe liver impairment: a clinicopathological correlation study. Liver Int 2006; 26 (10) 1225-1233
  • 19 Lu P, Sodhi CP, Jia H. , et al. Animal models of gastrointestinal and liver diseases. Animal models of necrotizing enterocolitis: pathophysiology, translational relevance, and challenges. Am J Physiol Gastrointest Liver Physiol 2014; 306 (11) G917-G928
  • 20 Li B, Lee C, Cadete M, Miyake H, Lee D, Pierro A. Neonatal intestinal organoids as an ex vivo approach to study early intestinal epithelial disorders. Pediatr Surg Int 2019; 35 (01) 3-7