Top

Published in:

01-11-2021 | Original Article

Defined serum-free culture of human infant small intestinal organoids with predetermined doses of Wnt3a and R-spondin1 from surgical specimens

Authors: Yuka Matsumoto, Hiroyuki Koga, Mirei Takahashi, Kazuto Suda, Takanori Ochi, Shogo Seo, Go Miyano, Yuichiro Miyake, Hideaki Nakajima, Shiho Yoshida, Takafumi Mikami, Tadaharu Okazaki, Nobutaka Hattori, Atsuyuki Yamataka, Tetsuya Nakamura

Published in: Pediatric Surgery International | Issue 11/2021

Abstract

Purpose

Refinement of organoid technology is important for studying physiology and disease of the intestine. We aimed to optimize defined serum-free conditions for human infant small intestinal (SI) organoid culture with predetermined doses of Wnt3a and Rspo1 from surgical specimens. We further assessed whether intestinal specimens could be stored before use as a source of organoids.

Methods

Different doses of Wnt3a and Rspo1 in a serum-free medium were tested to establish a condition in which surgically resected SI cells grew as organoids over multiple passages. The expression of marker genes for stem and differentiated cells was assessed by quantitative polymerase chain reaction. We also investigated the organoid-forming efficiency of cells in degenerating intestines stored at 4 °C for various intervals post-resection.

Results

We determined the doses of Wnt3a and Rspo1 required for the continuous growth of infant SI organoids with multi-differentiation potential. We revealed that, despite the time-dependent loss of stem cells, tissues stored for up to 2 days preserved cells capable of generating amplifiable organoids.

Conclusion

SI cells can be grown as organoids under defined conditions. This could provide a reproducible and customizable method of using surgical specimens for the study of intestinal maturation and their relevance to pediatric diseases.

Clevers H (2013) The intestinal crypt, a prototype stem cell compartment. Cell 154(2):274–284. https://doi.org/10.1016/j.cell.2013.07.004CrossRefPubMed

Crosnier C, Stamataki D, Lewis J (2006) Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat Rev Genet 7(5):349–359. https://doi.org/10.1038/nrg1840CrossRefPubMed

Chin AM, Hill DR, Aurora M, Spence JR (2017) Morphogenesis and maturation of the embryonic and postnatal intestine. Semin Cell Dev Biol 66:81–93. https://doi.org/10.1016/j.semcdb.2017.01.011CrossRefPubMedPubMedCentral

Muncan V, Heijmans J, Krasinski SD, Büller NV, Wildenberg ME, Meisner S, Radonjic M, Stapleton KA, Lamers WH, Biemond I, van den Bergh Weerman MA, O’Carroll D, Hardwick JC, Hommes DW, van den Brink GR (2011) Blimp1 regulates the transition of neonatal to adult intestinal epithelium. Nat Commun 2:452. https://doi.org/10.1038/ncomms1463CrossRefPubMed

Harper J, Mould A, Andrews RM, Bikoff EK, Robertson EJ (2011) The transcriptional repressor Blimp1/Prdm1 regulates postnatal reprogramming of intestinal enterocytes. Proc Natl Acad Sci USA 108(26):10585–10590. https://doi.org/10.1073/pnas.1105852108CrossRefPubMedPubMedCentral

Duerr CU, Hornef MW (2012) The mammalian intestinal epithelium as integral player in the establishment and maintenance of host-microbial homeostasis. Semin Immunol 24(1):25–35. https://doi.org/10.1016/j.smim.2011.11.002CrossRefPubMed

Clark DA, Mitchell AL (2004) Development of gastrointestinal function: risk factors for necrotizing enterocolitis. J Pediatr Pharmacol Ther 9(2):96–103. https://doi.org/10.5863/1551-6776-9.2.96CrossRefPubMedPubMedCentral

Chusilp S, Li B, Lee D, Lee C, Vejchapipat P, Pierro A (2020) Intestinal organoids in infants and children. Pediatr Surg Int 36(1):1–10. https://doi.org/10.1007/s00383-019-04581-3CrossRefPubMed

Nanthakumar N, Meng D, Goldstein AM, Zhu W, Lu L, Uauy R, Llanos A, Claud EC, Walker WA (2011) The mechanism of excessive intestinal inflammation in necrotizing enterocolitis: an immature innate immune response. PLoS ONE 6(3):e17776. https://doi.org/10.1371/journal.pone.0017776CrossRefPubMedPubMedCentral

10.

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.2018.01.014CrossRefPubMedPubMedCentral

11.

Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244):262–265. https://doi.org/10.1038/nature07935CrossRefPubMed

12.

Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, Van Houdt WJ, Pronk A, Van Gorp J, Siersema PD, Clevers H (2011) Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141(5):1762–1772. https://doi.org/10.1053/j.gastro.2011.07.050CrossRefPubMed

13.

Suzuki K, Murano T, Shimizu H, Ito G, Nakata T, Fujii S, Ishibashi F, Kawamoto A, Anzai S, Kuno R, Kuwabara K, Takahashi J, Hama M, Nagata S, Hiraguri Y, Takenaka K, Yui S, Tsuchiya K, Nakamura T, Ohtsuka K, Watanabe M, Okamoto R (2018) Single cell analysis of Crohn’s disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties. J Gastroenterol 53(9):1035–1047. https://doi.org/10.1007/s00535-018-1437-3CrossRefPubMedPubMedCentral

14.

Kraiczy J, Nayak KM, Howell KJ, Ross A, Forbester J, Salvestrini C, Mustata R, Perkins S, Andersson-Rolf A, Leenen E, Liebert A, Vallier L, Rosenstiel PC, Stegle O, Dougan G, Heuschkel R, Koo BK, Zilbauer M (2019) DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development. Gut 68(1):49–61. https://doi.org/10.1136/gutjnl-2017-314817CrossRefPubMed

15.

Fujii S, Suzuki K, Kawamoto A, Ishibashi F, Nakata T, Murano T, Ito G, Shimizu H, Mizutani T, Oshima S, Tsuchiya K, Nakamura T, Araki A, Ohtsuka K, Okamoto R, Watanabe M (2016) PGE2 is a direct and robust mediator of anion/fluid secretion by human intestinal epithelial cells. Sci Rep 6:36795. https://doi.org/10.1038/srep36795CrossRefPubMedPubMedCentral

16.

Tüysüz N, van Bloois L, van den Brink S, Begthel H, Verstegen MM, Cruz LJ, Hui L, van der Laan LJ, de Jonge J, Vries R, Braakman E, Mastrobattista E, Cornelissen JJ, Clevers H, Ten Berge D (2017) Lipid-mediated Wnt protein stabilization enables serum-free culture of human organ stem cells. Nat Commun 8:14578. https://doi.org/10.1038/ncomms14578CrossRefPubMedPubMedCentral

17.

VanDussen KL, Marinshaw JM, Shaikh N, Miyoshi H, Moon C, Tarr PI, Ciorba MA, Stappenbeck TS (2015) Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays. Gut 64(6):911–920. https://doi.org/10.1136/gutjnl-2013-306651CrossRefPubMed

18.

Pleguezuelos-Manzano C, Puschhof J, van den Brink S, Geurts V, Beumer J, Clevers H (2020) Establishment and culture of human intestinal organoids derived from adult stem cells. Curr Protoc Immunol 130(1):e106. https://doi.org/10.1002/cpim.106CrossRefPubMed

19.

Mustata RC, Vasile G, Fernandez-Vallone V, Strollo S, Lefort A, Libert F, Monteyne D, Pérez-Morga D, Vassart G, Garcia MI (2013) Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium. Cell Rep 5(2):421–432. https://doi.org/10.1016/j.celrep.2013.09.005CrossRefPubMed

20.

Fordham RP, Yui S, Hannan NR, Soendergaard C, Madgwick A, Schweiger PJ, Nielsen OH, Vallier L, Pedersen RA, Nakamura T, Watanabe M, Jensen KB (2013) Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury. Cell Stem Cell 13(6):734–744. https://doi.org/10.1016/j.stem.2013.09.015CrossRefPubMedPubMedCentral

21.

Holloway EM, Czerwinski M, Tsai YH, Wu JH, Wu A, Childs CJ, Walton KD, Sweet CW, Yu Q, Glass I, Treutlein B, Camp JG, Spence JR (2021) Mapping development of the human intestinal niche at single-cell resolution. Cell Stem Cell 28(3):568-580.e4. https://doi.org/10.1016/j.stem.2020.11.008CrossRefPubMed

22.

Guiu J, Jensen KB (2015) From definitive endoderm to gut-a process of growth and maturation. Stem Cells Dev 24(17):1972–1983. https://doi.org/10.1089/scd.2015.0017CrossRefPubMed

23.

Wells JM, Spence JR (2014) How to make an intestine. Development 141(4):752–760. https://doi.org/10.1242/dev.097386CrossRefPubMedPubMedCentral

24.

Ares GJ, Buonpane C, Yuan C, Wood D, Hunter CJ (2019) A novel human epithelial enteroid model of necrotizing enterocolitis. J Vis Exp. https://doi.org/10.3791/59194CrossRefPubMed

25.

Li B, Lee C, Cadete M, Zhu H, Koike Y, Hock A, Wu RY, Botts SR, Minich A, Alganabi M, Chi L, Zani-Ruttenstock E, Miyake H, Chen Y, Mutanen A, Ngan B, Johnson-Henry KC, De Coppi P, Eaton S, Määttänen P, Delgado-Olguin P, Sherman PM, Zani A, Pierro A (2019) Impaired Wnt/β-catenin pathway leads to dysfunction of intestinal regeneration during necrotizing enterocolitis. Cell Death Dis 10(10):743. https://doi.org/10.1038/s41419-019-1987-1CrossRefPubMedPubMedCentral

26.

Zhou J, Li C, Liu X, Chiu MC, Zhao X, Wang D, Wei Y, Lee A, Zhang AJ, Chu H, Cai JP, Yip CC, Chan IH, Wong KK, Tsang OT, Chan KH, Chan JF, To KK, Chen H, Yuen KY (2020) Infection of bat and human intestinal organoids by SARS-CoV-2. Nat Med 26(7):1077–1083. https://doi.org/10.1038/s41591-020-0912-6CrossRefPubMed

27.

Wu RY, Li B, Koike Y, Määttänen P, Miyake H, Cadete M, Johnson-Henry KC, Botts SR, Lee C, Abrahamsson TR, Landberg E, Pierro A, Sherman PM (2019) Human milk oligosaccharides increase mucin expression in experimental necrotizing enterocolitis. Mol Nutr Food Res 63(3):e1800658. https://doi.org/10.1002/mnfr.201800658CrossRefPubMed

28.

Merenda A, Fenderico N, Maurice MM (2020) Wnt signaling in 3D: recent advances in the applications of intestinal organoids. Trends Cell Biol 30(1):60–73. https://doi.org/10.1016/j.tcb.2019.10.003CrossRefPubMed

29.

Li Y, Liu Y, Liu B, Wang J, Wei S, Qi Z, Wang S, Fu W, Chen YG (2018) A growth factor-free culture system underscores the coordination between Wnt and BMP signaling in Lgr5+ intestinal stem cell maintenance. Cell Discov 4:49. https://doi.org/10.1038/s41421-018-0051-0CrossRefPubMedPubMedCentral

30.

Zhao J, Zeng Z, Sun J, Zhang Y, Li D, Zhang X, Liu M, Wang X (2017) A novel model of P-glycoprotein inhibitor screening using human small intestinal organoids. Basic Clin Pharmacol Toxicol 120(3):250–255. https://doi.org/10.1111/bcpt.12680CrossRefPubMed

31.

Matsumoto T, Mochizuki W, Nibe Y, Akiyama S, Matsumoto Y, Nozaki K, Fukuda M, Hayashi A, Mizutani T, Oshima S, Watanabe M, Nakamura T (2016) Retinol promotes in vitro growth of proximal colon organoids through a retinoic acid-independent mechanism. PLoS ONE 11(8):e0162049. https://doi.org/10.1371/journal.pone.0162049CrossRefPubMedPubMedCentral

32.

Matsumoto Y, Mochizuki W, Akiyama S, Matsumoto T, Nozaki K, Watanabe M, Nakamura T (2017) Distinct intestinal adaptation for vitamin B12 and bile acid absorption revealed in a new mouse model of massive ileocecal resection. Biol Open 6(9):1364–1374. https://doi.org/10.1242/bio.024927CrossRefPubMedPubMedCentral

33.

Drost J, van Jaarsveld RH, Ponsioen B, Zimberlin C, van Boxtel R, Buijs A, Sachs N, Overmeer RM, Offerhaus GJ, Begthel H, Korving J, van de Wetering M, Schwank G, Logtenberg M, Cuppen E, Snippert HJ, Medema JP, Kops GJ, Clevers H (2015) Sequential cancer mutations in cultured human intestinal stem cells. Nature 521(7550):43–47. https://doi.org/10.1038/nature14415CrossRefPubMed

34.

Fujii M, Matano M, Nanki K, Sato T (2015) Efficient genetic engineering of human intestinal organoids using electroporation. Nat Protoc 10(10):1474–1485. https://doi.org/10.1038/nprot.2015.088CrossRefPubMed

35.

Gjorevski N, Sachs N, Manfrin A, Giger S, Bragina ME, Ordóñez-Morán P, Clevers H, Lutolf MP (2016) Designer matrices for intestinal stem cell and organoid culture. Nature 539(7630):560–564. https://doi.org/10.1038/nature20168CrossRefPubMed

36.

Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, Barker N, Shroyer NF, van de Wetering M, Clevers H (2011) Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469(7330):415–418. https://doi.org/10.1038/nature09637CrossRefPubMed

37.

Boehnke K, Iversen PW, Schumacher D, Lallena MJ, Haro R, Amat J, Haybaeck J, Liebs S, Lange M, Schäfer R, Regenbrecht CR, Reinhard C, Velasco JA (2016) Assay establishment and validation of a high-throughput screening platform for three-dimensional patient-derived colon cancer organoid cultures. J Biomol Screen 21(9):931–941. https://doi.org/10.1177/1087057116650965CrossRefPubMedPubMedCentral

38.

Grossmann J, Maxson JM, Whitacre CM, Orosz DE, Berger NA, Fiocchi C, Levine AD (1998) New isolation technique to study apoptosis in human intestinal epithelial cells. Am J Pathol 153(1):53–62. https://doi.org/10.1016/S0002-9440(10)65545-9CrossRefPubMedPubMedCentral

39.

Grossmann J, Walther K, Artinger M, Kiessling S, Steinkamp M, Schmautz WK, Stadler F, Bataille F, Schultz M, Schölmerich J, Rogler G (2003) Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC). Eur J Cell Biol 82(5):262–270. https://doi.org/10.1078/0171-9335-00312CrossRefPubMed

40.

Tsai YH, Czerwinski M, Wu A, Dame MK, Attili D, Hill E, Colacino JA, Nowacki LM, Shroyer NF, Higgins PDR, Kao JY, Spence JR (2018) A method for cryogenic preservation of human biopsy specimens and subsequent organoid culture. Cell Mol Gastroenterol Hepatol 6(2):218-222.e7. https://doi.org/10.1016/j.jcmgh.2018.04.008CrossRefPubMedPubMedCentral

Title: Defined serum-free culture of human infant small intestinal organoids with predetermined doses of Wnt3a and R-spondin1 from surgical specimens
Authors: Yuka Matsumoto
Hiroyuki Koga
Mirei Takahashi
Kazuto Suda
Takanori Ochi
Shogo Seo
Go Miyano
Yuichiro Miyake
Hideaki Nakajima
Shiho Yoshida
Takafumi Mikami
Tadaharu Okazaki
Nobutaka Hattori
Atsuyuki Yamataka
Tetsuya Nakamura
Publication date: 01-11-2021
Publisher: Springer Berlin Heidelberg
Published in: Pediatric Surgery International / Issue 11/2021
Print ISSN: 0179-0358
Electronic ISSN: 1437-9813
DOI: https://doi.org/10.1007/s00383-021-04957-4

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Defined serum-free culture of human infant small intestinal organoids with predetermined doses of Wnt3a and R-spondin1 from surgical specimens

Abstract

Purpose

Methods

Results

Conclusion

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusion

Please log in to get access to this content

Other articles of this Issue 11/2021

Timing of adjuvant chemotherapy after laparotomy for Wilms tumor and neuroblastoma

Incidence of surgical procedures for gastrointestinal complications after abdominal wall closure in patients with gastroschisis and omphalocele

Different strategies, equivalent treatment approaches in terms of mortality in four university hospitals: a retrospective multicenter study of gastroschisis in Finland

The novel fetal MRI O/E CLV versus O/E LHR in predicting prognosis in congenital diaphragmatic hernias: can we teach an old dog new tricks?

Evaluation and management of rectovaginal fistula in anorectal malformation: an observational study

Comprehensive assessment of the quality and reliability of the ten most-viewed YouTube videos on thoracoscopic lobectomy in children: a comparison from the available videos on a peer-reviewed platform