Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids.
chemokines (cytokines)
differentiation
inflammatory bowel disease
intestinal epithelial cells (IECs)
oxygen
proliferation
single-cell RNA-sequencing (scRNAseq)
transcriptome
Journal
Frontiers in immunology
ISSN: 1664-3224
Titre abrégé: Front Immunol
Pays: Switzerland
ID NLM: 101560960
Informations de publication
Date de publication:
2023
2023
Historique:
received:
11
11
2022
accepted:
09
01
2023
entrez:
16
2
2023
pubmed:
17
2
2023
medline:
18
2
2023
Statut:
epublish
Résumé
The epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to <1% oxygen). We hypothesize that recapitulating the Growth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data. Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks. Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to
Sections du résumé
Background
The epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to <1% oxygen). We hypothesize that recapitulating the
Methods
Growth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data.
Results
Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks.
Conclusions
Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to
Identifiants
pubmed: 36793710
doi: 10.3389/fimmu.2023.1095812
pmc: PMC9922616
doi:
Substances chimiques
Lipocalin-2
0
Oxygen
S88TT14065
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1095812Informations de copyright
Copyright © 2023 Walaas, Gopalakrishnan, Bakke, Skovdahl, Flatberg, Østvik, Sandvik and Bruland.
Déclaration de conflit d'intérêts
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Références
Cell Death Differ. 2012 May;19(5):743-55
pubmed: 22139129
Cell. 2019 Jun 13;177(7):1888-1902.e21
pubmed: 31178118
Clin Exp Immunol. 2013 Sep;173(3):502-11
pubmed: 23668802
Transl Vis Sci Technol. 2022 Feb 01;11(2):33
pubmed: 35191961
Cell Rep. 2022 Sep 27;40(13):111439
pubmed: 36170836
Am J Physiol Gastrointest Liver Physiol. 2001 Jul;281(1):G144-50
pubmed: 11408266
Nat Rev Immunol. 2014 Mar;14(3):141-53
pubmed: 24566914
PLoS Comput Biol. 2021 Aug 24;17(8):e1009290
pubmed: 34428202
J Exp Med. 1998 Jan 5;187(1):129-34
pubmed: 9419219
J Immunol. 2017 Dec 1;199(11):3721-3730
pubmed: 29158348
Tumour Biol. 2016 Nov;37(11):14851-14861
pubmed: 27644243
PLoS Biol. 2022 Mar 31;20(3):e3001592
pubmed: 35358182
Cell Host Microbe. 2015 May 13;17(5):662-71
pubmed: 25865369
Gastroenterology. 2003 May;124(5):1381-94
pubmed: 12730878
Front Cell Infect Microbiol. 2020 May 15;10:227
pubmed: 32500042
Bioinformatics. 2009 Jan 15;25(2):288-9
pubmed: 19033274
Nature. 2009 May 14;459(7244):262-5
pubmed: 19329995
Front Immunol. 2021 Feb 12;11:547102
pubmed: 33643277
J Cell Biochem. 2006 Dec 15;99(6):1616-27
pubmed: 16823775
Stem Cell Res Ther. 2018 May 24;9(1):148
pubmed: 29793517
J Pathol. 2019 Jul;248(3):316-325
pubmed: 30746716
Cells. 2022 Oct 04;11(19):
pubmed: 36231085
Database (Oxford). 2019 Jan 1;2019:
pubmed: 30951143
Front Immunol. 2019 Apr 16;10:806
pubmed: 31040849
Nat Rev Genet. 2006 May;7(5):349-59
pubmed: 16619050
Nat Rev Mol Cell Biol. 2021 Jan;22(1):39-53
pubmed: 32958874
Aliment Pharmacol Ther. 2022 Feb;55(4):401-411
pubmed: 35014040
CA Cancer J Clin. 2021 May;71(3):209-249
pubmed: 33538338
Gastroenterology. 2020 Sep;159(3):984-998.e1
pubmed: 32433978
Physiol Rev. 2019 Jan 1;99(1):161-234
pubmed: 30354965
Nature. 1998 Apr 9;392(6676):565-8
pubmed: 9560152
Cancers (Basel). 2021 May 28;13(11):
pubmed: 34071313
World J Gastroenterol. 2019 Aug 14;25(30):4125-4147
pubmed: 31435168
Nat Genet. 2000 May;25(1):25-9
pubmed: 10802651
Stem Cells. 2022 Mar 16;40(2):123-132
pubmed: 35258629
J Gastroenterol. 2021 Oct;56(10):914-927
pubmed: 34414506
Annu Rev Immunol. 2020 Apr 26;38:341-363
pubmed: 31961750
Cell Rep. 2016 Feb 9;14(5):1062-1073
pubmed: 26832409
Nat Methods. 2012 Jun 28;9(7):676-82
pubmed: 22743772
Trends Immunol. 2018 Sep;39(9):677-696
pubmed: 29716793
Inflamm Bowel Dis. 2020 Feb 11;26(3):407-422
pubmed: 31751457
J Exp Med. 2001 May 7;193(9):1027-34
pubmed: 11342587
Cell Tissue Res. 2018 Nov;374(2):339-348
pubmed: 29869714
Trends Immunol. 2021 Jul;42(7):604-621
pubmed: 34171295
Cell. 1990 Jun 1;61(5):759-67
pubmed: 2188735
J Crohns Colitis. 2020 Jul 30;14(7):920-934
pubmed: 32020185
Cell Mol Biol (Noisy-le-grand). 2003 Feb;49(1):77-87
pubmed: 12839339
Cell Mol Gastroenterol Hepatol. 2017 Feb 20;3(3):303-315
pubmed: 28462372
PLoS Genet. 2012 Feb;8(2):e1002525
pubmed: 22346769
Nat Rev Gastroenterol Hepatol. 2019 Jan;16(1):19-34
pubmed: 30429586
Oncol Rep. 2013 Jan;29(1):149-54
pubmed: 23128569
Nature. 2019 Mar;567(7746):49-55
pubmed: 30814735
Inflamm Bowel Dis. 2013 Feb;19(2):265-74
pubmed: 22685032
Front Genet. 2020 Jan 17;10:1331
pubmed: 32010190
Front Endocrinol (Lausanne). 2020 Feb 21;11:57
pubmed: 32153502
PLoS One. 2009 Sep 15;4(9):e7045
pubmed: 19753114
Mol Cell Biol. 2019 Jun 27;39(14):
pubmed: 31061092
Nat Genet. 2017 May;49(5):708-718
pubmed: 28319088
N Engl J Med. 2011 Aug 11;365(6):537-47
pubmed: 21830968
Nat Commun. 2022 Jun 6;13(1):3135
pubmed: 35668108
Aliment Pharmacol Ther. 2008 Jan 15;27(2):104-19
pubmed: 17973645
Blood. 2011 Jan 13;117(2):459-69
pubmed: 20952688
Chem Biol Interact. 2022 Jul 1;361:109959
pubmed: 35533734
Proc Natl Acad Sci U S A. 2000 Jan 18;97(2):529-31
pubmed: 10639111
mBio. 2022 Jun 28;13(3):e0132122
pubmed: 35638758
Biofactors. 2016 Jan-Feb;42(1):93-105
pubmed: 26891020
Front Pharmacol. 2021 May 13;12:679741
pubmed: 34054553
Front Immunol. 2022 May 17;13:882277
pubmed: 35655783
PLoS One. 2015 Nov 04;10(11):e0141710
pubmed: 26536229
J Lab Clin Med. 1982 May;99(5):701-8
pubmed: 6279749
J Biol Chem. 2018 Apr 20;293(16):6039-6051
pubmed: 29487135
Mucosal Immunol. 2013 Nov;6(6):1110-8
pubmed: 23462909
Sci Rep. 2020 Jun 29;10(1):10533
pubmed: 32601325
Biomaterials. 2017 Nov;146:86-96
pubmed: 28898760
Stem Cells. 2020 Nov;38(11):1454-1466
pubmed: 32761664
Redox Biol. 2020 Feb;30:101420
pubmed: 31935648
Drug Discov Today. 2022 Apr;27(4):1167-1175
pubmed: 34896626
Nucleic Acids Res. 2019 Jan 8;47(D1):D330-D338
pubmed: 30395331
J Cell Physiol. 2000 Mar;182(3):311-22
pubmed: 10653597
Mol Biol Cell. 2019 May 1;30(10):1129-1137
pubmed: 31034354
J Gastroenterol Hepatol. 2017 Jan;32(1):128-135
pubmed: 27640344
Proc Natl Acad Sci U S A. 2013 Dec 3;110(49):19820-5
pubmed: 24248342
J Exp Med. 1993 Jun 1;177(6):1809-14
pubmed: 8496693
Sci Adv. 2022 Jan 14;8(2):eabh3375
pubmed: 35020422
Am J Physiol Cell Physiol. 2015 Sep 15;309(6):C350-60
pubmed: 26179603
Nat Commun. 2017 Feb 10;8:14262
pubmed: 28186126
Cell. 2019 Nov 14;179(5):1144-1159.e15
pubmed: 31708126
Drug Metab Dispos. 2014 Dec;42(12):2016-22
pubmed: 25233858
Int J Mol Sci. 2019 Mar 08;20(5):
pubmed: 30857245
BMC Cell Biol. 2010 Nov 02;11:85
pubmed: 21044324
Cell Stem Cell. 2010 Jan 8;6(1):25-36
pubmed: 20085740
Nat Rev Mol Cell Biol. 2014 Jan;15(1):19-33
pubmed: 24326621
Science. 2018 Nov 30;362(6418):
pubmed: 30498100