Background & Aims
Methods
Results
Conclusions
Graphical abstract

Keywords
Abbreviations used in this paper:
AE2 (anion exchanger 2), CA (carbonic anhydrase), cAMP (cyclic adenosine monophosphate), CFTR (cystic fibrosis transmembrane conductance regulator), ΔIsc (change in short-circuit current), DRA (down-regulated-in-adenoma), Isc (short-circuit current), KRB (Krebs–Ringer bicarbonate), mRNA (messenger ribonucleic acid), NBC (Na+/HCO3- co-transporter), NBCe1 (electrogenic Na+/HCO3- co-transporter 1), NHE (Na+/H+ exchanger), NKCC1 (Na+/K+/2Cl- co-transporter 1), qRT-PCR (quantitative real-time polymerase chain reaction), SDS (sodium dodecyl sulfate), SITS (4-Acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid disodium salt hydrate), TER (transepithelial electrical resistance)Materials and Methods
Propagation of Human Enteroid Cultures
- Heijmans J.
- van Lidth de Jeude J.F.
- Koo B.K.
- Rosekrans S.L.
- Wielenga M.C.
- van de Wetering M.
- Ferrante M.
- Lee A.S.
- Onderwater J.J.
- Paton J.C.
- Paton A.W.
- Mommaas A.M.
- Kodach L.L.
- Hardwick J.C.
- Hommes D.W.
- Clevers H.
- Muncan V.
- van den Brink G.R.
Enteroid Monolayer Formation and Differentiation
Quantitative Real-Time Polymerase Chain Reaction
Gene | Forward (5′-3′) | Reverse (5′-3′) |
---|---|---|
AE2 | TCCCTCTCCTTCCGCAGT | TGCTGGTCCAGATCCAAGA |
ATP1A1 | ACAGACTTGAGCCGGGGATTA | TCCATTCAGGAGTAGTGGGAG |
ATP1B1 | CCGGTGGCAGTTGGTTTAAGA | GCATCACTTGGATGGTTCCGA |
CA1 | TTGAGGACAACGATAACCGATCA | CTACGTGAAGCTCGGCAGAAT |
CA2 | CACCCCTCCTCTTCTGGAAT | AGTTTACGGAATTTCAACACCTG |
CA4 | CTGGTGCTACGAGGTTCAAGC | GAAGAAGAAGCGTCCCAGTTT |
CA9 | CCTTTGCCAGAGTTGACGAG | GCAACTGCTCATAGGCACTG |
CFTR | GAAGTAGTGATGGAGAATGTAACAGC | GCTTTCTCAAATAATTCCCCAAA |
DRA | CCATCATCGTGCTGATTGTC | AGCTGCCAGGACGGACTT |
GATA4 | GGAAGCCCAAGAACCTGAAT | GTTGCTGGAGTTGCTGGAA |
KCNE3 | TTAAGGGAGGTCGTCACTGG | ATGCACAAGGCTTCGGTCTA |
KCNQ1 | GCTTTTCCTAATAAACGTGGAGAA | GGAACCAAGGTGAGAGCAGT |
Ki67 | GAGGTGTGCAGAAAATCCAAA | CTGTCCCTATGACTTCTGGTTGT |
LGR5 | ACCAGACTATGCCTTTGGAAAC | TTCCCAGGGAGTGGATTCTAT |
NBCe1 | CCTCAAGCATGTGTGTGATGA | ACTCTTCGGCACATGGACTC |
NBCn1 | GCAAGAAACATTCTGACCCTCA | GCTTCCACCACTTCCATTACCT |
NHE1 | TCTTCACCGTCTTTGTGCAG | ATGGAGCGCTTCGTCTCTT |
NHE2 | CTTCCACTTCAACCTCCCGAT | GCTGCTATTGCCATCTGCAA |
NHE3 | GTCTTCCTCAGTGGGCTCAT | ATGAGGCTGCCAAACAGG |
NKCC1 | AAAGGAACATTCAAGCACAGC | CTAGACACAGCACCTTTTCGTG |
PAT-1 | TCTACCAGTTCATTGTTCAGAGGA | GAGAGGGTAGGTCTTCCAAGG |
SI | TTTTGGCATCCAGATTCGAC | ATCCAGGCAGCCAAGAATC |
18S | GCAATTATTCCCCATGAACG | GGGACTTAATCAACGCAAGC |
Immunoblotting
Antigen | Manufacturer | Catalog number | Host | Dilution |
---|---|---|---|---|
ATP1A1 | Developmental Studies Hybridoma Bank (Iowa City, IA) | a5 | Mouse | 1:1000 |
CA2 | Novus (Littleton, CO) | NB600-919 | Rabbit | 1:500 |
CFTR | Cystic Fibrosis Foundation Therapeutics (Chapel Hill, NC) | 217 | Mouse | 1:400 |
DRA | Santa Cruz | sc-376187 | Mouse | 1:200 |
GAPDH | Sigma-Aldrich | G8795 | Mouse | 1:5000 |
NBCe1 | Abcam (Cambridge, MA) | ab30322 | Rabbit | 1:500 |
NHE2 | Provided by Dr Chung-Ming Tse (Johns Hopkins University, Baltimore, MD) | N/A | Rabbit | 1:500 |
NHE3 | Novus (Littleton, CO) | NBP1-82574 | Rabbit | 1:500 |
NKCC1 | Developmental Studies Hybridoma Bank (Iowa City, IA) | T4-C | Mouse | 1:1000 |
Transepithelial Electrical Resistance
Ussing Chamber/Short-Circuit Current Measurement
Compound | Manufacturer | Final concentration | Side |
---|---|---|---|
Acetazolamide | Sigma-Aldrich | 250 μmol/L | AP + BL |
Bumetanide | Sigma-Aldrich | 100 μmol/L | BL |
CFTRinh-172 | EMD Millipore (Burlington, MA) | 5 or 25 μmol/L | AP |
Chromanol 293B | Sigma-Aldrich | 10 μmol/L | BL |
Forskolin | Sigma-Aldrich | 10 μmol/L | BL |
Ouabain | Sigma-Aldrich | 100 μmol/L | BL |
S0859 | Sigma-Aldrich | 30 μmol/L | BL |
SITS | Sigma-Aldrich | 1 mmol/L | BL |
Tenapanor | Ardelyx (Fremont, CA) | 0.1–1 μmol/L | AP |
Statistical Analysis
Results
Phenotypic Changes After Differentiation

Expression of Ion Transporters and Carbonic Anhydrase Isoforms Upon Differentiation



Reduced cAMP-Stimulated Anion Secretion Upon Differentiation

Apical Ion Transporters in cAMP-Stimulated Anion Secretion


Basolateral Ion Transporters and Carbonic Anhydrase(s) in cAMP-Stimulated Anion Secretion

Basolateral Ion Transporters in cAMP-Stimulated Cl- Secretion

Discussion
- Gorrieri G.
- Scudieri P.
- Caci E.
- Schiavon M.
- Tomati V.
- Sirci F.
- Napolitano F.
- Carrella D.
- Gianotti A.
- Musante I.
- Favia M.
- Casavola V.
- Guerra L.
- Rea F.
- Ravazzolo R.
- Di Bernardo D.
- Galietta L.J.
Acknowledgments
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Footnotes
Author contributions Jianyi Yin, Chung-Ming Tse, and Mark Donowitz designed the experiments; Jianyi Yin, Chung-Ming Tse, Leela Rani Avula, Varsha Singh, and Jennifer Foulke-Abel performed experiments and analyzed data; Jianyi Yin drafted the manuscript; and Chung-Ming Tse, Hugo R. de Jonge, and Mark Donowitz revised the manuscript.
Conflicts of interest The authors disclose no conflicts.
Funding This study was supported in part by National Institutes of Health grants RO1 DK26523, RO1 DK61765, P01 DK072084, P30 DK089502, UO1AI125181, UH3TR00003, U18TR000552, and R24 DK99803.
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