title: Development of Functional Microfold (M) Cells from Intestinal Stem Cells in Primary Human Enteroids.
abstract: OBJECTIVE
  Intestinal microfold (M) cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer's patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs), and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting.
  
  
  METHODS
  Human intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium.
  
  
  RESULTS
  Functional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2) in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells.
  
  
  CONCLUSIONS
  Human intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an in vitro setting. We anticipate that this model can be used to generate large numbers of M cells for further functional studies of these key cells of intestinal immune induction and their impact on controlling enteric pathogens and the intestinal microbiome.
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Rouch
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Andrew
authors.initials: A
authors.lastname: Scott
authors.affiliation: Department of Bioengineering, Henry Samueli School of Engineering, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Nan Ye
authors.initials: NY
authors.lastname: Lei
authors.affiliation: Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital and the David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: R Sergio
authors.initials: RS
authors.lastname: Solorzano-Vargas
authors.affiliation: Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital and the David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Jiafang
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Medicine, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Elaine M
authors.initials: EM
authors.lastname: Hanson
authors.affiliation: Department of Bioengineering, Henry Samueli School of Engineering, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Masae
authors.initials: M
authors.lastname: Kobayashi
authors.affiliation: Department of Pathology, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Lewis
authors.affiliation: Department of Surgery, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America.
authors.forename: Matthias G
authors.initials: MG
authors.lastname: Stelzner
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.|Department of Bioengineering, Henry Samueli School of Engineering, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: James C Y
authors.initials: JC
authors.lastname: Dunn
authors.affiliation: Department of Medicine, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Lars
authors.initials: L
authors.lastname: Eckmann
authors.affiliation: Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital and the David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.|Eli and Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Martín G
authors.initials: MG
authors.lastname: Martín
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002478:Cells, Cultured; D006801:Humans; D018928:Immunity, Mucosal; D007413:Intestinal Mucosa / Q000166:cytology* / Q000276:immunology / Q000382:microbiology; D007421:Intestine, Small / Q000166:cytology* / Q000276:immunology / Q000382:microbiology; D010581:Peyer's Patches / Q000166:cytology* / Q000276:immunology / Q000382:microbiology; D053245:RANK Ligand / Q000276:immunology; D012486:Salmonella typhimurium / Q000276:immunology; D013234:Stem Cells / Q000166:cytology* / Q000276:immunology
chemical_list: D053245:RANK Ligand
references.citation: Annu Rev Cell Dev Biol. 2000;16:301-32
references.pmid: 11031239
references.citation: Mucosal Immunol. 2013 Sep;6(5):1027-37
references.pmid: 23360902
references.citation: Science. 2001 Dec 7;294(5549):2155-8
references.pmid: 11739954
references.citation: J Virol. 2014 Jun;88(12):6934-43
references.pmid: 24696493
references.citation: Gastroenterology. 2013 Aug;145(2):383-95.e1-21
references.pmid: 23644405
references.citation: J Cell Biochem. 2008 Jun 1;104(3):733-44
references.pmid: 18253935
references.citation: Gut. 1990 Sep;31(9):1011-5
references.pmid: 2210445
references.citation: Nat Cell Biol. 2004 Oct;6(10):931-40
references.pmid: 15448698
references.citation: J Acquir Immune Defic Syndr. 1991;4(8):760-5
references.pmid: 1856788
references.citation: Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9297-301
references.pmid: 1718004
references.citation: Clin Exp Immunol. 1989 Apr;76(1):144-8
references.pmid: 2661061
references.citation: PLoS One. 2011;6(4):e18083
references.pmid: 21483738
references.citation: Nature. 2003 May 22;423(6938):448-52
references.pmid: 12717451
references.citation: Nat Immunol. 2012 Aug;13(8):729-36
references.pmid: 22706340
references.citation: Genome Biol. 2012;13(3):242
references.pmid: 22439850
references.citation: Cell. 1996 Aug 9;86(3):345-8
references.pmid: 8756716
references.citation: Cell Host Microbe. 2012 Nov 15;12(5):645-56
references.pmid: 23159054
references.citation: Science. 1997 Aug 15;277(5328):949-52
references.pmid: 9252325
references.citation: Nature. 2005 Jun 16;435(7044):964-8
references.pmid: 15959516
references.citation: PLoS One. 2014;9(9):e107814
references.pmid: 25222024
references.citation: PLoS One. 2011;6(11):e26898
references.pmid: 22125602
references.citation: Tissue Eng Part C Methods. 2013 Dec;19(12):961-9
references.pmid: 23566043
references.citation: J Immunol. 2008 Jun 15;180(12):7840-6
references.pmid: 18523247
references.citation: Mol Cell Biol. 2012 Sep;32(18):3639-47
references.pmid: 22778137
references.citation: J Biol Chem. 2001 Aug 10;276(32):30350-8
references.pmid: 11402039
references.citation: Can J Microbiol. 1988 Oct;34(10):1142-7
references.pmid: 3196964
references.citation: Adv Drug Deliv Rev. 1998 Dec 1;34(2-3):261-283
references.pmid: 10837681
references.citation: Infect Immun. 1993 Dec;61(12):5279-85
references.pmid: 8225601
references.citation: Pharm Res. 1996 Dec;13(12):1838-45
references.pmid: 8987081
references.citation: J Immunol. 2009 Nov 1;183(9):5738-47
references.pmid: 19828638
references.citation: Ann N Y Acad Sci. 1992;664:18-26
references.pmid: 1456648
references.citation: Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9410-4
references.pmid: 12093918
references.citation: Cell Res. 2008 Aug;18(8):858-70
references.pmid: 18645583
references.citation: Nature. 2009 May 14;459(7244):262-5
references.pmid: 19329995
references.citation: J Control Release. 2001 Sep 11;76(1-2):149-68
references.pmid: 11532321
references.citation: Nat Genet. 2000 Jan;24(1):36-44
references.pmid: 10615124
references.citation: Nature. 2013 Mar 7;495(7439):65-9
references.pmid: 23446353