Identification of a cKit + colonic crypt base secretory cell that supports Lgr5 + stem cells in mice

Michael E. Rothenberg, Ysbrand Nusse, Tomer Kalisky, John J. Lee, Piero Dalerba, Ferenc Scheeren, Neethan Lobo, Subhash Kulkarni, Sopheak Sim, Dalong Qian, Philip A. Beachy, Pankaj Jay Pasricha, Stephen R. Quake, Michael F. Clarke

Research output: Contribution to journalArticle

Abstract

Background & Aims: Paneth cells contribute to the small intestinal niche of Lgr5 + stem cells. Although the colon also contains Lgr5 + stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5 + stem cells. Methods: We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. Results: Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117 + crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit + goblet cells were interdigitated with Lgr5 + stem cells. In vivo, this colonic cKit + population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit + cells. When isolated from mouse colon, cKit + cells promoted formation of organoids from Lgr5 + stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit + cells using a toxin-conjugated antibody, organoid formation decreased. Conclusions: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5 + stem cells.

Original languageEnglish (US)
JournalGastroenterology
Volume142
Issue number5
DOIs
StatePublished - May 2012
Externally publishedYes

Fingerprint

Paneth Cells
Organoids
Stem Cells
Goblet Cells
Colon
Flow Cytometry
Stem Cell Niche
Gene Expression
Amyloid Precursor Protein Secretases
Stem Cell Factor
Reverse Transcriptase Polymerase Chain Reaction
Epidermal Growth Factor
Population
Antibody Formation
Cluster Analysis
Epithelium
Cell Count
Ligands

Keywords

  • Cancer
  • Inflammatory Bowel Disease
  • Intestine
  • Regenerate

ASJC Scopus subject areas

  • Gastroenterology

Cite this

Rothenberg, M. E., Nusse, Y., Kalisky, T., Lee, J. J., Dalerba, P., Scheeren, F., ... Clarke, M. F. (2012). Identification of a cKit + colonic crypt base secretory cell that supports Lgr5 + stem cells in mice. Gastroenterology, 142(5). https://doi.org/10.1053/j.gastro.2012.02.006

Identification of a cKit + colonic crypt base secretory cell that supports Lgr5 + stem cells in mice. / Rothenberg, Michael E.; Nusse, Ysbrand; Kalisky, Tomer; Lee, John J.; Dalerba, Piero; Scheeren, Ferenc; Lobo, Neethan; Kulkarni, Subhash; Sim, Sopheak; Qian, Dalong; Beachy, Philip A.; Pasricha, Pankaj Jay; Quake, Stephen R.; Clarke, Michael F.

In: Gastroenterology, Vol. 142, No. 5, 05.2012.

Research output: Contribution to journalArticle

Rothenberg, ME, Nusse, Y, Kalisky, T, Lee, JJ, Dalerba, P, Scheeren, F, Lobo, N, Kulkarni, S, Sim, S, Qian, D, Beachy, PA, Pasricha, PJ, Quake, SR & Clarke, MF 2012, 'Identification of a cKit + colonic crypt base secretory cell that supports Lgr5 + stem cells in mice', Gastroenterology, vol. 142, no. 5. https://doi.org/10.1053/j.gastro.2012.02.006
Rothenberg, Michael E. ; Nusse, Ysbrand ; Kalisky, Tomer ; Lee, John J. ; Dalerba, Piero ; Scheeren, Ferenc ; Lobo, Neethan ; Kulkarni, Subhash ; Sim, Sopheak ; Qian, Dalong ; Beachy, Philip A. ; Pasricha, Pankaj Jay ; Quake, Stephen R. ; Clarke, Michael F. / Identification of a cKit + colonic crypt base secretory cell that supports Lgr5 + stem cells in mice. In: Gastroenterology. 2012 ; Vol. 142, No. 5.
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abstract = "Background & Aims: Paneth cells contribute to the small intestinal niche of Lgr5 + stem cells. Although the colon also contains Lgr5 + stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5 + stem cells. Methods: We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. Results: Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117 + crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit + goblet cells were interdigitated with Lgr5 + stem cells. In vivo, this colonic cKit + population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit + cells. When isolated from mouse colon, cKit + cells promoted formation of organoids from Lgr5 + stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit + cells using a toxin-conjugated antibody, organoid formation decreased. Conclusions: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5 + stem cells.",
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T1 - Identification of a cKit + colonic crypt base secretory cell that supports Lgr5 + stem cells in mice

AU - Rothenberg, Michael E.

AU - Nusse, Ysbrand

AU - Kalisky, Tomer

AU - Lee, John J.

AU - Dalerba, Piero

AU - Scheeren, Ferenc

AU - Lobo, Neethan

AU - Kulkarni, Subhash

AU - Sim, Sopheak

AU - Qian, Dalong

AU - Beachy, Philip A.

AU - Pasricha, Pankaj Jay

AU - Quake, Stephen R.

AU - Clarke, Michael F.

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N2 - Background & Aims: Paneth cells contribute to the small intestinal niche of Lgr5 + stem cells. Although the colon also contains Lgr5 + stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5 + stem cells. Methods: We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. Results: Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117 + crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit + goblet cells were interdigitated with Lgr5 + stem cells. In vivo, this colonic cKit + population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit + cells. When isolated from mouse colon, cKit + cells promoted formation of organoids from Lgr5 + stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit + cells using a toxin-conjugated antibody, organoid formation decreased. Conclusions: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5 + stem cells.

AB - Background & Aims: Paneth cells contribute to the small intestinal niche of Lgr5 + stem cells. Although the colon also contains Lgr5 + stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5 + stem cells. Methods: We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. Results: Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117 + crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit + goblet cells were interdigitated with Lgr5 + stem cells. In vivo, this colonic cKit + population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit + cells. When isolated from mouse colon, cKit + cells promoted formation of organoids from Lgr5 + stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit + cells using a toxin-conjugated antibody, organoid formation decreased. Conclusions: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5 + stem cells.

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KW - Intestine

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