title: Side population sorting separates subfractions of cycling and non-cycling intestinal stem cells.
abstract: We report here that side population (SP) sorting allows for the simultaneous isolation of two intestinal stem cell (ISC) subsets from wild-type (WT) mice which are phenotypically different and represent cycling and non-cycling pools of cells. Following 5-ethynyl-2'-deoxyuridine (EdU) injection, in the upper side population (USP) the percentage of EdU+ was 36% showing this fraction to be highly proliferative. In the lower side population (LSP), only 0.4% of cells were EdU+, indicating this fraction to be predominantly non-cycling. Using Lgr5-EGFP mice, we show that Lgr5-EGFP(hi) cells, representing actively cycling ISCs, are essentially exclusive to the USP. In contrast, using histone 2B-YFP mice, SP analysis revealed YFP label retaining cells (LRCs) in both the USP and the LSP. Correspondingly, evaluation of the SP fractions for mRNA markers by qRT-PCR showed that the USP was enriched in transcripts associated with both quiescent and active ISCs. In contrast, the LSP expressed mRNA markers of quiescent ISCs while being de-enriched for those of the active ISC. Both the USP and LSP are capable of generating enteroids in culture which include the four intestinal lineages. We conclude that sorting of USP and LSP fractions represents a novel isolation of cycling and non-cycling ISCs from WT mice.
authors.affiliation: Department of Medicine, Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: von Furstenberg
authors.affiliation: Cancer Research UK, Cambridge Research Institute, Cambridge CB2 ORE, UK.
authors.forename: Simon J A
authors.initials: SJ
authors.lastname: Buczacki
authors.affiliation: Department of Medicine, Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Smith
authors.affiliation: Department of Medicine, Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Seiler
authors.affiliation: Cancer Research UK, Cambridge Research Institute, Cambridge CB2 ORE, UK.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Winton
authors.affiliation: Department of Medicine, Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: sjh@med.unc.edu.
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Henning
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D007422:Intestines / Q000166:cytology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D013234:Stem Cells / Q000166:cytology*