The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt1. In addition to the Lgr5 marker, intestinal stem cells have been associated with other markers that are expressed heterogeneously within the crypt base region1,2,3,4,5,6. Previous quantitative clonal fate analyses have led to the proposal that homeostasis occurs as the consequence of neutral competition between dividing stem cells7,8,9. However, the short-term behaviour of individual Lgr5+ cells positioned at different locations within the crypt base compartment has not been resolved. Here we establish the short-term dynamics of intestinal stem cells using the novel approach of continuous intravital imaging of Lgr5-Confetti mice. We find that Lgr5+ cells in the upper part of the niche (termed ‘border cells’) can be passively displaced into the transit-amplifying domain, after the division of proximate cells, implying that the determination of stem-cell fate can be uncoupled from division. Through quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed ‘central cells’, experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5+ cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem-cell maintenance in which a dynamically heterogeneous cell population is able to function long term as a single stem-cell pool.
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The authors would like to thank A. de Graaff from the Hubrecht Imaging Center for imaging support, all members of the van Rheenen group for useful discussions and the Hubrecht Institute animal caretakers for animal support. This work was supported by a Vidi fellowship (91710330; J.v.R.) and equipment grants (175.010.2007.00 and 834.11.002; J.v.R.) from the Dutch Organization of Scientific Research (NWO), a grant from the Dutch Cancer Society (KWF; HUBR 2009-4621; J.v.R.), a grant from the Association for International Cancer Research (AICR; 13-0297; J.v.R.), and the Wellcome Trust (grant number 098357/Z/12/Z; B.D.S.).
Extended data figures
Lgr5+ CBC cells are shown in green, non-CBC cells are shown in red and Collagen 1 is shown in blue.
Left, time series of Lgr5+ CBC cells in a crypt. In the cartoons, Lgr5+ CBC cells are highlighted and the moving cells are colour coded. In the right two videos cell tracks are shown by lines and the centre of cells is indicated by dots. The time is indicated in hours. Scale bar, 20 µm.
Three days after Tamoxifen administration, mice were imaged in real-time. Lgr5+ CBC cells within the entire stem-cell compartment of a single crypt are projected onto one plane and are shown over time. Note the outlined cells that divide and move.
Three days after Tamoxifen administration, mice were imaged in real-time. Lgr5+ CBC cells within the entire stem-cell compartment of a single crypt are projected onto one plane and they are shown over time. Note the outlined cells that divide, move, and disappear.
In mice where the human DT receptor (DTR) fused to eGFP was knocked in the Lgr5 locus (Lgr5DTR:eGFP), Lgr5+ cells were fully ablated using diphtheria toxin (DT) injection. The recovery was monitored by acquiring a z-stack at 58hrs. The video shows the z-stack. The empty crypts are indicated with yellow. The crypts containing one recovered cell are indicated with red. The crypts containing more than 1 recovered cell are indicated with grey.
In mice where the human DT receptor (DTR) fused to eGFP was knocked in the Lgr5 locus (Lgr5DTR:eGFP), Lgr5+ cells were fully ablated using diphtheria toxin (DT) injection. The video shows a 3D reconstruction of clusters of recovered Lgr5+ cells from different sizes found 72hrs after ablation. Note the cohesion of these clusters, which is suggesting their origin lies in clonal expansion of cells.
About this article
The FEBS Journal (2019)