Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging

Abstract

SummaryThe rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt1. Alongside Lgr5, intestinal stem cells have been associated with various markers, which are expressed heterogeneously within the crypt base region1-6. Previous quantitative clonal fate analyses have proposed that homeostasis occurs as the consequence of neutral competition between dividing stem cells7-9. However, the short-term behaviour of individual Lgr5+ cells positioned at different locations within the crypt base compartment has not been resolved. Here, we established the short-term dynamics of intestinal stem cells using a 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 (TA) domain, following division of proximate cells, implying that determination of stem cell fate can be uncoupled from division. Through the 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.

Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids

“…To perform intravital microscopy, human in vitro engineered organoids were infected (30) with pLKO-1.UbC.Dendra2.puro lentivirus. Ten to 12 wk after orthotopic organoid transplantation, an abdominal imaging window was surgically implanted on the primary tumor as described (12,31). The surgical procedure was performed under isoflurane inhalation anesthesia.…”

“…The converted-red isoform of the protein was collected to monitor cell migration. The imaging areas were retraced in subsequent imaging sessions by storing the stage coordinates of the imaging areas and tracking photomarked Dendra2 cells as described (31). Custom-designed Visual Basic and ImageJ software were used to enhance the red signal from the photoswitched cells.…”

Wnt some lose some: transcriptional governance of stem cells by Wnt/β-catenin signaling

“…Another interesting parallel between HFSCs and ISCs is that they each exist in two discrete states based in part on their sensitivity to Wnt signaling: In both cases, the more quiescent stem cells (hair follicle bulge and crypt +4 ISCs) are in a more Wnt-restricted microenvironment, while the primed progenitors (HG) or active stem cells (0 to +3 ISCs) have a reduced threshold for activation upon canonical Wnt stimulation (Sangiorgi and Capecchi 2008;Greco et al 2009;Tian et al 2011;Buczacki et al 2013;Ritsma et al 2014). In both cases, Wnt/b-catenin activity is associated with proliferation and cell fate determination, and, as discussed above, these two features might be inseparable if Wnt/b-catenin turns out to function in these tissues at least in part to promote asymmetric cell divisions.…”

Microenvironmental control of stem cell fate in intestinal homeostasis and disease

“…Recent advances in biotechnology have led to a surge of studies that have characterized the properties and interactions of putative intestinal stem cell populations in vivo 4, 5. Transgenic activation of heritable fluorescent labels in cells expressing candidate stem cell markers allows accurate cell fate mapping and progeny lineage tracing over time, to satisfy the defining stem cell characteristics of self‐renewal and multipotency.…”