Monoclonal Antibodies Reveal Dynamic Plasticity Between Lgr5- and Bmi1-Expressing Intestinal Cell Populations

Abstract

Background & AimsContinual renewal of the intestinal epithelium is dependent on active- and slow-cycling stem cells that are confined to the crypt base. Tight regulation of these stem cell populations maintains homeostasis by balancing proliferation and differentiation to support critical intestinal functions. The hierarchical relation of discrete stem cell populations in homeostasis or during regenerative epithelial repair remains controversial. Although recent studies have supported a model for the active-cycling leucine-rich repeat-containing G-protein–coupled receptor 5 (Lgr5)+ intestinal stem cell () functioning upstream of the slow-cycling B lymphoma Mo-MLV insertion region 1 homolog (Bmi1)-expressing cell, other studies have reported the opposite relation. Tools that facilitate simultaneous analyses of these populations are required to evaluate their coordinated function.MethodsWe used novel monoclonal antibodies (mAbs) raised against murine intestinal epithelial cells in conjunction with ISC–green fluorescent protein (GFP) reporter mice to analyze relations between ISC populations by microscopy. Ex vivo 3-dimensional cultures, flow cytometry, and quantitative reverse-transcription polymerase chain reaction analyses were performed.ResultsTwo novel mAbs recognized distinct subpopulations of the intestinal epithelium and when used in combination permitted isolation of discrete Lgr5GFP and Bmi1GFP-enriched populations with stem activity. Growth from singly isolated Lgr5GFP ISCs gave rise to small spheroids. Spheroids did not express Lgr5GFP and instead up-regulated Bmi1GFP expression. Conversely, Bmi1-derived spheroids initiated Lgr5GFP expression as crypt domains were established.ConclusionsThese data showed the functional utility of murine mAbs in the isolation and investigation of Lgr5GFP and Bmi1GFP ISC-enriched populations. Ex vivo analyses showed hierarchical plasticity between different ISC-expressing states; specifically Lgr5GFP ISCs gave rise to Bmi1GFP cells, and vice versa. These data highlight the impact of temporal and physiological context on unappreciated interactions between Lgr5GFP and Bmi1GFP cells during crypt formation.

Heterogeneity and dynamics of active Kras-induced dysplastic lineages from mouse corpus stomach

“…To identify the DSCs and determine the extent of heterogeneity of Meta4 organoids, the Meta4 cells were dissociated and subjected to high-throughput functional analysis by micro-CellRaft Arrays (CRAs) 42,43 . Flow cytometry data demonstrated two populations of DSCs: CD44neg/CD133+/CD166+ (Double-positive) and CD44+/CD133+/CD166+cells (Triple-positive) (Fig.…”

Lgr5 and stem/progenitor gene expression in gastric/gastroesophageal junction carcinoma – significance of potentially retained stemness

“…one more intestinal (cluster #1, LGR5-high) and one more MSC-like (cluster #2, THY1-high). However, this is likely to be a simplified version of the story, considering the complex hierarchy between the different stem/progenitor cells in the GI tract [35,36]. Future studies may thus benefit from examining co-expression patterns of multiple markers, including Bmi1, which appear to mark slow-cycling stem/progenitor cells that can revert to express Lgr5 [35,37,38], while noting the different sub-cellular Lgr5 expression patterns.…”

Effect of Antifreeze Glycoproteins on Organoid Survival during and after Hypothermic Storage

“…Cystic organoids, sometimes referred to as “enterospheres”, consist of undifferentiated cells with stem cell-like properties. They differ from mature intestinal stem cells in having a distinct set of gene expression patterns as well as different sets of active signaling pathways [21,22,23]. These characteristics enable cystic organoids to recapitulate the essential features of intestinal tissue under a state of repair after injury [21,22].…”