Background & aims: Continual 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 (ISC) 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.
Methods: We 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.
Results: Two 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.
Conclusions: These 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.
Keywords: 3D, 3-dimensional; 4-OHT, 4-hydroxytamoxifen; APC, allophycocyanin; Bmi1; Bmi1, B lymphoma Mo-MLV insertion region 1 homolog; Egf, epidermal growth factor; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; HBSS, Hank’s balanced salt solution; Hierarchy; ISC, intestinal stem cell; Intestinal Stem Cells; Lgr5; Lgr5, leucine-rich repeat-containing G-protein–coupled receptor 5; Lyz, lysozyme; OHSU, Oregon Health and Science University; PBS, phosphate-buffered saline; PE, Phycoerythrin; Plasticity; Rspo1, R-spondin1; TdT, tdTomato; Wnt, wingless-type MMTV (mouse mammary tumor virus) integration site; cDNA, complementary DNA; mAb, monoclonal antibody; mRNA, messenger RNA; qRT-PCR, quantitative reverse-transcription polymerase chain reaction.