@article{a48adb64e90845e1a999d3bb3c877f49,
title = "Monoclonal Antibodies Reveal Dynamic Plasticity Between Lgr5- and Bmi1-Expressing Intestinal Cell Populations",
abstract = "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 = "Bmi1, Hierarchy, Intestinal Stem Cells, Lgr5, Plasticity",
author = "Smith, {Nicholas R.} and Swain, {John R.} and Davies, {Paige S.} and Gallagher, {Alexandra C.} and Parappilly, {Michael S.} and Beach, {Catherine Z.} and Streeter, {Philip R.} and Williamson, {Ian A.} and Magness, {Scott T.} and Wong, {Melissa H.}",
note = "Funding Information: To fully appreciate relationships between ISC populations, tools to concurrently analyze these discrete populations in tissue or in ex vivo cultures are required. Antibodies that support identification and isolation of Bmi1+ and Lgr5+ ISCs are lacking, therefore we generated antibodies against murine intestinal epithelial cells and screened more than 6000 clones for antibodies with potential overlap with ISC populations within the crypt. One novel mAb clone, F5C12, identified in our screen showed specific expression within the ISC zone. This mAb robustly stained the apical surface of epithelial cells in the lower region of the crypt (Figure 2A), along the entire proximal-distal axis of the small intestine from duodenum, jejunum, ileum, and within the colon (Figure 3 A and B). Co-staining of Lgr5GFP and F5C12 in reporter mouse intestines showed intriguing overlapping expression (Figure 2B); importantly, the F5C12 mAb was expressed in cells from all crypts, unlike the mosaic expression of the Lgr5-GFP reporter, indicating uniform expression of the F5C12 epitope. Despite this, the Lgr5-GFP reporter and the F5C12 staining pattern had subtle differences that immunofluorescent analyses were not able to resolve on a cellular level. Therefore, we FACS-isolated F5C12+ crypt epithelial cells and performed qRT-PCR for crypt-based and differentiated cell lineage gene expression. The F5C12 mAb reproducibly fractionated 20.7% ± 1.2% of crypt-enriched single epithelial cells (Figure 2C). Consistent with the crypt-base expression pattern, the F5C12+ cell population showed gene expression enrichment of the crypt markers CD44, Sox9, Ki67, and cyclinD1, and was de-enriched for differentiated lineage markers Fabpl, Muc2, and ChgA relative to a crypt-enriched cell population34 (Figure 2F). Consistent with overlapping protein expression with Lgr5GFP, F5C12+ cells were enriched for Lgr5 gene expression, but not for Bmi1 or the Paneth cell marker Lyz1 (Figure 2G). Bmi1 mRNA is reported to be diffusely expressed in all crypt-based cells, 14,39,40 however, it is expressed at lower levels in Lgr5GFP cells relative to all cells within the crypt (Figure 2G) and therefore provides an additional approach to assay for discrete crypt-based cell populations. To evaluate the extent to which the F5C12 mAb was co-expressed on Lgr5GFP ISCs, we analyzed Lgr5-GFP reporter mouse intestines by flow cytometry (Figure 2D). Consistent with the observed in vivo staining pattern, the majority of all Lgr5GFP cells expressed F5C12 epitopes (66.5% ± 1.5%) (Figure 2D, left). However, this population increased when analyzing only the Lgr5GFP-hi stem cell population (85.7% ± 2.0%) (Figure 2D, right), consistent with enrichment of Lgr5+ ISCs within the F5C12+ population. When we performed a similar experiment using Bmi1-GFP reporter mice, we found that Bmi1GFP cells were rarely marked by F5C12 staining (4.5% ± 0.4%) (Figure 2E). These data support that F5C12 is expressed on Lgr5+ ISCs, and not robustly expressed on the Bmi1GFP population or the supportive Paneth cell. Publisher Copyright: {\textcopyright} 2018 The Authors",
year = "2018",
month = jan,
day = "1",
doi = "10.1016/j.jcmgh.2018.02.007",
language = "English (US)",
volume = "6",
pages = "79--96",
journal = "CMGH",
issn = "2352-345X",
publisher = "Elsevier Inc.",
number = "1",
}