Centre de Génétique et de Physiologie Moléculaire et Cellulaire, Université Claude Bernard, France
Correspondence: Michelina Plateroti, Ph.D., Centre de Génétique et de Physiologie Moléculaire et Cellulaire, Université Lyon 1, 16 Rue Raphael Dubois, 69622 Villeurbanne, France. Telephone: 33–472431595; Fax: 33–472432685; e-mail: email@example.com
Author contributions: F.C. and A.R.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; J.N.: collection and assembly of data; M.P.: conception and design, financial support, assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript. All authors approved the manuscript. F.M.C. and A.R. contributed equally to this article.
The intestinal epithelium self-renews rapidly and continuously throughout life, due to the presence of crypt stem cells. Two pools of these cells have been identified in the small intestine, which differ in position (“+4” or the bottom of the crypts), expression of specific markers (Bmi1/mTert or Lgr5/Ascl2), and cell cycle characteristics. Interestingly, the RNA-binding protein Musashi1 is expressed in both populations and therefore a potential marker for both stem cell types. In order to locate, isolate, and study Musashi1-expressing cells within the intestinal epithelium, we generated transgenic mice expressing GFP fluorescent protein under the control of a 7-kb Msi1 promoter. The expression pattern of GFP in the intestinal crypts of both small and large intestines completely overlapped that of Musashi1, validating our model. By using fluorescence-activated cell sorting, cellular, and molecular analyses, we showed that GFP-positive Msi1-expressing cells are divided into two major pools corresponding to the Lgr5- and mTert-expressing stem cells. Interestingly, monitoring the cell cycle activity of the two sorted populations reveals that they are both actively cycling, although differences in cell cycle length were confirmed. Altogether, our new reporter mouse model based upon Musashi1 expression is a useful tool to isolate and study stem cells of the intestinal epithelium. Moreover, these mice uniquely enable the concomitant study of two pools of intestinal stem cells within the same animal model. Stem Cells2013;31:2273–2278
Continuous renewal of the intestinal epithelium depends on stem cells located in the crypts of Lieberkühn . Different studies have suggested that two pools of stem cells exist: one located at the very bottom of the crypts, the crypt basal columnar (CBC) stem cells, actively cycling and expressing Lgr5 and Ascl2 markers [2, 3], and one considered quiescent but more resistant to irradiation [4-6], located at the “+4” position from the crypt bottom and expressing m-Tert, Lrig1, and D-Camkl1 markers [2, 6-9]. The “+4”-stem cells are required to maintain intestinal crypts, and thus epithelial homeostasis . However, studies showed that the best-characterized stem cell markers are expressed in gradient throughout a “stem zone,” and not exclusively in a single stem cell pool [10, 11]. In addition, a recent paper showed that the quiescent label-retaining cells (LRC) in the crypts express CBC-associated genes, can serve as reserve stem cells upon injury, and are Paneth and enteroendocrine precursors , but their precise link to the classically defined “+4”-stem cells remains unclear. Therefore, the exact cell hierarchy at the crypt bottom is still elusive, and the debate concerning the location and physiology of gut stem cells has been reopened . Interestingly, several studies reported Musashi1 (Msi1) as an intestinal epithelial stem cell marker for both populations of small intestine stem cells, which also identifies colon stem cells [4, 11, 14]. Msi1 is an RNA-binding protein originally described to control stemness in Drosophila . We have recently shown that its overexpression in intestinal epithelial progenitors enhances their proliferative capacity through activation of Wnt and Notch pathways , key regulators of gut cell fate and stem cell biology [17-19]. In order to study Msi1-expressing cells and their potential stem cell-like properties, we describe here the generation of Msi1-eGFP transgenic mice as a new and efficient tool to concomitantly label and study intestinal crypts stem cells.
Materials and Methods
Materials and Methods are described in the Supporting Information.
Results and Discussion
Msi1-eGFP Mice Express GFP in the Gut Stem Cell Compartment
We generated Msi1-eGFP mice (Supporting Information Fig. S1) to study Msi1-expressing cells and their associated stemness. Green fluorescent protein (GFP) immunostaining on sections revealed specific expression in small intestine at the level of the CBCs and around the +4 position (Fig. 1A, 1C, 1D; Supporting Information Fig. S2) and at the bottom of colonic crypts (Supporting Information Fig. S3). Importantly, GFP labeling strongly overlapped the expression pattern of Msi1 in all Msi1-eGFP lines (Fig. 1A; Supporting Information Fig. S2, S3). Coimmunolabeling experiments showed the presence of GFP+ cells between and above Paneth cells (Supporting Information Fig. S4A). Moreover, GFP colocalized with a restricted subset of β-catenin or CD44-expressing cells at the crypt bottom (Supporting Information Fig. S4B, S4C). To further characterize this new model, small intestinal epithelia of Msi1-eGFP mice were fractionated (Fig. 2; Supporting Information Fig. S5) and the presence of GFP in the fractions was evaluated by fluorescent microscopy. As expected GFP+ cells were detected only in F4 from F19 and F20 Msi1-eGFP mice (Fig. 2C; Supporting Information Fig. S5B). Moreover, F4 cells were enriched for GFP and Msi1 mRNAs and for the established stem cell markers Lgr5 and Bmi1 mRNAs [2, 5] (Fig. 2D; Supporting Information Fig. S5C). These data show that our model expresses GFP specifically in the stem cell compartment of the small intestinal epithelium.
Different Levels of GFP in Msi1-eGFP Mice Distinguish Two Intestinal Stem Cell Pools
To identify and study GFP+ cells from Msi1-eGFP small intestinal crypts, we used a fluorescence-activated cell sorting approach. Given that GFP+/GFP− populations showed strong similarities between founders (not shown), detailed analysis focused on F19 line. GFP levels and the common crypt-cell surface marker CD24 [12, 20] were used to isolate different cell populations. GFPHi and GFPLo cells were distinguishable (Fig. 3A), both being CD24Lo (Fig. 3B). Four populations were sorted: GFP−/CD24Hi, GFP−/CD24Lo, GFPLo/CD24Lo, and GFPHi/CD24Lo (Fig. 3B), and quantitative reverse transcriptase polymerase chain reaction (RTqPCR) was performed (Fig. 3C; Supporting Information Fig. S6). GFPHi/CD24Lo cells (from here GFPHi) expressed high levels of Msi1, mTert, Hopx, and Lrig1 mRNAs, and low levels of Lgr5, Ascl2, Olfm4, and Smoc2 mRNAs, suggesting an enrichment of “+4”-stem cells in this population. No specific enrichment of Bmi1 was observed, consistent with its expression throughout the crypts [10, 21, 22]. GFPLo/CD24Lo cells (from here GFPLo) expressed low levels of Msi1, mTert, Hopx, and Lrig1 but high amounts of Lgr5, Ascl2, Olfm4, and Smoc2 mRNAs, reflecting the presence of CBC-stem cells in this population. GFPLo cells also expressed high levels of Wnt-targets Axin2 and c-Myc (Supporting Information Fig. S6C), in accordance with high Wnt activity in CBCs . Mmp7, recently described as a LRC marker , was absent from both GFP+ populations (Supporting Information Fig. S6D). GFP−/CD24Lo and GFP−/CD24Hi cells are clearly mixed populations (Fig. 3C; Supporting Information Fig. S6D). No contaminants from differentiated epithelial cells were detected in GFPHi or GFPLo cells (Supporting Information Fig. S6D). Analysis of Msi1 expression in intestinal crypts of Lgr5-GFP mice  confirmed a gradient of Msi1 expression in the different GFP+ cell populations (not shown), as reported .
Altogether, these results strongly indicate that Msi1 expression characterizes the whole crypt stem cell zone and suggest the existence of two distinct pools of stem cells, as originally proposed . Moreover, the differential GFP expression in Msi1-eGFP mice distinguishes CBCs- and “+4”-stem cells, as also suggested by the frequency profile (Fig. 1D).
GFPHi and GFPLo Cells Have Different Cell Cycle Activity
Previous studies on cycling activity, evaluated by BrdU incorporation and immunohistochemistry, suggested that “+4”-stem cells are essentially quiescent while CBCs are actively cycling [2, 4, 6]. To quantitatively analyze the proliferative characteristics of the different populations, bromo-deoxy-uridine (BrdU) was administered to Msi1-eGFP mice and its incorporation was analyzed by flow cytometry (Fig. 4; Supporting Information Fig. S7). Three incorporation protocols were used: one pulse with a 2-hour chase to label cells in S-phase or with a 48-hour chase to follow crypt cell dynamics, and a continuous oral administration for 48-hour to label every replicating/replicated cell (Fig. 4A). After a 2-hour chase, 7.3% (±0.28, n = 2) of total crypt cells incorporated BrdU, whereas only 2.55% (±0.35, n = 2) remained BrdU+ after a chase period of 48 hours (p < .01). After continuous administration for 48 hours, 12.85% (±0.49, n = 2) of crypt cells were BrdU+ (p < .05). GFPHi and GFPLo populations were enriched in BrdU+ cells compared to total crypt cells after a 2-hour chase (Fig. 4B) but these percentages were not significantly different contrary to the assumption of “+4”-stem cell quiescence [5, 6]. After a 48-hour chase, the percentage of BrdU+ cells was significantly higher in GFPHi than in GFPLo cells, clearly indicating a slower cell cycle. This result was confirmed by the continuous BrdU administration, where the GFPHi population showed significantly fewer BrdU+ cells than the GFPLo population (Fig. 4B). These data show that the GFPHi population is enriched in cells with a longer cycle than GFPLo cells, suggesting that “+4”-stem cells, that is, Msi1-GFPHi cells, are slow cycling compared to rapidly cycling CBCs, that is, Msi1-GFPLo cells.
We show here that newly generated Msi1-eGFP mice express different levels of GFP specifically within different pools of intestinal stem cells: GFPHi cells preferentially express “+4”-stem cell markers and GFPLo, CBC-stem cell markers. These populations are clearly distinct regarding gene expression and cell cycle characteristics. However, contrary to previous reports, Msi1-GFPHi “+4”-stem cells are not quiescent but do have a slower cell cycle than CBCs. Additionally, Msi1-GFPHi cells appear different from LRCs.
Altogether, Msi1-eGFP mice represent a useful model to study Msi1-expressing cells and their potential stem cell properties. Future investigations will aim to better characterize these cells and the specific hierarchy between CBCs, Msi1-GFPHi “+4”-stem cells and LRCs. This new model will also be useful to explore whether Msi1-expressing cells are involved in gut tumor development, as it has been shown for Lgr5- and Bmi1-expressing cells.
We thank Nadine Aguilera for animal handling, Sebastien Dussurgey and Thibault Andrieu for their invaluable help with fluorescence-activated cell sorting analysis. We are indebted with Dr. Philippe Jay for Lgr5-GFP animals and with Dr. Maria Sirakov and Rachel Sennett for critical reading of the manuscript. This work was supported by the Institut National pour le Cancer (Grant INCA-2009-175), the ANR Blanc ThRaSt (ANR-11-BSV2-019), and the Ligue contre le cancer Department du Rhone (N-074937). F.C. was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) and the Association pour la Recherche sur le Cancer (ARC); A.R. was supported by the Ligue Nationale Contre le Cancer and the Fondation pour la Recherche Medicale. A.R. is currently affiliated with Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY.
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflict of interest.