Cryptal
Selina Basua
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Mesenchymal cells in the crypt play indispensable roles in the maintenance of intestinal epithelial homeostasis through their contribution to the preservation of stem cells. However, the acquisition properties of the production of stem cell niche factors by the mesenchymal cells have not been well elucidated, due to technical limitations regarding the isolation and subsequent molecular and cellular analyses of cryptal mesenchymal cells. To evaluate the function of mesenchymal cells located at the large intestinal crypt, we established a novel method through which cells are harvested according to the histologic layers of mouse colon, and we compared cellular properties between microenvironmental niches, the luminal mucosa and crypts. The gene expression pattern in the cryptal mesenchymal cells showed that receptors of the hormone/cytokine leptin were highly expressed, and we found a decrease in Wnt2b expression under conditions of leptin receptor deficiency, which also induced a delay in cryptal epithelial proliferation. Our novel stratified layer isolation strategies thus revealed new microenvironmental characteristics of colonic mesenchymal cells, including the intrinsic involvement of leptin in the control of mucosal homeostasis.
Accumulated evidence had indicated the importance of the mesenchymal network-based stem cell microenvironment for the creation and maintenance of intestinal homeostasis6. Epithelial regeneration is achieved through the proliferation of LGR-5+ stem cells in the epithelial cryptal base7,8, and the maintenance and differentiation of those stem cells are tightly regulated by the mesenchymal cells around those stem cells8,9,10. Various subpopulations of mesenchymal cells (e.g., intra-sub-epithelial myofibroblasts, GLI family zinc finger 1+ [Gli1+] fibroblasts, and telocytes) provide niche factors involved in the Wnt/β-catenin pathway, including WNTs, Gremlin (GREM), and Bone Morphogenetic Proteins (BMP)s, that are essential for intestinal renewal and stem cell maintenance11,12. However, the mechanisms how the cryptal mesenchymal cells acquire the ability to maintain the stem cell niche are not well elucidated due to the limited methods for isolating cryptal mesenchymal cells for molecular and cellular analyses. To evaluate the precise function of mesenchymal cells located at the large intestinal crypt, reliable isolation methods of cryptal cells based on the histological layers of the colon are required.
The alimentary canal is anatomically and histologically composed of four broad layers: the mucosa, submucosa, muscularis externa (MEx), and serosa13. The mucosal layer is subdivided further into three layers: epithelium, lamina propria, and muscularis mucosae (MM)13. The compositions of these layers are similar throughout the digestive tract, with partial specialization among mucosal tissues13. Importantly, in gut mucosa, mesenchymal cells or stroma cells exist in each layer of the intestinal compartments; therefore, a new technique that facilitates stratified isolation is needed for the study of cryptal mesenchymal cells6.
In this study, we sought to uncover the anatomical and histological characteristics of the mesenchymal cells in colonic cryptal microenvironments by using a newly developed intestinal layer isolation method with experimental verification. The subsequent molecular and cellular analysis newly identified several receptors highly expressed in cryptal mesenchymal cells, including receptors for leptin, which is a homeostatic hormone/cytokine derived from adipose tissues. Here we present our novel gut mucosal layer isolation method for the characterization of the mesenchymal cell population in murine colon. Using this strategy, we discovered important roles of intrinsic leptin and its involvement in intestinal mucosal regeneration.
That unique mesenchymal populations support cryptal niches in the small intestine and colon is widely accepted14. In addition, the roles of mesenchymal cells differ among their histological and microenvironmental locations, such as villus tips and crypt8,15. To evaluate the characteristics and function of crypt mesenchymal cells in colon as a first step in uncovering mesenchymal cell-mediated mucosal homeostatic pathways, we developed a stratified isolation method for murine colon that achieved optimal separation of the lumen and crypt parts of the colonic mucosa. To this end, murine colon tissues were isolated according to the 6 histologic components: epithelium, upper mucosa with lamina propria, crypt, MM, submucosa, and MEx13.
First, we separated the MEx from the mucosa and submucosa by using microtweezers (Fig. 1a, Supplemental Fig. 1, and Supplemental Video); these tissues were partly de-epithelialized through incubation in cell dissociation solution (Fig. 1b). Under a stereo-microscope, we then used a scraper and microtweezers to sub-divide the upper mucosal and cryptal compartments with the MM and submucosal compartments (Fig. 1b, Supplemental Fig. 1, and Supplemental Video). Finally, we isolated the cryptal regions from the MM and submucosal compartment (Fig. 1b, Supplemental Fig. 1, and Supplemental Video). Staining with hematoxylin and eosin confirmed the isolation of the various layers and revealed that, through the separation procedure, the various layers of the colonic mucosa had been anatomically and histologically correctly dissected (Fig. 1a,b).
To confirm the validity of our novel isolation method by using an alternative means of assessment, we next used FACS to confirm the immunological cellular landscape of the isolated layers (Fig. 2). Epithelial and hematopoietic cells were defined as EpCAM+ and CD45+ cells, respectively. FACS analysis revealed that EpCAM+ cells were rich in the epithelial layer that had been removed through de-epithelialization, and the proportion of epithelial cells gradually decreased in the histological preparations of the crypt and MM compartments (Fig. 2a). CD45+ cells were consistently present throughout the mucosa, but the submucosal microenvironment with the MM contained a relatively lower proportion of CD45+ cells (Fig. 2a). Even though the stratified isolation procedure extended the tissue processing time, cell viability in each layer was not critically decreased compared with that after traditional (unseparated whole-colon) cell-isolation methods (Supplemental Fig. 3a). Next, to clarify the methodological robustness, we assessed the cellular properties in independent experiments (Supplemental Fig. 3b). Importantly, the ratio of TCRγδ cells, recognized by their localization in the intraepithelial compartment, within CD45+ population was high in the epithelial layer, whereas CD4+ cell counts were higher in both the upper mucosa and cryptal layer16 (Supplemental Fig. 4). These results further proved the methodological validity of our stratified isolation strategies and indicate its versatility for further research.
Importantly, gp38+ cells, including most of the mesenchymal cells and lymphatic endothelial cells17, were preferentially distributed in the preparations from the upper mucosa compared with the MM with submucosal compartment (Fig. 2). Together, these results further indicate that this method achieved sufficient stratified isolation for spatial and phenotypic characterization of mesenchymal cells.
To precisely evaluate the colonic mesenchymal cellular populations by flow cytometry, we focused on gp38-expressing cellular populations. Specifically, we gated on gp38+ cells and then examined the expression of CD90 and CD34 (Fig. 2b). According to previous studies, CD90 and CD34 are expressed in the mesenchymal cells located in the crypt8,18; however, those markers were expressed in the mesenchymal cells in other microenvironments, including MM, which mostly consists of smooth muscle cells. Indeed, no previous study has examined the murine colon with exclusion of the submucosa, MM, and MEx; therefore, our results represent the first cellular profiling of the histologically different layers of the colonic mucosa.
Our current evaluation uncovered enrichment of CD34+ mesenchymal cells in not only the crypt but also in the MM (Fig. 2c), indicating the need for cellular profiling of the cryptal region after the use of stratified isolation methods to separate the upper mucosa, crypt, and MM.
These evidences possibly indicate the critical role of intrinsic leptin for functional regulation of mesenchymal cells, especially in crypts. Intriguingly, when cryptal mesenchymal cells were isolated from germ-free mice and examined for the expression of LepR, the degree of the receptor expression was unaltered regardless of the presence or absence of commensal microbiota (Supplemental Fig. 6). This result indicated the de novo programmed expression of the LepR in mesenchymal cells in the crypt and the expressions were dispensable of signaling from commensal bacteria.
Because the receptors for leptin were dominantly expressed in the colonic cryptal mesenchymal cells (Fig. 3), we further evaluated the biological role of leptin and its receptors in terms of the function of cryptal mesenchymal cells. Cryptal mesenchymal cells are considered to be important in mucosal regeneration and homeostasis18; therefore, we examined whether the leptin system is involved in the production of cryptal niche factors.
Lactate has been reported to have a regulatory function in mucosal regeneration and mobility24,27. After intestinal injury due to either radiation or chemotherapy, lactate stimulation of receptors promotes production of niche factors, including WNTs, and stimulates stem cell proliferation27. We thus first evaluated whether leptin stimulation resulted in the production of crypt niche-related factors, such as Wnt2b, Wnt5a, Rspo3, Grem1, and Bmp1, by comparing the results before and after leptin stimulation. We found that Wnt2b was indeed induced after leptin stimulation, thus indicating the potential involvement of leptin in mucosal protection and repair from injury (Fig. 4a).
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