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    • Several strategies have been recently developed

    2018-10-20

    Several strategies have been recently developed for CBC cell isolation via cell surface markers and fluorescence-activated cell sorting (FACS; Gracz et al., 2013; King et al., 2012; Merlos-Suarez et al., 2011; Wang et al., 2013). Although they represent considerable advances in the isolation of CBC cells independently of transgenic reporter alleles, these methodologies are suggested to be contaminated with other cell types and have not been fully characterized at the molecular level. The approach by Merlos-Suarez et al. (2011) mainly relies on extracting a subset of EPHB2 high cells from EPCAM+ epithelial cells (named SM2 in our study). However, the EPHB2 receptor is not only expressed at high levels in CBC cells but also in committed progenitor cells (Merlos-Suarez et al., 2011). In another study, Wang et al. (2013) used three crypt APO866 cost markers (CD24/CD166/CD44) while depleting for GRP78+ progenitor cells (named SM4 in our study). Nonetheless, the resultant population was found to be contaminated by endocrine cells (Wang et al., 2013).
    Results and Discussion To investigate in a comprehensive way how these different cell surface markers are expressed in the different cell populations of the intestinal crypt, we employed two recently developed tools that allow mapping of high-dimensional cytometry data onto two dimensions, yet conserving its high-dimensional structure (Amir el et al., 2013; Qiu et al., 2011). Spanning-tree progression analysis of density-normalized events (SPADE) clusters phenotypically similar cells into nodes (Qiu et al., 2011), while viSNE displays individual cells on a map that preserves their multidimensional separation (Amir el et al., 2013). SPADE and viSNE have been used to interrogate, infer and visualize cellular hierarchies and transitions based on expression of cell surface markers in diverse systems including nuclear reprogramming (Lujan et al., 2015) and hematopoiesis (Qiu et al., 2011). For the generation of high-dimensional flow cytometry data, intestinal epithelial cells from Lgr5-Gfp reporter mice were labeled with a broad range of intestinal crypt markers including markers of CBC cells (EPHB2, CD24med, CD44, CD166), transit-amplifying cells (GRP78), Paneth cells (CD24high, UEA-1), epithelial cells (EPCAM), and non-epithelial contaminating cells (CD45, CD31) (Figure S1A) (Merlos-Suarez et al., 2011; Wang et al., 2013; Wong et al., 2012). Analysis revealed that CBC cells, as identified by high levels of Lgr5-GFP expression (Lgr5-GFPhigh) (Figures S1B and S1C), clustered together in SPADE trees and on viSNE maps (Figure 1A), and that the expression of EPHB2, CD44, CD166, and CD24 overlapped with this population to various degrees. Interestingly, when nodes/cells of the SPADE trees/viSNE maps were categorized into stem cells (Lgr5-GFPhigh), Paneth cells (CD24high, UEA1high, SSChigh) (Sato et al., 2011), transient amplifying cells (GRP78high), and other mature epithelial cell types (EPCAM-positive or EPCAM-negative, or low for CBC cell markers, negative for Paneth cell markers), the known intestinal cell hierarchy could be inferred (Figures 1B and 1C). The pool of Lgr5-GFPhigh stem cells was closely associated with the niche cells (Paneth cells) and, via a stream of transient amplifying cells, was connected to the other mature epithelial cell types. Therefore, this suggested that the combination of surface markers with multidimensional analysis could be used to identify sorting strategies for the purification of CBC cells. As both SM2 (based on EPHB2 and EPCAM markers) and SM4 (a combination of CD24, CD44, CD166, and GRP-78 markers) strategies (Figures S1B, S1D, and S1E) failed to isolate a pure CBC cell population (Merlos-Suarez et al., 2011; Wang et al., 2013) and importantly as their key cell surface makers (EPHB2, CD44, CD166) have different expression patterns (Figure 1A), we utilized viSNE to explore whether a reporter-free sorting strategy combining the different intestinal crypt surface markers, termed SM6 (Figure 1D), could improve the purity of the CBC cell population to a level comparable with the Lgr5-GFPhigh cells. Briefly, cells were depleted for contaminating CD31 and CD45 cells (endothelial and hematopoietic cells) and enriched for a specific population of CD166low CD24med cells. These cells were subsequently gated into CD44high GRP78neg-low cells and then only the EPCAMhigh/EPHB2high cells were sorted (see Supplemental Experimental Procedures for more details).