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  • By using viSNE maps the degree of overlap between SM

    2018-10-26

    By using viSNE maps, the degree of overlap between SM2, SM4, and SM6 populations and the reference Lgr5-GFPhigh Myoseverin was investigated (Figure 1E). Interestingly, the SM2 gating strategy was not able to exclude a considerable number of cells that clustered outside of the region occupied by the Lgr5-GFPhigh population. However, both SM4 and SM6 strategies produced homogeneous appearing populations that overlapped well with Lgr5-GFPhigh cells. As previously mentioned, the expression of the Lgr5-Gfp cassette is mosaic and, accordingly, many CBC cells are not labeled by GFP. To investigate whether the SM6 gating strategy was superior at purifying a homogeneous population of CBC cells, a Lgr5-GFP back gating analysis was conducted on SM2, SM4, and SM6 populations. The enrichment of both Lgr5-GFPhigh cells and Lgr5-GFPlow cells within SM2, SM4, and SM6 cell populations was assessed. It is generally accepted that only Lgr5-GFPhigh cells represent CBC cells, while Lgr5-GFPlow cells are committed progenitors of Lgr5-GFPhigh cells. In agreement, single-cell PCR for Lgr5 demonstrated that nearly all Lgr5-GFPhigh cells express the transcript in contrast to only a small fraction of Lgr5-GFPlow cells (Figures S1G and S1H). Our analysis showed that the SM6 strategy was better than SM2 and SM4 cell isolation strategies in enriching for Lgr5-GFPhigh cells, while depleting for Lgr5-GFPlow cells. However, these differences were only significant between SM6 and SM4 (Figures S1I and S1J). In order to adequately benchmark the quality of our method with the existing methods, we first performed RNA sequencing with the Lgr5-Gfp line on five FACS-purified groups: SM2, SM4, SM6, Lgr5-GFPhigh reference population, and cells negative or low for all of the cell surface markers used (negative) (Figure 2A, Figures S1B–S1F, Table S1). All the cell populations, with the exception of negative cells, had a similar transcriptional signature (Figure 2B). We used principal component analysis (PCA) to compare the sequencing data of the different isolation strategies. Importantly, the transcriptional signatures of SM6 and Lgr5-GFPhigh cells overlapped, indicating that these two populations were highly similar. SM2 and SM4 cell populations clustered further away and were therefore more different (Figure 2C) although still relatively close to the Lgr5-GFPhigh population. Unsupervised hierarchical clustering on a population level also confirmed that CBC cell-enriched populations (SM2, SM4, Lgr5-GFPhigh, and SM6) were clustered and distinct from the negative population (Figure 2D). Lgr5-GFPhigh and SM6 cells formed a separate subgroup within this CBC cell-enriched branch (Figure 2D) confirming high similarity. Moreover, we could not find any genes that were significantly differentially expressed between the SM6 and Lgr5-GFPhigh populations (Figure 2E) (2-fold, Benjamin-Hockberg correction). However, several genes were upregulated in SM2 and SM4 populations, mostly related to secretory cell lineage identity as already reported by Wang et al. (2013) for the SM4 approach (Figure 2E and Table S2). Together, these results indicate that cells isolated using our FACS sorting strategy are highly similar to the Lgr5-GFPhigh cells from a transcriptional viewpoint. Expression of the Lgr5-Gfp reporter is mosaic in the intestine and only marks around a third of all CBC cells. The SM6 and Lgr5-GFPhigh approaches allow the isolation of comparable cell numbers (SM6, 2.7% ± 0.4%; Lgr5-GFPhigh, 2.6% ± 0.2% of all live cells) because the loss of a proportion of CBC cells via the SM6 method is a necessary trade-off between cell number and purity. In order to exclude the majority of the Lgr5-GFPlow cells, very stringent gating for EPHB2 is required (Figure 1D). We performed single-cell transcriptional profiling for a broad panel of CBC and +4 reserve stem cell markers to determine the degree of homogeneity of the SM6 and Lgr5-GFPhigh isolated cell populations (Figures S2A–S2F). PCA revealed that SM6 and Lgr5-GFPhigh single-cell signatures overlapped and were highly homogeneous as indicated by the ellipses, representing 67% of the cells in each population (Figures 3A and 3B). The other strategies (SM2 and SM4) were more different. Violin plot analysis, which shows the distribution of gene expression per cell for any given population, demonstrated that all the different cell isolation methods were enriched for cells expressing ISC cell marker genes (Lgr5, Olfm4, Bmi1, Lrig1, HopX, Sox9, CD44, EphB2) (Figures 3C and S2G). Notably, this analysis also established that SM6 and Lgr5-GFPhigh single cells had an analogous gene expression pattern at the individual cell level (Figures 3C and S2G). Co-expression of the key CBC markers Lgr5, EphB2, and CD44 was detected in 90.1% and 90.3% of the individual cells from SM6 (n = 61) and Lgr5-GFPhigh (n = 62) isolation methods, respectively, compared with only 61% for SM4 (n = 31) and 79% for SM2 (n = 29) (Figures 3D and S2H). Analysis of the co-expression of +4 ISC marker genes demonstrated a similar trend, where the majority of these genes were co-expressed in each cell in the SM6 and Lgr5-GFPhigh populations (Figures 3D and S2H), as previously described (Li et al., 2014). However, we noted slight differences between SM6 and Lgr5-GFPhigh in the numbers of cells positive for the ISC marker Sox9. SM6 cells were more enriched for Sox9-positive cells (95.1%) compared with the Lgr5-GFPhigh strategy (79%) (Figure S2H). In summary, our single-cell transcriptional analysis, based on these key genes, demonstrates that our isolation method gives rise to a homogeneous population of CBC cells which co-express key stem cell markers in a similar way to the well-established Lgr5-Gfp model.