Transcriptional Profiling Of Intestine Stem Cells Populations [RNA-Seq] (mouse)

The isolation of pure populations of mouse intestinal stem cells (ISCs) is essential to facilitate functional studies of tissue homeostasis, tissue regeneration and intestinal diseases. However, the purification of ISCs has relied predominantly on the use of transgenic reporter alleles in mice. Here, we introduce a new combinational cell surface marker mediated strategy that allows the isolation of an ISC population transcriptionally and functionally equivalent to the gold standard Lgr5-GFP ISCs. We tested the ability of three cell surface marker mediated isolated strategies (termed SM2, SM4 and SM6 according to the number of key cell surface markers used) to purify ISCs and transcriptionally compared them to established standards, Lgr5-GFP high cells and cells negative for any ISC markers (Negative). The best cell surface marker mediated strategy (SM6) allowed the isolation of ISCs from reporter free mice (SM6-WT) that were functionally and transcriptionally distinct from cells isolated from transgenic mice (SM6-TG) due to Lgr5 haploinsufficiency. Overall design: To adequately benchmark the quality of our method with the existing methods, we performed first RNA sequencing with the Lgr5-GFP strain (C57/Bl6 background) on 5 FACS purified groups: SM2, SM4, SM6, Lgr5-GFPhigh reference population and cells negative or low for all of the cell surface markers used. We also performed RNA sequencing of SM6-TG and SM6-WT cells to investigate in detail potential transcriptional differences between them.

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