Fig. 1.

Multipotent stem cells reside within clonal CCO⁻ adenomatous crypts. (A) (i) H&E staining showing tubular adenoma C157 with low-grade dysplasia. (ii) CCO enzyme histochemistry identifying two patches of multiple, blue, CCO⁻ crypts. (iii and iv) Laser-capture microdissection of areas in ii outlined in red and (v) mtDNA sequencing of single cells from multiple blue crypts and within the same blue crypts versus adjacent brown, wild-type crypts demonstrated that all blue crypts shared a common, clonal point mutation in their mtDNA that was not present in adjacent brown crypts. (B) Immunofluorescence staining of serial sections from adenoma 160. Clonal CCO⁻ crypts contained cells positive for markers of neuroendocrine cells (chromogranin A) and secretory cells [mucin2 (MUC2) and mucin5AC (MUC5AC)], indicating these crypts contained a multipotential stem cell that had produced these distinct cell types. Detection of CCO expression was conducted on the same section as chromogranin A after visualization for chromogranin A expression. Detection of MUC2 and MUC5AC expression was conducted on the same section simultaneously. Negative controls were isotype-matched at the same concentration as the corresponding primary antibody. Asterisks indicate the crypt enlarged in high-power images. (Scale bars: ∼50 μm in low-power images and 25 μm in high-power images. (C) LGR5 mRNA detection in FFPE tissue from patients with familial adenomatous polyposis (left pair) and sporadic adenocarcinoma (right pair). Expression is detectable in bases of unaffected crypts, in patches of adenomatous epithelium, and, in this example, extensively through invasive adenocarcinoma. Bright-field and dark-field reflected light image pairs with Giemsa counterstain.