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Strategy to perform lineage tracing from slowly dividing or post-mitotic label-retaining cells (LRCs) in the murine small intestine. Cre-recombinase is expressed as two parts: the ubiquitously produced C-terminal CreB (in yellow) and the N-terminal CreA that is fused to histone 2B (H2B) (in blue). CreA expression can be induced by the addition of beta-naphthoflavone (betaNF), followed by cell-cycle-dependent dilution of the H2B-CreA protein. Both Cre-halves are additionally fused to an FKHB domain, of which fusion can be induced by a small molecule in order to reconstitute a functional enzyme and induce lineage tracing (traced cells will be red). CreB is constitutively expressed (yellow cells, left). Induction of CreA expression by betaNF followed by a 3-13 days dilution will lead to a scenario in which only slowly dividing LRCs and post-mitotic cells retain both parts of the enzyme (blue and yellow cells, middle). The induction of dimerization does not yield long-term clone formation, suggesting that slowly dividing cells are not homeostatic stem cells (red cells, top). When epithelial damage is induced before dimerization of the Cre-domains, long-term tracing can be observed (bottom). These experiments evidence that LRCs are not homeostatic stem cells, but regain stemness upon damage.

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