Isolation of nuclei from label-retaining cells and measurement of their turnover rates in rat colon.

We describe here a new technique for isolating nuclei from long-term label-retaining cells (LRCs), a subpopulation enriched with stem cells from colon, and for measuring their proliferation rates in vivo. A double-label approach was developed, combining the use of bromodeoxyuridine (BrdU) and (2)H(2)O. Male Fisher 344 rats were administered BrdU in drinking water continuously for 2-8 wk. BrdU was then discontinued (BrdU washout), and animals (n = 33) were switched to (2)H(2)O in drinking water and killed after 2, 4, and 8 wk. Nuclei from BrdU-positive cells (LRCs) were collected by flow cytometry. The percentages of LRCs were 7 and 3.8% after 4 and 8 wk of BrdU washout, respectively. Turnover rates of LRCs were measured on the basis of deuterium incorporation from (2)H(2)O into DNA of LRC nuclei, as determined by mass spectrometry. The proliferation rate of the LRCs collected was 0.33-0.90% per day (half-life of 77-210 days). Significant contamination from other potentially long-lived colon cells was excluded. In conclusion, this double-labeling method allows both physical isolation of nuclei from colon epithelial LRCs and measurement of their in vivo proliferation rates. Use of this approach may allow better understanding of mechanisms by which agents induce or protect against colon carcinogenesis.