Lgr5 intestinal stem cells have high telomerase activity and randomly segregate their chromosomes

Abstract

Somatic cells have been proposed to be limited in the number of cell divisions they can undergo. This is thought to be a mechanism by which stem cells retain their integrity preventing disease. However, we have recently discovered intestinal crypt stem cells that persist for the lifetime of a mouse, yet divide every day. We now demonstrate biochemically that primary isolated Lgr5+ve stem cells contain significant telomerase activity. Telomerase activity rapidly decreases in the undifferentiated progeny of these stem cells and is entirely lost in differentiated villus cells. Conversely, asymmetric segregation of chromosomes has been proposed as a mechanism for stem cells to protect their genomes against damage. We determined the average cell cycle length of Lgr5+ve stem cells at 21.5 h and find that Lgr5+ve intestinal stem cells randomly segregate newly synthesized DNA strands, opposing the 'immortal strand' hypothesis.

Figure 1

Three different Lgr5-EGFP populations can be distinguished. (A) Isolated EGFP-Lgr5+ crypts. (B) FACS isolation of three different Lgr5+ve populations. (C) Single sorted cells are all GFP-positive. (D) FACS analysis of wild-type intestinal cells. (E) Gene expression analysis comparing the three sorted GFP populations. Expression of stem cell markers is highest in the GFP-Hi population.

Figure 2

Telomerase activity in stem cells but not in villus cells. (A). mTert expression analysed by RT–PCR. Stem cells express higher levels of mTert than average crypt or villus cells. Normalized for Lgr5-high stem cells, error bars represent s.e.m. (B) Telomerase activity of cell extracts measured by TRAPeze reaction, normalized for the telomerase-positive control cell line provided with the kit. Extract from the colorectal cancer cell line, DLD1, gives high activity. Lgr5-high cells have a higher telomerase activity than Lgr5-low cells, whereas villus cells possess no telomerase activity. Error bars represent s.e.m.

Figure 3

Telomeres in stem cells and daughters shorten over time. Telomere length is represented relative to telomere length of DLD1. Bars represent average values of relative telomere length. Error bars represent s.e.m.

Figure 4

Crypt base columnar cells cycle once every 21.5 h. (A) Outline of experiment. (B) Co-staining of Lgr5-GFP antibodies (green), PH3 antibodies (white), which marks cells in mitosis, and incorporated EdU (red) together with DAPI. (C) Differential interference contrast visualization of cells in two crypts labelled with PH3 antibodies (green), which marks cells in mitosis, and incorporated EdU (red). CBCs in mitosis can be seen with (arrowhead) or without (arrow) incorporated EdU. (D) Graphic overview of counted stem cells. Individual data points were obtained from different mice. The line connecting the data points (optimal polynomial, R²=0.958) shows two peaks, representing the first and second wave of mitosis. The difference between the peaks gives the average cell cycle length.

Figure 5

Random segregation of EdU label during stem cell mitosis. (A) Three colour overlay of intestinal crypt showing membrane marker E-cadherin (green), incorporated EdU label (red) and DNA (blue). A simplified representation of the division is given to accentuate the cell that is dividing. (B–I) Divisions at multiple positions in the crypt all show symmetric segregation of label.