Intestinal stem cells reside in a paused, unlicensed G₁ phase. (A) Quantification of cell-cycle stages for cells in the stem cell (SC) compartment (<40 µm from the crypt base) and in the early TA compartment (40–80 µm from the crypt base), as described in Fig. 3 E (n = 49 crypts). (B) Representative image of an extracted crypt base isolated 1 h after a pulse of EdU (green) and stained with Hoechst (blue), UEA (red), and antibodies against Mcm2 (white). Bar, 10 µm. Nuclear morphology and UEA signals were used to distinguish between UEA⁺ Paneth cells (outlined by dashed lines and nuclei marked with blue stars) and UEA⁻ stem cells (situated between outlined Paneth cells, with nuclei marked by red stars). (C) The mean percentage of UEA⁻ stem cells that fall into the previously defined cell-cycle bins: unlicensed; G₁ licensed; S phase; and S/G₂ phase (n = 68 crypts). Displayed are the means ± SEM. (D) Representative flow cytometry profiles of isolated Lgr5^Hi intestinal stem, showing DNA-bound Mcm2 and DNA content (n = 3 mice; panel i). The 2N (G₁) cells of the same population are also shown (ii). The respective gated populations for unlicensed G₁ (black), licensed (red), S phase (yellow), and G₂/M (green) are shown. The Ki67 content of the licensed and unlicensed G₁ cells are also shown (iii). (E) The frequency distribution of mean DNA-bound Mcm2 intensities for Mcm2⁺ cells in G₁ cells. Shown are pooled data from three mice. (F) Simulated ergodic-rate analysis of origin licensing during G₁, with the licensing rate varied and a significant paused period incorporated (unlicensed G₁). The displayed histograms show the frequency distribution of DNA-bound Mcm2 of G₁ cells (n = 10,000). Please refer to Materials and methods for further information. However, an analogy to explain the model is as follows, as described in Matson et al. (2017): Origin licensing through G₁ can be thought of as cars traveling along a long stretch of motorway; if cars enter the motorway at a fixed rate (i.e., unsynchronized cell cycles, cells entering G₁), the density of the cars at any one point is inversely proportional to the speed at which they are traveling (“speed” in this case means the rate of licensing). We have added the concept of delays at the start and end, like tollbooths. In our model, there is a minimum drive time (minimum length of G₁ required for cells to grow to a critical size before they enter the S phase; arbitrarily set at 100%). If the speed of the cars means they don’t reach the end of the motorway before that time is up, cars maintain a constant speed along the entire road and exit the motorway as soon as they reach the end. If cars drive faster, and they reach the end before that time is up (>100%), they have to wait at the end of the motorway until the critical time has expired, resulting in a peak accumulation at the fully licensed point. Unlicensed G₁ is an enforced time that cars have to wait once they enter the motorway before they are allowed to drive along it. This creates a peak on the left (unlicensed cells). If the cars then drive slowly, when they get to the end of the motorway, they exit immediately because the critical time has expired (center), but if they drive fast enough, they reach the end of the motorway before the critical time has expired. This results in a peak accumulation at both minimally and maximally licensed points. (G) Comparison of DNA-bound Mcm2 content of Lgr5⁺ and Lgr5⁻ G₁ cells and of cells in the very early S phase. Shown are pooled data from three mice.