Investigating stem cells in human colon by using methylation patterns

Abstract

The stem cells that maintain human colon crypts are poorly characterized. To better determine stem cell numbers and how they divide, epigenetic patterns were used as cell fate markers. Methylation exhibits somatic inheritance and random changes that potentially record lifelong stem cell division histories as binary strings or tags in adjacent CpG sites. Methylation tag contents of individual crypts were sampled with bisulfite sequencing at three presumably neutral loci. Methylation increased with aging but varied between crypts and was mosaic within single crypts. Some crypts appeared to be quasi-clonal as they contained more unique tags than expected if crypts were maintained by single immortal stem cells. The complex epigenetic patterns were more consistent with a crypt niche model wherein multiple stem cells were present and replaced through periodic symmetric divisions. Methylation tags provide evidence that normal human crypts are long-lived, accumulate random methylation errors, and contain multiple stem cells that go through "bottlenecks" during life.

Figure 1

Methylation tags. CpG sites are indicated by + marks and transcription start sites are indicated by arrows. The tags are in different locations: MYOD1 5′ to the promoter, CSX in the 3′ untranslated region, and BGN 3′ to the promoter. Cumulative percent methylation from all patients and tags are indicated by numbers within circles representing the 5, 8, and 9 CpG sites of the three tags. Bisulfite-treated sequences are illustrated with converted Cs indicated by capital Ts and CpG sites in bold.

Figure 2

(A) Methylation tags represented as binary strings (0 = unmethylated and 1 = methylated) are sampled from individual crypts. Crypts contain unmethylated tags at birth and if tags do not drift. (B) When tags drift, they may differ within and between adult crypts. Differences depend on stem cell numbers and whether they are immortal or can be lost with replacement (stem cell niche). Trees illustrate these different scenarios. If stem cells are immortal, tags will reflect lifelong divergence, and numbers of unique tags will be proportional to numbers of crypt stem cells. In contrast, stochastic bottlenecks recur with a stem cell niche, and some unique tags will be lost. Numbers of unique tags and differences between tags within a crypt will be variable and reflect the time since the last bottleneck. Quantitative comparisons of methylation tags between and within crypts can distinguish between these scenarios.

Figure 3

Mosaic crypts. Each horizontal line represents one tag and each group represents tags from one crypt. Methylated sites are filled circles. Arrows separate tags from upper and lower bisected crypt regions.

Figure 4

Fluctuations in crypt values with sampling. Methylation and distances within crypts are relatively stable after five tags are sampled. In contrast, unique tags are still found as more molecules are sampled.

Figure 5

Tag variability between and within individual crypts. (A) Numbers of unique tags were variable between crypts although average numbers of unique tags were relatively stable with aging. (B) Methylation increases with aging. (C) Intracrypt distances were smaller compared with intercrypt distances. Individual crypt values (“X”) and patient averages (“O”) are illustrated. Simulated average values (solid lines) with 64 stem cells and 95% asymmetric divisions are superimposed on the experimental data (see Fig. 7).

Figure 6

Lack of correlation between average methylation of CSX and MYOD1 within single crypts. Correlation coefficient is −0.17.

Figure 7

Simulations modeled 2,048 cell crypts with varying numbers of stem cells, modes of stem cell division (asymmetric or symmetric), methylation error rates, and numbers of divisions (0–35,000). Methylation was site-autonomous and the same for all divisions (including non-stem cell divisions) with an error rate (either methylation or demethylation) of 2 × 10⁻⁵ per CpG site per division. The experimental process of sampling eight molecules from a crypt was also simulated. A 64-stem cell niche with 95% asymmetric divisions is illustrated.