Human hair genealogies and stem cell latency

Abstract

Background: Stem cells divide to reproduce themselves and produce differentiated progeny. A fundamental problem in human biology has been the inability to measure how often stem cells divide. Although it is impossible to observe every division directly, one method for counting divisions is to count replication errors; the greater the number of divisions, the greater the numbers of errors. Stem cells with more divisions should produce progeny with more replication errors.

Methods: To test this approach, epigenetic errors (methylation) in CpG-rich molecular clocks were measured from human hairs. Hairs exhibit growth and replacement cycles and "new" hairs physically reappear even on "old" heads. Errors may accumulate in long-lived stem cells, or in their differentiated progeny that are eventually shed.

Results: Average hair errors increased until two years of age, and then were constant despite decades of replacement, consistent with new hairs arising from infrequently dividing bulge stem cells. Errors were significantly more frequent in longer hairs, consistent with long-lived but eventually shed mitotic follicle cells.

Conclusion: Constant average hair methylation regardless of age contrasts with the age-related methylation observed in human intestine, suggesting that error accumulation and therefore stem cell latency differs among tissues. Epigenetic molecular clocks imply similar mitotic ages for hairs on young and old human heads, consistent with a restart with each new hair, and with genealogies surreptitiously written within somatic cell genomes.

Figure 1

Human hair genealogy. a. Hair follicle genealogy can be divided into three phenotypic phases: neogenesis, bulge stem cell latency, and anagen differentiation and division. Mitotic age includes divisions during all three phases and may be inferred from numbers of replication errors (methylation) in sampled follicle cells. Short hairs should have fewer anagen divisions relative to longer hairs and therefore fewer errors (less methylation). Hairs from individuals of different ages have similar neogenesis and anagen intervals, and therefore any age-related increase in average methylation reflects lifelong stem cell mitotic activity. Average mitotic ages would be similar for hairs plucked from different aged individuals because of stem cell bulge latency. b. Sequences (after bisulfite treatment) of the CSX and SOX10 methylation tags. CpG sites that may be methylated are in bold, and converted Cs in non-CpG sites are capital T's. PCR primers are underlined. c. Examples of CSX tags sampled from two follicles (eight tags per hair) from a 37 year old. Circles represent the 5' to 3' order of adjacent CpG sites and filled circles are methylated.

Figure 2

Methylation with age. a. CSX tags sampled from anagen follicles. Individual hair values ("X") were scattered but averages ("O") from individuals exhibited no increase after two years of age. Tag diversity (number of unique tags found in the 8 sampled follicle tags) also exhibited an increase before two years of age. Graphs on the right give an expanded view of values early in life. The earliest hairs are from fetuses of 22 and 32 weeks estimated gestational ages, and a term stillborn. Symbols and trend lines are green up to two years of age, and red after infancy. b. Average SOX10 methylation also exhibited relatively constant values after two years of age.

Figure 3

Summary of CSX tag methylation and diversity between different hair phenotypes. Average hair methylation increased from short to long hairs, and there were no significant differences between pigmented and white hairs. Tag diversity was significantly lower for short hairs. (Bar is overall average and "X" indicates individual hairs.)

Figure 4

Validation and reproducibility. a. Comparison between bisulfite sequencing and pyrosequencing for the first five CSX CpG sites with different hairs. Correlation coefficient is 0.65. b. Comparisons of bisulfite sequencing of eight more bacterial clones from the same PCR (black "X's" and trend line) or from repeat PCR of the same hair (red crosses and trend line). Identical methylation patterns were not obtained with resampling, probably because hair follicles contain complex mixtures of alleles, but correlation coefficients are respectively 0.86 and 0.93.

Figure 5

The CSX molecular clock across different human tissues. a. Comparisons of CSX tag methylation between human hair (green or red), small intestines (blue) and colon (black). Intestinal data [3, 5, 21] include additional unpublished individuals. Plotted are average values from each individual. In contrast to hair, the intestines show an age-related increase in methylation. Regression analysis of intestinal data were consistent with positive slopes (F_1,25 = 21.58, P < 0.0001 for the colon, and F_1,10 = 21.03, P = 0.001 for the small intestines), but not with hair data after two years of age (F_1,21 = 0.0009, P = 0.98). b. Hair and intestinal genealogies can be divided into three phases (neogenesis (green), stem cell (red) and differentiation (blue)). Anagen cycles allow for episodic changes in hair mitotic ages due to lower follicle divisions, but average mitotic age remains constant with chronological age because bulge stem cell divisions are infrequent. Differentiated intestinal cells divide only a few times and survive about a week, and therefore the age-related increase in mitotic age represents crypt stem cell divisions. Crypt stem cells appear to divide more often than bulge stem cells. c. Hair has two distinct mitotic compartments (lower follicle and bulge, indicated in yellow) whereas the intestines have a single mitotic compartment at the crypt base. The lower follicle physically disappears at telogen and previously mitotic lineages become extinct. This telogen bottleneck allows infrequently dividing bulge stem cells to become common ancestors because their progeny repopulate the new lower follicle. The telogen bottleneck effectively resets the mitotic age of the follicle. A similar bottleneck does not occur in intestinal crypts and therefore a cell must divide frequently to become a common ancestor. Without a bottleneck, crypt mitotic age increases with age. A quiescent crypt cell may have stem cell potential, but is not the common ancestor for present day differentiated cells.