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. Author manuscript; available in PMC: 2016 Mar 1.
Published in final edited form as: Stem Cell Res. 2015 Jan 24;14(2):238–241. doi: 10.1016/j.scr.2015.01.005

The (not so) Immortal Strand Hypothesis

Cristian Tomasetti 1,3, Ivana Bozic 2
PMCID: PMC4405781  NIHMSID: NIHMS665541  PMID: 25700960

Abstract

Background

Non-random segregation of DNA strands during stem cell replication has been proposed as a mechanism to minimize accumulated genetic errors in stem cells of rapidly dividing tissues. According to this hypothesis, an “immortal” DNA strand is passed to the stem cell daughter and not the more differentiated cell, keeping the stem cell lineage replication error-free. After it was introduced, experimental evidence both in favor and against the hypothesis has been presented.

Principal Findings

Using a novel methodology that utilizes cancer sequencing data we are able to estimate the rate of accumulation of mutations in healthy stem cells of the colon, blood and head and neck tissues. We to find that in these tissues mutations in stem cells accumulate at rates strikingly similar to those expected without the protection from the immortal strand mechanism.

Significance

Utilizing an approach that is fundamentally different from previous efforts to confirm or refute the immortal strand hypothesis, we provide strong evidence against non-random segregation of DNA during stem cell replication. Our results strongly suggest that parental DNA is passed randomly to stem cell daughters and provides new insight into the mechanism of DNA replication in stem cells.

Introduction

Before a cell divides, its DNA content is replicated and then segregated during mitosis. Whether the DNA strands are passed randomly to the two daughter cells or according to some other mechanism is however not clear.

The immortal DNA strand hypothesis has been proposed1 as a mechanism used by stem cells in order to minimize the accumulation of mutations in their genomes. This hypothesis proposes that stem cells divide predominantly asymmetrically, producing one stem cell and one differentiated cell, and that at each division, a template set of DNA strands (known as the “parental” or “immortal strands”) is transferred to the daughter stem cell and not to the non-stem cell daughter. The fundamental idea behind this hypothesis is that, by retaining the immortal strands within the stem cell progeny, the accumulation of random DNA replication errors in the stem cell compartment would be greatly reduced, therefore decreasing the risk of genetic disorders such as cancer. Indeed, errors in DNA duplication would be passed on to non-stem, more differentiated, and shorter-lived daughter cells.

The earliest experimental evidence for the non-random segregation of stem cell DNA strands in mammalian tissues comes from studies by Potten et al. of tongue epithelia and intestinal crypts2. Although some further experimental work appears to support this hypothesis3-8, whether such a mechanism is operational in adult stem cells in vivo or not is still controversial9, with reports arguing against the existence of immortal strands in intestinal crypts10 and hematopoietic stem cells11. Most of the studies arguing for or against the immortal strand relied on labeling early stem cells and testing whether segregation of label followed a random or non-random pattern. Here we use a different approach that utilizes genetic sequencing data and provide new evidence against the immortal strand hypothesis, by showing that stem cells in hematopoietic, colorectal and head and neck epithelial tissue contain as many somatic mutations as would be expected if no protection mechanism were in place.

Results

Tomasetti et al.12 recently provided a new methodology for estimating the number of mutations that accumulate in healthy self-renewing tissues. Their results were obtained via the analysis of The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) whole-exome sequencing data on somatic mutations from patients with a given tumor type. Tomasetti et al. found statistically significant strong positive correlations between age and number of somatic mutations in tumors of several self-renewing tissues, in patients with matched tumor stages. For example, the data show that colorectal cancers from 85-year-old patients have, on average, 47 mutations more than colorectal cancers at the same stage from 45-year-old patients. Since the cancer stage is the same, those extra 47 mutations are, on average, not due to the cancer phase, but to the normal accumulation of somatic mutations occurring in the healthy tissue during the extra time the older patients had before tumorigenesis started. This allows the estimation of the rate at which mutations accumulate in healthy cells, prior to the first driver mutation hit. This method represents a unique way of indirect single cell sequencing, since all tumor cells carry the changes present in their last healthy ancestor in addition to changes accumulated during tumor evolution (Fig. 1). Importantly, their results yielded estimates for the rate of accumulation of somatic mutations in healthy tissues that are remarkably in line with estimates obtained via completely different methodologies12.

Figure 1. Number of mutations in a patient's tumor is a function of age.

Figure 1

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A) Tomasetti et al.12 found that the number of mutations in cancer tissues correlates significantly with the age of the patient in chronic lymphocytic leukemia, uterine corpus endometrioid carcinoma and colorectal cancer, independently of the cancer stage. For example, colorectal cancers in 85-year olds harbor on average 121 mutations, in contrast to 74 mutations in colorectal cancers of 45-year old patients. B) Difference in the numbers of somatic mutations detected in the cancers of patients of different ages can be explained by the difference in the length of the self-renewal phase in the lineage that lead to cancer. Thus the average difference in the numbers of mutations in colorectal cancer patients ages 85 and 45 provides a good estimate for the number of somatic mutations that accumulated during 40 years of healthy self-renewal. This allows us to infer the normal mutation rate in healthy stem cells.

The first initiating event that starts the process of tumorigenesis must originate in a healthy cell. It is possible that this healthy cell may not be a stem cell13. However, given any cell of a healthy self-renewing tissue, the number of divisions that separates this cell from its mother stem cell is not very large. And even a hundred divisions will not create any relevant difference between the mutational load of the stem cell mother and its healthy differentiated daughter cell. In fact, normal somatic mutation rates are of the order of 10−10 per base per cell division12, and therefore 100 divisions will not cause, on average, even one extra point mutation in the exome of the differentiated cell. Thus, we can safely assume that the mutational load found in healthy cells is an accurate estimate for the mutational load of their mother stem cells.

We are then able to provide estimates for the number of somatic mutations accumulating in healthy stem cells of self-renewing tissues as a function of time. In Figure 2a, we show the average number of somatic mutations accumulating with age in stem cells of the colonic crypt (red line), plus or minus 2 standard errors (shaded red area), as it was estimated from TCGA sequencing data in Tomasetti et al.12. We next compare these estimates with the number of somatic mutations expected to be present in healthy stem cells of the colonic crypt if we assume that the epithelial lining of the colon regenerates approximately every week14, and that, contrary to the immortal strand hypothesis, the parental DNA strands were randomly assigned to either of the daughter cells (blue and green lines).

Figure 2. Estimates for mutational load obtained from sequencing data are strikingly similar to the expected mutational load in healthy cells without the protection of an immortal strand.

Figure 2

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Red line represents the average number of somatic mutations accumulating with age in stem cells of the colonic crypt, plus or minus 2 standard errors (shaded red area), as it was estimated from TCGA sequencing data in Tomasetti et al.12. Expected mutational load in healthy stem cells without the protection from the immortal strand is represented by the grey cone in-between the blue and green lines (see Materials and Methods). For evidence of the stem cells enjoying a protection mechanism reducing the accumulation of random mutations, the red line with its confidence interval (red cone) would have to be significantly below the grey cone, which is never the case.

The estimates for the mutational load from sequencing data are strikingly similar to the expected mutational load in healthy cells without the protection of an immortal strand12,15-17 (Fig. 2a). Thus we see no evidence for the protection afforded by immortal strands on the rate of mutation accumulation in healthy colon tissue. Further comparisons are provided in Figure 2b,c for blood, where there is evidence of stem cell asymmetric divisions18, as well as head and neck epithelial stem cells of never-smokers, since tongue epithelia was one of the two first tissues on which evidence in favor of the immortal strand hypothesis was provided2. Similarly as in the case for colon, the predicted mutational load assuming no protection from the immortal strand is very similar to the mutational load inferred from sequencing data for these two tissues.

Conclusions

Our results provide new evidence against the immortal DNA strand hypothesis, specifically for colon, blood and head and neck epithelial stem cells. We find evidence that the healthy stem cell divisions did not include asymmetric segregation of an “immortal strand”, since the stem cells responsible for maintaining the tissue accumulated replication errors at normal rates. We cannot rule out the existence of a separate pool of tissue-maintaining stem cells that are retaining immortal strands and whose progeny never (or very rarely) give rise to tumors, as our methodology would not be able to sample them, but there is no evidence that such a stem cell pool exists. Similarly, our analysis cannot not rule out the existence of a pool of quiescent stem cells that rarely divide on which this mechanism might operate, but there is no evidence from our data that such cells are involved in tissue maintenance in colon, blood and epithelial cells of the head and neck.

Materials and Methods

The mutational load as a function of age was obtained from the publicly available The Cancer Genome Atlas (TCGA) database for the indicated tissues. More specifically, the red lines in Fig. 2 represent the average number of somatic mutations accumulating with age in stem cells of the colonic crypt, plus or minus 2 standard errors (shaded red area), as estimated from TCGA sequencing data using the robust regression described in Tomasetti et al.12.

The expected mutational load in healthy stem cells without the protection from the immortal strand are instead represented by the blue and green lines in Fig. 2. In order to obtain these estimates, the formulas for the expected values, as found in Tomasetti et al.12 were used. In particular, the somatic mutation rate per cell division per nucleotide base has been estimated via various orthogonal methods12,15-17 to be 1.4 ·10−10 in human fibroblasts, and 0.66 ·10−10 in B-lymphoblastoid. We have used a mutation rate equal to 5 ·10−10 (blue line)12 and, to be conservative, an even higher 10−9 (green line) per cell division per nucleotide base. Stem cells division rates were assumed to be once a month in blood11 and once a week in colon14 and head and neck epithelium19.

Highlights.

  • Novel methodology to estimate the accumulation of mutations in normal stem cells.

  • Mutations rates found are comparable to expected without protection mechanism.

  • Strong evidence against non-random segregation of DNA during stem cell replication.

Footnotes

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