Immortal Strand Hypothesis Research Articles

SACK-expanded hair follicle stem cells display asymmetric nuclear Lgr5 expression with non-random sister chromatid segregation.

We investigated the properties of clonally-expanded mouse hair follicle stem cells (HF-SCs) in culture. The expansion method, suppression of asymmetric cell kinetics (SACK), is non-toxic and reversible, allowing evaluation of the cells' asymmetric production of differentiating progeny cells. A tight association was discovered between non-random sister chromatid segregation, a unique property of distributed stem cells (DSCs), like HF-SCs, and a recently described biomarker, Lgr5. We found that nuclear Lgr5 expression was limited to the HF-SC sister of asymmetric self-renewal divisions that retained non-randomly co-segregated chromosomes, which contain the oldest cellular DNA strands, called immortal DNA strands. This pattern-specific Lgr5 association poses a potential highly specific new biomarker for delineation of DSCs. The expanded HF-SCs also maintained the ability to make differentiated hair follicle cells spontaneously, as well as under conditions that induced cell differentiation. In future human cell studies, this capability would improve skin grafts and hair replacement therapies.

Molecular Cloaking of H2A.Z on Mortal DNA Chromosomes During Nonrandom Segregation

Although nonrandom sister chromatid segregation is a singular property of distributed stem cells (DSCs) that are responsible for renewing and repairing mature vertebrate tissues, both its cellular function and its molecular mechanism remain unknown. This situation persists in part because of the lack of facile methods for detecting and quantifying nonrandom segregating cells and for identifying chromosomes with immortal DNA strands, the cellular molecules that signify nonrandom segregation. During nonrandom segregation, at each mitosis, asymmetrically self-renewing DSCs continuously cosegregate to themselves the set of chromosomes that contain immortal DNA strands, which are the oldest DNA strands. Here, we report the discovery of a molecular asymmetry between segregating sets of immortal chromosomes and opposed mortal chromosomes (i.e., containing the younger set of DNA template strands) that constitutes a new convenient biomarker for detection of cells undergoing nonrandom segregation and direct delineation of chromosomes that bear immortal DNA strands. In both cells engineered with DSC-specific properties and ex vivo-expanded mouse hair follicle stem cells, the histone H2A variant H2A.Z shows specific immunodetection on immortal DNA chromosomes. Cell fixation analyses indicate that H2A.Z is present on mortal chromosomes as well but is cloaked from immunodetection, and the cloaking entity is acid labile. The H2A.Z chromosomal asymmetry produced by molecular cloaking provides a first direct assay for nonrandom segregation and for chromosomes with immortal DNA strands. It also seems likely to manifest an important aspect of the underlying mechanism(s) responsible for nonrandom sister chromatid segregation in DSCs.

Label Retaining Cells and Cutaneous Stem Cells

This is a comprehensive review on label retaining cells (LRC) in epidermal development and homeostasis. The precise in vivo identification and location of epidermal stem cells is a crucial issue in cutaneous biology. We discuss here the following problems: (1) Identification and location of LRC in the interfollicular epithelium and hair follicle; (2) The proliferative potential of LRC and their role in cutaneous homeostasis (3); LRC phenomenon and the Immortal Strand Hypothesis, which suggests an alternative mechanism for retention of genetic information; (4) Significance of LRC studies for development of stem cell concept. Now, it seems evident that LRC are a frequent feature of stem cell niches and revealing highly dormant LRC may be used for identification of stem cell niches in different tissues. LRC were used for screening specific markers of epidermal stem cells. Within a given tissue stem cells have different proliferative characteristics. There are more frequently cycling stem cells which function primarily in homeostasis, while LRC form a reserve of dormant, may be ultimate, stem cells, which are set aside for regeneration of injury or unforeseen need. The authors suggest that LRC dormancy described in Mammalia has much in common with developmental quiescence found in some other animals. For example in C. elegans reproductive system, vulval precursor cells have developmentally programmed cell-cycle arrest in the first larval stage, and then undergo an extended period of quiescence before resuming proliferation. Another example of developmental quiescence is the diapause, a widespread phenomenon exhibited by animals ranging from nematodes to mammals, often occurring at genetically predetermined life history stage.

TRAPping telomerase within the intestinal stem cell niche

Adult intestinal stem cells (ISCs) reside at the crypt base, where they continuously proliferate to maintain homeostasis of the intestinal epithelium. As a result of a lifetime of cell division, these LGR5+ ISCs face the combined risk of genomic mutations and telomere attrition. In this issue of EMBOJ , [Schepers et al (2011)][1] examine whether ISCs employ telomerase expression and asymmetric chromosome segregation to protect their genome. [1]: #ref-9

Drosophila male germline stem cells do not asymmetrically segregate chromosome strands

Adult stem cells continuously supply differentiated cells throughout the life of organisms. This increases the risk of replicative senescence or neoplastic transformation due to mutations that accumulate over many rounds of DNA replication. The immortal strand hypothesis proposes that stem cells reduce the accumulation of replication-induced mutations by retaining the older template DNA strands. Other models have also been proposed in which stem cells asymmetrically segregate chromosome strands for other reasons, such as retention of epigenetic memories. Recently, the idea has emerged that the mother centrosome, which is stereotypically retained within some asymmetrically dividing stem cells, might be utilized as a means of asymmetrically segregating chromosome strands. We have tested this hypothesis in germline stem cells (GSCs) from Drosophila melanogaster testis, which undergo asymmetric divisions marked by the asymmetric segregation of centrosomes and the acquisition of distinct daughter cell fates (stem cell self-renewal versus differentiation). Using 5-bromo-2-deoxyuridine labeling combined with direct visualization of GSC-gonialblast (differentiating daughter) pairs, we directly scored the outcome of chromosome strand segregation. Our data show that, in male GSCs in the Drosophila testis, chromosome strands are not asymmetrically segregated, despite asymmetrically segregating centrosomes. Our data demonstrate that asymmetric centrosome segregation in stem cells does not necessarily lead to asymmetric chromosome strand segregation.

Drosophila male germline stem cells do not asymmetrically segregate chromosome strands

Adult stem cells continuously supply differentiated cells throughout the life of organisms. This increases the risk of replicative senescence or neoplastic transformation due to mutations that accumulate over many rounds of DNA replication. The immortal strand hypothesis proposes that stem cells reduce the accumulation of replication-induced mutations by retaining the older template DNA strands. Other models have also been proposed in which stem cells asymmetrically segregate chromosome strands for other reasons, such as retention of epigenetic memories. Recently, the idea has emerged that the mother centrosome, which is stereotypically retained within some asymmetrically dividing stem cells, might be utilized as a means of asymmetrically segregating chromosome strands. We have tested this hypothesis in germline stem cells (GSCs) from Drosophila melanogaster testis, which undergo asymmetric divisions marked by the asymmetric segregation of centrosomes and the acquisition of distinct daughter cell fates (stem cell self-renewal versus differentiation). Using 5-bromo-2-deoxyuridine labeling combined with direct visualization of GSC-gonialblast (differentiating daughter) pairs, we directly scored the outcome of chromosome strand segregation. Our data show that, in male GSCs in the Drosophila testis, chromosome strands are not asymmetrically segregated, despite asymmetrically segregating centrosomes. Our data demonstrate that asymmetric centrosome segregation in stem cells does not necessarily lead to asymmetric chromosome strand segregation.

Semiconservative quasispecies equations for polysomic genomes: The general case

This paper develops a formulation of the quasispecies equations appropriate for polysomic, semiconservatively replicating genomes. This paper is an extension of previous work on the subject, which considered the case of haploid genomes. Here, we develop a more general formulation of the quasispecies equations that is applicable to diploid and even polyploid genomes. Interestingly, with an appropriate classification of population fractions, we obtain a system of equations that is formally identical to the haploid case. As with the work for haploid genomes, we consider both random and immortal DNA strand chromosome segregation mechanisms. However, in contrast to the haploid case, we have found that an analytical solution for the mean fitness is considerably more difficult to obtain for the polyploid case. Accordingly, whereas for the haploid case we obtained expressions for the mean fitness for the case of an analog of the single-fitness-peak landscape for arbitrary lesion repair probabilities (thereby allowing for noncomplementary genomes), here we solve for the mean fitness for the restricted case of perfect lesion repair.

T1748 Comparative Studies of Label-Retaining Epithelial Cells in the Small and Large Intestine: Implications for the Concept of Asymmetric Stem Cell Division in the Colon

Introduction Irradiation induces stem cell apoptosis and exposure to DNA label (eg, tritiated thymidine; [3H]dT) when new stem cells are being generated during recovery, leads to retention of the label in cells located above the Paneth cell region in crypts of small intestine (SI). Persistence of label retaining cells (LRC), despite many rounds of cell division, occurs because of asymmetric segregation of DNA strands in which the old/template strands are selectively retained in daughter cells that are destined to remain stem cells (immortal strand hypothesis). The aims of our studies were to (i) determine whether asymmetric DNA strand segregation occurs in stem cells (located at crypt base) of colon (CO) and (ii) assess radiosensitivity of newly generated stem cells in SI. Methods (A) Following 12 Gy irradiation, [3H]dT was administered to BDF1 mice over 5 days. SI and CO were collected at 40 min and 4, 7 and 15 days after the last dose of [3H]dT (chase period). (B) As in (A) above, and additionally, second dose of radiation (1 Gy) after 7-day chase, followed by tissue collection after 4.5 h to assess for apoptotic epithelial cells that had taken up [3H]dT. [3H]dT-labelled (autoradiography) and apototic (morphology) cells were assigned a cell position (cp) along the crypt (cell number increasing sequentially from cp 1 at the centre of crypt base). Percentage of [3H]dT-labelled cells at a particular cell position is expressed as the labelling index (LI). Data are expressed as mean (SEM). Results (A). In contrast to CO, peaks of LRC were consistently seen in SI at cp 4–9 at chase periods of 4, 7 and 15 days (Abstract 040). After 15-day chase, only occasional LRC were seen in different regions of crypts of CO. (B). In SI, LRC undergoing apoptosis were seen predominantly at cp 4–6 (LI 2.2(0.4)) and cp 7–9 (1.0(0.3)). Conclusion Following irradiation, LRC are predominant in the stem cell region above Paneth cells in SI crypts and these newly generated stem cells are sensitive to radiation-induced apoptosis. LRC are not consistently seen in the stem cell region of colonic crypts. This implies lack of asymmetric segregation of DNA strands in colonic stem cells, which may explain the increased risk of cancer in the colon (compared to SI).

OC-040 Comparative studies of label-retaining epithelial cells in the small and large intestine: implications for the concept of asymmetric stem cell division in the colon: Abstract OC-040

<h3>Introduction</h3> Irradiation induces stem cell apoptosis and exposure to DNA label (eg, tritiated thymidine; [³H]dT) when new stem cells are being generated during recovery, leads to retention of the label in cells located above the Paneth cell region in crypts of small intestine (SI). Persistence of label retaining cells (LRC), despite many rounds of cell division, occurs because of asymmetric segregation of DNA strands in which the old/template strands are selectively retained in daughter cells that are destined to remain stem cells (immortal strand hypothesis). The aims of our studies were to (i) determine whether asymmetric DNA strand segregation occurs in stem cells (located at crypt base) of colon (CO) and (ii) assess radiosensitivity of newly generated stem cells in SI. <h3>Methods</h3> (A) Following 12 Gy irradiation, [³H]dT was administered to BDF1 mice over 5 days. SI and CO were collected at 40 min and 4, 7 and 15 days after the last dose of [³H]dT (chase period). (B) As in (A) above, and additionally, second dose of radiation (1 Gy) after 7-day chase, followed by tissue collection after 4.5 h to assess for apoptotic epithelial cells that had taken up [³H]dT. [³H]dT-labelled (autoradiography) and apototic (morphology) cells were assigned a cell position (cp) along the crypt (cell number increasing sequentially from cp 1 at the centre of crypt base). Percentage of [³H]dT-labelled cells at a particular cell position is expressed as the labelling index (LI). Data are expressed as mean (SEM). <h3>Results</h3> (A). In contrast to CO, peaks of LRC were consistently seen in SI at cp 4–9 at chase periods of 4, 7 and 15 days (Abstract 040). After 15-day chase, only occasional LRC were seen in different regions of crypts of CO. (B). In SI, LRC undergoing apoptosis were seen predominantly at cp 4–6 (LI 2.2(0.4)) and cp 7–9 (1.0(0.3)). <h3>Conclusion</h3> Following irradiation, LRC are predominant in the stem cell region above Paneth cells in SI crypts and these newly generated stem cells are sensitive to radiation-induced apoptosis. LRC are not consistently seen in the stem cell region of colonic crypts. This implies lack of asymmetric segregation of DNA strands in colonic stem cells, which may explain the increased risk of cancer in the colon (compared to SI).

John Updike Packing

Immortal DNA offers as cold a comfort as the transmigration of souls. This is an invited review. If we can't take our memories with us, why go? (Updike, 1997, p. 74) John Updike succinctly summariz...

Colonic stem cell data are consistent with the immortal model of stem cell division under non‐random strand segregation

Colonic stem cells are thought to reside towards the base of crypts of the colon, but their numbers and proliferation mechanisms are not well characterized. A defining property of stem cells is that they are able to divide asymmetrically, but it is not known whether they always divide asymmetrically (immortal model) or whether there are occasional symmetrical divisions (stochastic model). By measuring diversity of methylation patterns in colon crypt samples, a recent study found evidence in favour of the stochastic model, assuming random segregation of stem cell DNA strands during cell division. Here, the effect of preferential segregation of the template strand is considered to be consistent with the 'immortal strand hypothesis', and explore the effect on conclusions of previously published results. For a sample of crypts, it is shown how, under the immortal model, to calculate mean and variance of the number of unique methylation patterns allowing for non-random strand segregation and compare them with those observed. The calculated mean and variance are consistent with an immortal model that incorporates non-random strand segregation for a range of stem cell numbers and levels of preferential strand segregation. Allowing for preferential strand segregation considerably alters previously published conclusions relating to stem cell numbers and turnover mechanisms. Evidence in favour of the stochastic model may not be as strong as previously thought.

The germline stem cells of Drosophila melanogaster partition DNA non-randomly

The Immortal Strand Hypothesis proposes that asymmetrically dividing stem cells cosegregate chromatids to retain ancestral DNA templates. Using both pulse-chase and label retention assays, we show that non-random partitioning of DNA occurs in germline stem cells (GSCs) in the Drosophila ovary as these divide asymmetrically to generate a new GSC and a differentiating cystoblast. This process is disrupted when GSCs are forced to differentiate through the overexpression of Bag of Marbles, a factor that impels the terminal differentiation of cystoblasts. When Decapentaplegic, a ligand which maintains the undifferentiated state of GSCs, is expressed ectopically the non-random partitioning of DNA is similarly disrupted. Our data suggest asymmetric chromatid segregation is coupled to mechanisms specifying cellular differentiation via asymmetric stem cell division.

Nonselective Sister Chromatid Segregation in Mouse Embryonic Neocortical Precursor Cells

We have investigated whether the precursor cells that give rise to the neurons of the neocortex during mouse embryonic development segregate sister chromatids nonrandomly upon mitosis, as would be predicted by the immortal strand hypothesis. Using various protocols of 5-bromo-2-deoxyuridine (BrdU) labeling and chase, we were unable to detect BrdU label-retaining neocortical precursor cells at any of the embryonic stages analyzed, even when the entire brain was analyzed by serial sectioning. Analysis of mitotic neuroepithelial and radial glial cells revealed BrdU-labeled sister chromatid segregation to both nascent daughter cells, which showed a mirror-symmetrical pattern in the first and a non-mirror-symmetrical pattern in the second division after BrdU labeling. Taken together, our data are incompatible with embryonic neocortical precursor cells segregating the sister chromatids selectively to one daughter cell upon mitosis and hence argue against the existence of immortal DNA strands in these cells. In light of the previously reported existence of immortal DNA strands in adult neural stem cells, we discuss that either 1) embryonic and adult neural stem cells in the cortex are distinct or 2) that most, if not all, of the embryonic precursor cells to neocortical neurons are progenitor cells rather than true neural stem cells.

Hematopoietic Stem Cells Reversibly Switch from Dormancy to Self-Renewal during Homeostasis and Repair

Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

A New Mechanism for Aging: Chemical “Age Spots” in Immortal DNA Strands in Distributed Stem Cells

The existence of immortal DNA strands (IDSs) in distributed stem cells (DSCs) of adult human tissues was first inferred by Cairns. Cairns deduced the existence of IDSs by connecting two seemingly disparate observations - one his own and the other belonging to Lark. Cairns noted a mathematical discrepancy between predicted human tissue cell mutation rates and human cancer incidence. He integrated this insight with Lark's earlier discovery of non-random mitotic chromosome segregation in both plant root tip cells and mouse fetal fibroblast cultures to predict the existence of IDSs as the essential elements of a mutation-defense mechanism in DSCs. Since Cairns' seminal prediction, several laboratories have identified IDSs in diverse mammalian cells with DSC properties. Past studies focused on the potential roles of IDSs as originally envisioned in DSC genetic fidelity or in the maintenance of the DSC phenotype. Another possible consequence of IDSs, aging, has received little attention. Herein, the potential for cumulative chemical modifications and decompositions (i.e., "age spots") of IDSs in DSCs to act as a major determinant of human aging is considered. If accrued chemical alterations of IDSs prove to be essential determinants of aging, then a means to restore IDSs may yield new strategies for tissue rejuvenation.

Abstract 3422: Asymmetric Division of Human Cardiac Progenitor Cells Involves Immortal DNA Strand Cosegregation

The objective of this study was to determine whether asymmetric division of human cardiac progenitor cells (hCPCs) occurs by: random DNA template segregation; selective retention of the old template DNA strand; or a combination of both processes. Myocardial samples were enzymatically dissociated and hCPCs were sorted for the stem cell antigen c-kit. During in vitro expansion, hCPCs were exposed to BrdU for 36 hours to reach a 90% degree of labeling. BrdU-tagged hCPCs were plated at limiting dilution to obtain single cell-derived clones. Sixty clones comprising 10 –125 c-kit-positive hCPCs developed in 7–10 days. In four cases, one single BrdU-bright hCPC was identified while the remaining clonogenic cells were negative for the halogenated nucleotide. In these clones, the number of BrdU-negative hCPCs was 25, 85, 95, and 111. Conversely, in 56 clones hCPCs were uniformly labeled and showed very low levels of BrdU. In a second group of experiments, hCPCs in late-anaphase initial-telophase were identified and the distribution of BrdU in the two clusters of chromosomes was analyzed. In 5% of mitotic cells, three-dimensional reconstruction by confocal microscopy documented that BrdU-labeling was restricted at one pole only of the dividing hCPCs. PCNA which is highly expressed in newly synthesized DNA was restricted to the BrdU-negative chromosomes. In a third set of studies, hCPCs were loaded with quantum dots, cultured for 36 hours in the presence of BrdU and examined 96 hours later. Quantum dots are progressively diluted by cell division independently from the modality of DNA template segregation. Thus, hCPCs with minimal levels of quantum dots and bright BrdU localization were interpreted as replicating cells which retained the old DNA strand. By this approach, 5% hCPCs displayed these two critical properties. The uneven distribution of the cell fate determinants Numb and α-adaptin confirmed that hCPCs underwent asymmetric division. In conclusion, these data support the hypothesis that immortal DNA strand cosegregation participates in asymmetric kinetics of hCPCs although random-segregation of DNA template is the prevailing mechanism of hCPC growth.

The Majority of Multipotent Epidermal Stem Cells Do Not Protect Their Genome by Asymmetrical Chromosome Segregation

The maintenance of genome integrity in stem cells (SCs) is critical for preventing cancer formation and cellular senescence. The immortal strand hypothesis postulates that SCs protect their genome by keeping the same DNA strand throughout life by asymmetrical cell divisions, thus avoiding accumulation of mutations that can arise during DNA replication. The in vivo relevance of this model remains to date a matter of intense debate. In this study, we revisited this long-standing hypothesis, by analyzing how multipotent hair follicle (HF) SCs segregate their DNA strands during morphogenesis, skin homeostasis, and SC activation. We used three different in vivo approaches to determine how HF SCs segregate their DNA strand during cell divisions. Double-labeling studies using pulse-chase experiments during morphogenesis and the first adult hair cycle showed that HF SCs incorporate two different nucleotide analogs, contradictory to the immortal strand hypothesis. The co-segregation of DNA and chromatin labeling during pulse-chase experiments demonstrated that label retention in HF SCs is rather a mark of relative quiescence. Moreover, DNA labeling of adult SCs, similar to labeling during morphogenesis, also resulted in label retention in HF SCs, indicating that chromosome segregation occurs randomly in most of these cells. Altogether, our results demonstrate that DNA strand segregation occurs randomly in the majority of HF SCs during development, tissue homeostasis, and following SC activation. Disclosure of potential conflicts of interest is found at the end of this article.

Quantitative proliferation dynamics and random chromosome segregation of hair follicle stem cells

Regulation of stem cell (SC) proliferation is central to tissue homoeostasis, injury repair, and cancer development. Accumulation of replication errors in SCs is limited by either infrequent division and/or by chromosome sorting to retain preferentially the oldest 'immortal' DNA strand. The frequency of SC divisions and the chromosome-sorting phenomenon are difficult to examine accurately with existing methods. To address this question, we developed a strategy to count divisions of hair follicle (HF) SCs over time, and provide the first quantitative proliferation history of a tissue SC during its normal homoeostasis. We uncovered an unexpectedly high cellular turnover in the SC compartment in one round of activation. Our study provides quantitative data in support of the long-standing infrequent SC division model, and shows that HF SCs do not retain the older DNA strands or sort their chromosome. This new ability to count divisions in vivo has relevance for obtaining basic knowledge of tissue kinetics.

Mortal and Immortal DNA: Craig Venter and Keats, “Lamia”

Mortal and Immortal DNA: Craig Venter and Keats, “Lamia”

Does 'Immortal DNA strand' exist in 'immortal' stem cells?

Stem cells function to generate differentiated cells, and, at the same time, are maintained as 'immortal' cells through self-renewal. Accumulated evidence indicates that adult stem cells may be the most common precursor for cancers in adult mammalian tissue 1, and this concept is gaining increasing support 2, 3. Meanwhile, this also raises the question of how stem cells, which have a much longer lifespan compared to other progenitor and mature cells, can avoid accumulating mutations from DNA replication errors, thereby reducing the risk of tumorigenesis. In 1975, J Cairns proposed an intriguing hypothesis from an evolutionary point of view to explain this phenomenon in organisms with long life spans. According to this 'immortal DNA strand' model, adult stem cells might be able to retain template (old) DNA strands in each asymmetric division to avoid accumulation of mutations during the process of DNA replication 4.