High Incidence of Non-Random Template Strand Segregation and Asymmetric Fate Determination In Dividing Stem Cells and their Progeny

“Cell pairs were analyzed for the expression of Syndecan-4 and Pax7, myogenic lineage markers [9,27], and for the marker of cell proliferation, Ki67.”

“Second, our data require a careful analysis of the use of ‘‘label retention’’ to identify stem cells in tissues, based on the assumption that label retention is equated with very long cell cycle times or quiescence [21–24].”

“According to the ‘‘immortal strand hypothesis’’ [1], as the stem cell divides asymmetrically, it selectively retains those sister chromatids containing the older template DNA strands in the daughter destined to be the renewed stem cell, thus passing the younger strands (with any mutations acquired during replication),…”

“As previously described [25], muscle was dissected, digested in 0.”

“Antibody staining was performed as previously described [15].”

“25% Triton-X-100, digested with DNAse1 in F-10 medium [8], and immunostained for CldU and IdU as above, using Alexa 488 or Rphycoerythrin anti-rat secondary antibodies, and PE- or FITCconjugated mouse anti-BrdU (IdU) clone B44.”

“Cells obtained by this methods are more than 95% positive for the myogenic cell markers CD34 and M-cadherin and less than 2% positive for the endothelial cell marker PECAM [18,25].”

“We also examined the inheritance of labeled DNA in an ex vivo system in which satellite cells activate and proliferate while still associated with individual muscle fibers in culture [10,11].”

“We also examined the inheritance of labeled DNA in an ex vivo system in which satellite cells activate and proliferate while still associated with individual muscle fibers in culture [10,11].”

“Cell pairs were analyzed for the expression of Syndecan-4 and Pax7, myogenic lineage markers [9,27], and for the marker of cell proliferation, Ki67.”

“This phenomenon of template strand segregation was originally based on observations in embryonic fibroblasts [2] and supported by evidence from dividing cells in the intestinal epithelium [3].”

“5 U/ml dispase and 80 U/ml collagenase in media and then filtration and subsequent washing of cells in PBS, or by trituration in media through a 20-gauge needle [26].”

“The high frequency observed in our in vivo studies may relate to the fact that we analyzed cells for asymmetric inheritance of template strands during the process of tissue repair, whereas other in vivo studies have sought evidence of this process during normal homeostatic turnover of tissues [6,7].”

“First, the assessment of the proliferation kinetics of stem and progenitor cells has been carried out in many tissues by analyzing the dilution of label incorporated into DNA [19,20].”

“According to the immortal strand hypothesis, the daughter cell inheriting the older template DNA is the renewing stem cell, whereas the other daughter acquires a more differentiated phenotype. However, our experimental protocol was not designed to test specifically for self-renewal, but rather focused on the prolifera…”

“This phenomenon of template strand segregation was originally based on observations in embryonic fibroblasts [2] and supported by evidence from dividing cells in the intestinal epithelium [3].”

“First, the assessment of the proliferation kinetics of stem and progenitor cells has been carried out in many tissues by analyzing the dilution of label incorporated into DNA [19,20].”

“For Sca-1 and Syndecan-4 labeling, cells were fixed in 4% paraformaldehyde, washed, and incubated with primary antibody (in PBS/5% fetal bovine serum for Sca-1; and in 10% BlockHen [Aves Labs, Tigard, Oregon, United States] for Syndecan-4) overnight without any detergent permeabilization [17].”

“Second, our data require a careful analysis of the use of ‘‘label retention’’ to identify stem cells in tissues, based on the assumption that label retention is equated with very long cell cycle times or quiescence [21–24].”

“Several studies have identified Sca-1 as a marker of undifferentiated progenitors derived from skeletal muscle, demonstrating that satellite cells can give rise to progeny that express Sca-1 at least transiently [16–18].”

“Second, our data require a careful analysis of the use of ‘‘label retention’’ to identify stem cells in tissues, based on the assumption that label retention is equated with very long cell cycle times or quiescence [21–24].”

“Consistent with this notion, multiple studies reported that only a subset of satellite cells undergo asymmetric division in vivo or in vitro (108, 109, 489).”

“Indeed, asymmetric cosegregation of DNA strands into different daughter cells has been documented for satellite cells (Shinin et al. 2006; Conboy et al. 2007).”

“Heterogeneities reported among satellite cells (Biressi and Rando, 2010; Conboy et al., 2007; Day et al., 2010; Shinin et al., 2006; Tajbakhsh, 2009) also point to the existence of stem and progenitor cells within this population.”

“…by BrdU incorporation showed that it is also associated with the asymmetric inheritance of cell fate determinants, with the daughter cell that inherits the old templateDNAexpressing SCA-1 (Ly6a) and the cell that receives the new template DNA being positive for desmin (Conboy et al., 2007).”

“While this teleological function has not been demonstrated and there may be detrimental consequences of template strand segregation during aging (Charville and Rando, 2011), it is supported by the fact that the oldest template strands segregate with the less differentiated satellite cell daughter (Conboy et al., 2007;…”

“Perhaps the best evidence for asymmetric DNA segregation comes from experiments in which use of two different DNA labels during two subsequent rounds of replication allowed staining artefacts to be excluded, and revealed that it is always the less differentiated daughter cell that retains the incorporated label (Conbo…”

“Satellite cell heterogeneity (Kuang et al., 2007), asymmetric inheritance of fate determinants (Conboy et al., 2007; Shinin et al., 2009) and modulation of signaling cascades such as Notch, Wnt and MAPK pathways (Jones et al., 2005; Kuang et al., 2007; Perez-Ruiz et al., 2008; Abou-Khalil et al.,…”

“In vitro studies, showed that the frequency of asymmetric cell division of SC-derived myoblasts diminishes in time (Shinin et al., 2006; Conboy et al., 2007), and that SC-derived myoblasts have a limited proliferation capacity compared to in vivo (Machida et al., 2004).”

“However, in most stem cell compartments, the number of stem cells does not decline significantly with age (Booth and Potten, 2000; Brack and Rando, 2007; Giangreco et al., 2008); rather, these stem cells experience a change in cell fate with age.”

“Retention of BrdU has been attributed to nonrandom DNA strand segregation (Shinin et al., 2006; Conboy et al., 2007; Rocheteau et al., 2012), and thus we employed an alternative technique to determine whether PAX7+ reserve cells exit the cell cycle.”

“The behavior of the satellite-SP cells in intact myofiber cultures suggest that the factors controlling their cell cycle entry are distinct from those of the majority of satellite cells since they appear to cycle once and then either exit the cell cycle completely or cycle very slowly, a trait that has been ascribed t…”

“3F; Supplemental Table S1; Bracken et al. 2007; Kotake et al. 2007; Chen et al. 2009; Ezhkova et al. 2009; Pereira et al. 2010).”

“A proportion of satellite-cell divisions in vivo and in vitro have been observed to be asymmetric, with the labelled DNA being transferred to the daughter cell that has the self-renewal phenotype (Conboy et al., 2007; Shinin et al., 2006).”

“…DNA strands, inheritance of the cell fate determinant Numb, and expression of the myogenic commitment marker Myf5 or the self-renewal maker Pax7 have been reported in previous studies (Conboy and Rando, 2002; Conboy et al., 2007; Kuang et al., 2007; Rocheteau et al., 2012; Shinin et al., 2006).”

“Recently, it has been demonstrated that the ‘‘immortal strand hypothesis’’ is not exclusive of self-renewing cells, but also of divisions in which daughter cells adopt divergent fates [45].”

“Recent reports have provided further data that appear to support the immortal strand hypothesis (Conboy et al., 2007; Karpowicz et al., 2005; Potten et al., 2002; Shinin et al., 2006; Smith, 2005).”

“For bromodeoxyuridine (BrdU) experiments, the mice received two injections of BrdU (50 mg/Kg body weight each) 4 hrs apart on the day before analysis [42].”

“Over the past few years, it has become apparent that functional heterogeneity exists within the satellite cell pool [13, 44–46].”

“Mouse satellite cells (muscle stem cells) and human/mouse embryonic stem cells exhibit a high frequency of biased sister chromatid segregation when old versus new sister chromatids are distinguished by pulse labeling of DNA with bromodeoxyuridine (Conboy et al., 2007; Rocheteau et al., 2012; Elabd et al., 2013).”

“…self-renewal (Cairns, 1975) or generalized to include asymmetric cell divisions associated with divergent cell-fate decisions in stem cell progeny (Conboy et al., 2007), there is nonrandom chromatid segregation requiring the recognition and alignment of all chromatids containing the older…”

“Cell adhesion regulates DNA segregation and satellite cell fate during asymmetric division in situ and in vitro [35,51,52]; thus, monitoring satellite cell division dynamics within their native microenvironment is of critical importance.”

“The retention or loss of BrdU can also reflect asymmetric chromosome segregation within a single mitosis (Shinin et al., 2006; Conboy et al., 2007).”

“The frequencies reported for this henomenon vary from a conservative figure of at least 7% [104] o a higher value of about 50% [105] for satellite cells in vivo.”

“Asymmetric division with respect to cell fate can involve asymmetric segregation of newly synthesized DNA strands (Conboy et al., 2007), and this type of asymmetric division has been phenotypically associated with a hierarchical organization and cell fate in lung cancer models (Pine et al.”

“[PubMed: 16799552] Shinin V, Gayraud-Morel B, Tajbakhsh S. Template DNA-strand co-segregation and asymmetric cell division in skeletal muscle stem cells.”

“Regulation of satellite cell number has been viewed largely in terms of intrinsic, cell lineage-defined, satellite cell qualities that bias the proportion of symmetric versus asymmetric divisions, as influenced by spatial relationships to the basement membrane [46-50].”

“Previous studies have suggested that asymmetric division of template DNA occurs along multiple successive divisions (5, 6, 8).”

“…(Cairns, 2006; Lansdorp, 2007; Rando, 2007) supported by data from tissues such as mammary gland (Smith, 2005), muscle (Shinin et al, 2006; Conboy et al, 2007), nervous system (Karpowicz et al, 2005), HFs (Morris and Potten, 1999), and intestine (Potten et al, 2002), as well as from a few…”

“It has also been proposed that stem cells can reduce their mutational load by differentially segregating chromosomes during asymmetric divisions, such that the chromosomes which contain older, potentially replication-error free, DNA strands are retained in the stem cell daughter (Cairns, 1975; Conboy et al., 2007; Kar…”

“es the structural elements, myofibers (29-31),”

“The loss of muscle mass, reduced regenerative capacity, and diminished function that occur upon aging, recently attributed to an inhibitory environment (20, 49), have been viewed as inevitable (5, 7, 8).”

“The observation of Wnt ACD encouraged us to assess nonrandom DNA segregation that is a well-established feature in stem cell ACD (18).”

“DNA strand was more often found in cells that had acquired a more differentiated phenotype.(35) The concept of HSC niches was first proposed by Schofield.”

“Fast-dividing and slow-dividing cell populations within activated satellite cells in growing and regenerating muscles have also been observed (Baroffio et al., 1996; Schultz, 1996; Rouger et al., 2004; Shinin et al., 2006; Conboy et al., 2007).”

“This conclusion was drawn from the fact that the probability of cure decreases as the size of the tumor increases, and also from the prediction that larger tumors present a higher level of heterogeneity, which is inversely related to the probability of successful therapy (Goldie & Coldman 1984);…”

“…stem cells have developed a mechanism selectively to retain the older set of template DNA within the daughter cell destined to become the stem cell, a process termed immortal strand co-segregation (Cairns 1975, 2006; Merok et al. 2002; Rambhatla et al. 2005; Shinin et al. 2006; Conboy et al. 2007).”

“%) perform biased DNA segregation during mitosis where one daughter cell inherits older template strands and the other daughter cell newly replicated DNA strands (Conboy et al., 2007; Shinin et al., 2006, Rocheteau et al., 2012).”

“To maintain a quiescent pool of SCs through multiple regenerative cycles, SCs have been shown to undergo asymmetric cell division, which gives rise to one daughter cell destined to self-renew and another committed to differentiation [119–123].”

“That said, the behaviours of stem cells in murine skeletal muscle and in the Drosophila female germline seem to fit the mode”

“This division can occur through random or non-random segregation (Conboy et al., 2007).”

“…to ensure the identification of daughter cell pairs, as this drug does not appear to overtly interfere with non-random DNA segregation when applied for a short interval (Conboy et al., 2007; Huh and Sherley, 2011; Kar- Cell Reports 7, 961– powicz et al., 2005; S.Y. and S.T., unpublished data).”

“Some reports suggest that nonrandom segregation of DNA strands or asymmetric distribution of Numb, an inhibitor of Notch signaling, in the daughters of dividing satellite cells (66, 67) may drive asymmetric division of satellite cells and preservation of the satellite cell pool.”

“[64] damaged mouse muscles and injected them with a thymidine analog compound two days later, which the cells used during replication.”

“Experiments in both adult muscle stem cells ('satellite' cells) and mouse embryonic stem cells showed that non-random chromosome segregation occur”

“Myogenic markers Myf5, MyoD, Myosin Heavy Chain (MHC), MCadherin, Desmin, and Pax 7 increase expression within the first week following DHR (121-127).”

“In several mammalian tissues, including the small intestine 4 , breast 5 , brain 6 and muscle 7,8 , nonrandom CS has been linked to self-renewing divisions of a fraction of stem cells.”

“It has been suggested that the frequency of asymmetrical inheritance of template strands increase considerably during muscle regeneration, which involved both symmetric and asymmetric cell divisions [ 30 ].”

“(15), who tracked DNA strands during tissue regeneration of adult muscle by employing three different modified nucleotides (bromo-, chloro-, and iodouridine).”

“This study left unanswered whether small intestinal stem cells are stimulated to undergo asymmetric division after injury, as described in both the intestine [49,50] and skeletal muscle [51,52], and it does not definitively exclude very subtle asymmetry of chromosomal segregation as suggested in the large intestine [5…”

“This hypothesis is supported by the fact that the expression of the DGC components is loss when primary myoblasts are cultured in vitro(18) together with their ability to perform asymmetric cell division.(57) The DGC complex was also shown by proximity ligation assay to physically interact in myofibers and in satellit…”

“Whereas most proliferating myoblasts divide symmetrically, a subpopulation of cells can divide asymmetrically to give rise to both self-renewal and differentiating progenies [14-16].”

“For example, using sequential pulses of halogenated thymidine analogues, high frequencies of segregation of older and younger template strands during proliferative expansion of mouse muscle stem cells was observed [260].”

“…Huttner, 2009; Karpowicz et al., 2005), mammary epithelial cells (Smith, 2005), fibroblasts (Merok et al., 2002), skeletal muscle satellite cells (Conboy et al., 2007; Shinin et al., 2006), human lung cancer cells (Pine et al., 2010) and female germline stem cells in the Drosophila ovaries…”

“Mechanisms such as these raise the possibility of immortal chromosome attachments and recall observations of immortal DNA strands in asymmetrically dividing cells (reviewed by Cairns 2006; see also Conboy et al. 2007).”

“This differential strand segregation has been reported in a number of adult tissues (12, 27, 28).”

“…(ii) altered deposition of collagen and other non-contractile tissue in the muscles [77], and (iii) progressive loss of individual muscle fibers [6], including a decrease in the proportion of fast-twitch type II muscle fibers, while the size of type I muscle fibers remain largely unaffected…”

“Therefore, whether symmetric and asymmetric divisions, attributed to distinct subsets of SCs [37,46,47,64,68], belongs to LPC, HPC or both, still have to be investigated.”

“Activated muscle stem cells through symmetric/ asymmetric divisions give rise to daughter cells that maintain the muscle stem cell population (satellite cells) and committed myogenic progenitor cells (MPC) that fuse to form the differentiated skeletal muscle [6, 9, 10].”

“Daughter stem cells inherit the original DNA from the mother cell, maintaining superior DNA information fidelity, while the newly synthesised DNA is allocated to daughter cells with higher differentiation potential [33].”