Frequency Of Division Research Articles

Cell Trivision of Hyperploid Cells

Malignant transformation is likely to render cells hyperploid, primarily tetraploid. We have measured the frequency of division into three rather than two daughter cells as a function of ploidy. Such trivisions were followed in near-tetraploid uveal melanoma (UM), hypotetraploid HaCaT (<4 N), hypertriploid HeLa (>3 N), and in near-diploid (∼2 N) lung epithelial cell lines by time-lapse image analyses. A stepwise analysis of cytokinesis revealed higher frequency of cell trivisions relative to divisions in hyperploid HeLa (1:24, 4%), HaCaT (1:126, 8%), and UM (1:186, 0.5%) cells. The occurrence of trivision was significantly lower in near-diploid endothelial cells (1:1400, 0.07%). We have previously observed the phenomenon of trivision in HaCaT cells treated with heavy metal lead, and here we describe that trivision is a spontaneous process taking place without genotoxic treatment. Beside re-diploidization by trivision, the hyperploid state decreases the cell size of the daughter cells and is likely to increase the time of cytokinesis. On the basis of the results, it is hypothesized that among other cancer-related causes, hyperploidy could be related to cell trivision, could cause random aneuploidy, and could generate new cancer-specific karyotypes.

Monitoring cell proliferation in vitro with different cellular fluorescent dyes

There are few methods for quantifying cell proliferation. Those tests describe the proliferation kinetics of a cell population, but they do not report the history of single cells, the number and frequency of cell divisions, or the precursor cell frequency. Cell-tracking assays based on dilution of the green fluorescent protein labelling dye, CFSE, has become the standard for monitoring cell proliferation. Other labelling dyes, e.g. CellTrace Violet and CellVue Claret, are also used for the same purpose. This study aimed to compare these three cell labelling methods for analysing the kinetics of cell viability, proliferation, and precursor cell frequency. Human peripheral blood mononuclear cells stimulated with Concanavalin A (ConA) were used as a model system. After labelling with a cell-tracking dye cells were divided into groups with and without ConA stimulation. From the 5th to 8th day, cells were collected and analysed with flow cytometry. Cell viability was not significantly different between labelled and unlabelled cells that received ConA stimulation. The proliferative fraction, proliferation index, and nonproliferative fraction were not significantly different among lymphocytes labelled with different dyes. Precursor cell frequency was also similar among cells labelled with the three cell-tracing dyes. The practical conclusion from our observations is that the results from cells labelled with different tracers may be compared directly and discussed jointly.

Notes from the Underground: Receptor‐Like Kinases in Arabidopsis Root Development

During plant development, the frequency and context of cell division must be controlled, and cells must differentiate properly to perform their mature functions. In addition, stem cell niches need to be maintained as a reservoir for new cells. All of these processes require intercellular signaling, whether it is a cell relaying its position to other cells, or more mature cells signaling to the stem cell niche to regulate the rate of growth. Receptor-like kinases have emerged as a major component in these diverse roles, especially within the Arabidopsis root. In this review, the functions of receptor-like kinase signaling in regulating Arabidopsis root development will be examined in the areas of root apical meristem maintenance, regulation of epidermal cell fate, lateral root development and vascular differentiation. [Figure: see text] Frans E. Tax (Corresponding author).

Genomics of corneal wound healing: a review of the literature

Corneal wound healing is a complex process: its mechanisms and the underlying genetic control are not fully understood. It involves the integrated actions of multiple growth factors, cytokines and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells and lacrimal gland cells. Following an epithelial insult, multiple cytokines are released triggering a cascade of events that leads to repair the epithelial defect and remodelling of the stroma to minimize the loss of transparency and function. In this review, we examine the literature surrounding the genomics of corneal wound healing with respect to the following topics: epithelial and stromal wound healing (including inhibition); corneal neovascularisation; the role of corneal nerves in wound healing; the endothelium; the role of aquaporins and aptamers. We also examine the effect of ectasia on corneal wound healing with regard to keratoconus and following corneal surgery. A better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design treatments that selectively modulate key phases of the healing process resulting in scars that more closely resemble normal corneal architecture.

Phased cell division, specific division rates and other biological observations of Dinophysis populations in sub-surface layers off the south coast of Ireland

The proportions of viable cells of Dinophysis spp. that were paired (dividing) and recently divided during a cell cycle were measured on populations of D. acuta and D. acuminata observed off the south coast of Ireland in July 2007 and July 2009. Both species exhibited phased cell division in 2009 with maximum frequency of division (fmax) 2h after sunrise. Different patterns of division (timing of fmax) were shown by D. acuta in 2007, when the population aggregated in a thin layer was transported by a coastal jet flow. High resolution (decimetre-scale) profiles within the thin layer showed large differences in the vertical distribution of biological properties (feeding status, mortality). Values of the specific growth rate µ were compared to estimates derived in similar fashion from observations on Dinophysis populations elsewhere. Different patterns exhibited by the same species in different regions may be attributed to adaptations to latitudinal differences (length of photoperiod). The question of whether phased cell division always occurs in Dinophysis populations, and the incorporation of the potential specific division rate into models of Dinophysis growth are discussed. Comprehensive field data sets demonstrate the impact of the results on the coherence of Dinophysis populations during their transport along the Irish coast in jet-like flows towards sites of intensive shellfish culture.

Division of Shape-Standardized Tobacco Cells Reveals a Limit to the Occurrence of Single-Criterion-Based Selection of the Plane of Symmetric Division

The plane of symmetric plant cell division tends to be selected so that the new cross-wall halving the cell volume has the least possible area, and several cases of such selection are best represented by a recently formulated model which promotes the view that the strength of the least area tendency is the only criterion for selecting the plane. To test this model, the present study examined the divisions of two types of shape-standardized tobacco BY-2 cell, oblate-spheroidal (os) cells prepared from protoplasts and spheri-cylindrical (sc) cells with unusual double-wall structures prepared from plasmolyzed cells. Measurements of cell shape parameters and division angles revealed that both cell types most frequently divide nearly along their short axes. While os cells did not exhibit any other division angle bias, sc cell division was characterized by another bias which made the frequency of longitudinal divisions secondarily high. The geometry of sc cells barely allows the longitudinal cross-walls to have locally minimum areas. Nevertheless, a comparison of detected and hypothetical standard divisions indicates that the frequency of longitudinal sc cell division can be significantly higher than that predicted when the longitudinal cross-walls are assumed to have locally minimum areas smaller than their original areas. These results suggest that, even in isolated plant cell types, the strength of the least area tendency is not the only criterion for selecting the division plane. The possibility that there is another basic, though often hidden, criterion is discussed.

Asymmetric division: a marker for cancer stem cells?

Asymmetric division: a marker for cancer stem cells?

Highly stable CD8 populations are maintained by systemic, low level division across multiple clones during MCMV memory inflation. (P6067)

Abstract Murine cytomegalovirus (MCMV) establishes a persistent/latent infection that induces a characteristic “memory inflation” of CD8 T cells specific for some viral epitopes. The inflationary CD8 populations are maintained at high numbers for life in spite of the fact that inflationary effectors turnover with a half-life of 45-60 days. The current model to describe memory inflation suggests that waves of memory CD8s divide in the lymph nodes to produce effectors that migrate to the tissues to control viral activity. We used an adoptive transfer of two congenically marked OT1 populations to show that, in spite of the constant turnover, two inflationary populations were maintained in parallel with minimal fluctuation over time. Likewise, within the endogenous antigen population, different clones identified by V-beta staining divided at a similar frequency. Division of antigen specific CD8s occurred at a low level systemically, including lymph nodes, spleen and non-lymphoid organs, as measured by Ki67 expression and BrdU incorporation. Although the frequency of division was the highest in draining lymph nodes, large numbers of dividing cells were still found in non-lymphoid organs, even when lymphocyte egress was blocked from lymph nodes with FTY720 treatment. These results suggest that inflationary populations are maintained by systemic, stable, low-level division across multiple clones and that division is not restricted to small numbers of cells dividing in the lymph nodes.

Modelling for rearrangement of fusiform initials during radial growth of the vascular cambium in Pinus sylvestris L.

In contrast to common belief, recent studies have confirmed that intrusive growth of fusiform cambial initials has a significant role in the rearrangement of the initials, but does not contribute to the cambial circumference increment. We observed a rapid rearrangement of cambial initials on a long series of transverse sections of the vascular cambium and the wood of a 50-year-old pine (Pinus sylvestris L.) tree. A comparison of cell arrangement in consecutive sections, as well as a critical analysis of tangential reconstructions, has confirmed that changes in cell locations in a group of cells on the tangential surface caused no change in the total tangential width of the whole group. Models illustrating changes in locations of the initials have been proposed, assuming that intrusive growth, which makes the growing initials intrude between the neighbouring initials and their immediate derivatives, is localized on the longitudinal edges of cells. We infer that intrusive growth of the cambial initials in P. sylvestris is not involved in the cambial circumference increment, but plays a significant role in the rearrangement of the initials, probably allowing for a relaxation of shearing strains generated during radial growth. The relationship of intrusive growth with the elimination of initials has been discussed with reference to the frequency of anticlinal divisions. It has been proposed that the occurrence of anticlinal divisions in excess over the actual requirement for increase in the cambial circumference could be due to internal shearing strains.

Cancer Stem Cells: A Minor Cancer Subpopulation that Redefines Global Cancer Features

In recent years cancer stem cells (CSCs) have been hypothesized to comprise only a minor subpopulation in solid tumors that drives tumor initiation, progression, and metastasis; the so-called “cancer stem cell hypothesis.” While a seemingly trivial statement about numbers, much is put at stake. If true, the conclusions of many studies of cancer cell populations could be challenged, as the bulk assay methods upon which they depend have, by, and large, taken for granted the notion that a “typical” cell of the population possesses the attributes of a cell capable of perpetuating the cancer, i.e., a CSC. In support of the CSC hypothesis, populations enriched for so-called “tumor-initiating” cells have demonstrated a corresponding increase in tumorigenicity as measured by dilution assay, although estimates have varied widely as to what the fractional contribution of tumor-initiating cells is in any given population. Some have taken this variability to suggest the CSC fraction may be nearly 100% after all, countering the CSC hypothesis, and that there are simply assay-dependent error rates in our ability to “reconfirm” CSC status at the cell level. To explore this controversy more quantitatively, we developed a simple cellular automaton model of CSC-driven tumor growth dynamics. Assuming CSC and non-stem cancer cells (CC) subpopulations coexist to some degree, we evaluated the impact of an environmentally dependent CSC symmetric division probability and a CC proliferation capacity on tumor progression and morphology. Our model predicts, as expected, that the frequency of CSC divisions that are symmetric highly influences the frequency of CSCs in the population, but goes on to predict the two frequencies can be widely divergent, and that spatial constraints will tend to increase the CSC fraction over time. Further, tumor progression times show a marked dependence on both the frequency of CSC divisions that are symmetric and on the proliferation capacities of CC. Together, these findings can explain, within the CSC hypothesis, the widely varying measures of stem cell fractions observed. In particular, although the CSC fraction is influenced by the (environmentally modifiable) CSC symmetric division probability, with the former converging to unity as the latter nears 100%, the CSC fraction becomes quite small even for symmetric division probabilities modestly lower than 100%. In the latter case, the tumor exhibits a clustered morphology and the CSC fraction steadily increases with time; more so on both counts when the death rate of CCs is higher. Such variations in CSC fraction and morphology are not only consistent with the CSC hypothesis, but lend support to it as one expected byproduct of the dynamical interactions that are predicted to take place among a relatively small CSC population, its CC counterpart, and the host compartment over time.

Meta-analysis of the cell cycle related C12orf48

The cell cycle is a conserved process from yeast to mammals and focuses on mechanisms that regulate the timing and frequency of DNA replication and cell division. The temporal and spatial expression of the genes is tightly regulated to ensure accurate replication and transmission of DNA to daughter cells during the cycle. Although the genes involved in interphase are well studied, most of the genes which are involved in mitotic events still remain unidentified. Since, the discovery of mitosis related genes is still incomplete, we performed a co-expression and gene ontology analysis for revealing novel mitosis regulated genes. In this study, we showed that C12orf48 is co-expressed with well-known mitotic genes. Moreover, it is also co-expressed with the genes that have roles in interphase such as DNA replication. Furthermore, our results showed that C12orf48 is also differentially expressed in various cancers. Therefore, the results presented in this study suggest that C12orf48 may be an important molecule for both interphase and mitosis. Since, the molecules involved in these mechanisms are crucial for proliferation as well as in carcinogenesis, C12orf48 should be considered as a novel cell cycle and carcinogenesis related gene.

Differential Localization of RAC1 and RAC2 Reflects Their Specific Functions in Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells.

AbstractAbstract 2302Hematopoietic stem cells (HSCs) depend on the bone marrow niche to provide signals for their survival, quiescence and differentiation. Many of these microenvironmental signals converge on RAC GTPases. In the hematopoietic system, two members of the RAC family are expressed, RAC1 and RAC2. Although RAC1 and RAC2 share a very high sequence homology, specific functions of these proteins have been suggested. However, little has been revealed about the downstream effectors and molecular mechanisms. In this study, we used multiple approaches to gain insight into the molecular biology of RAC1 and RAC2 in normal and leukemic human HSCs. Firstly, GFP-tagged constructs of RAC1 and RAC2 were used to study localization of these proteins in CD34+/CD38−/Lin− HSCs. Time-lapse confocal imaging of living cells plated on stroma revealed that RAC1 was strongly enriched in the plasma membrane. In contrast, RAC2 localized predominantly in the cytoplasm of both resting and dividing HSCs, whereby localization changed dramatically when cells progressed from S to the G2 phase of the cell cycle. This very distinct localization pattern implied different functions of RAC1 and RAC2. Therefore, we specifically downregulated RAC1 and/or RAC2 to study the effects of their depletion in normal and BCR-ABL-transduced leukemic HSCs. In normal HSCs, simultaneous downregulation of RAC1 and RAC2 resulted in a modest but significant decrease in proliferation and progenitor frequencies in the long term stromal co-cultures. However, in BCR-ABL-transduced HSCs depletion of RAC2 alone, but not RAC1, was sufficient to induce a marked proliferative disadvantage, decreased progenitor frequency, reduced leukemic cobblestone formation and diminished replating capacity. To elucidate the mechanisms involved in the observed phenotypes, we employed an in vivo biotin labeling strategy of Avi-tagged RAC1 and RAC2 followed by pull down and mass-spectrometry to identify specific interaction partners of RAC1 and RAC2 in BCR-ABL-expressing hematopoietic cells. Several of the RAC1-specific interaction partners were annotated as plasma membrane proteins, involved in cell adhesion, cytoskeleton assembly and regulation of endocytosis. In contrast, RAC2-interacting proteins were cytoplasmic and involved in processes such as cell cycle progression, mitosis and regulation of apoptosis. Consistently with these findings, confocal time-lapse imaging of living hematopoietic cells revealed that pharmacological inhibition of RAC2 activity resulted in greatly decreased frequency of cell division. Moreover, the average division time was significantly extended upon RAC2 inhibition. Further functional characterization of RAC1 and RAC2-specific interactions is currently ongoing and will be discussed, but our data clearly indicate that distinct subcellular localization of RAC1 and RAC2 dictates their interaction with specific sets of proteins and consequently their specific functions in hematopoietic cells. Disclosures:No relevant conflicts of interest to declare.

Development of cultured microspores of maize in the presence of n-butanol and 2-aminoethanol

The aim of the present study was to examine whether the induction of maize microspore embryogenesis could be triggered by the application of biogenic alcohols, as was reported earlier in wheat. A single cross hybrid (A 18) raised in the phytotron was used as anther donor for shed microspore cultures after cold pretreatment. At the onset of culturing, anthers in liquid YP medium were treated with 0.2 or 0.4% n-butanol or with 2 mM aminoethanol (2-AE) for 6 or 18 hours.The treatments caused a drastic (approx. 50%) decrease in the viability of the microspores. After a few days of culture in medium containing neither n-butanol nor 2-AE, 9-13% of the microspores remained alive and capable of switching to the sporophytic pathway of development.Treatment with 0.2% n-butanol for 6 h considerably increased the frequency of symmetric nuclear divisions (more than 3×) and of induced microspores (2×). The embryo yield was also elevated by 10%. The results showed that n-butanol could be used to improve the androgenic response and microspore embryogenesis in maize, but not as efficiently as in wheat. Further examination will be required to find the reasons for the different behaviour of microspores of the two species.

Cytogenetic analyses of Azadirachtin reveal absence of genotoxicity but marked antiproliferative effects in human lymphocytes and CHO cells in vitro

In this work we have examined the genotoxic potential of the bioinsecticide Azadirachtin A (AZA) and its influence on cell proliferation on human lymphocytes and Chinese Hamster ovary (CHO) cells. AZA genotoxicity was assessed by the analysis of chromosomal aberrations and sister chromatid exchanges (SCEs) in the absence and presence of rat liver S9 metabolism. Primary DNA damage was also investigated by means of the comet assay. The results obtained clearly indicate that AZA is not genotoxic in mammalian cells. On the other hand, AZA proved to interfere with cell cycle progression as shown by modulation of frequencies of first (M1) and second division (M2) metaphases detected by 5-Bromo-2′-deoxyuridine labeling. Accumulation of M1 metaphases were more pronounced in human lymphocytes. In the transformed CHO cell line, however, significant increases of multinucleated interphases and polyploid cells were observed at long treatment time. At higher dose-levels, the incidence of polyploidy was close to 100%. Identification of spindle structure and number of centrosomes by fluorescent immunostaining with α- and γ-tubulin antibodies revealed aberrant mitoses exhibiting multipolar spindles with several centrosomal signals.These findings suggest that AZA can act either through a stabilizing activity of microtubules or by inhibition of Aurora A, since both mechanisms are able to generate genetically unstable polyploid cells with multipolar spindles and multinucleated interphases.

Increased Asymmetric and Multi-Daughter Cell Division in Mechanically Confined Microenvironments

As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 µm) to low (7 µm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes.

Protein Segregation Between Dividing Hematopoietic Progenitor Cells in the Determination of the Symmetry/Asymmetry of Cell Division

In the present study, we investigated how the symmetry/asymmetry of cell division in mitotic CD34(+) cells can be evaluated by determining the plane of cell division and the potential distribution of proteins between daughter cells. The orientation of the mitotic spindle is dependent upon the positioning of the centrosomes, which determine the plane of cell division and the sharing of proteins. If the functions of unequally shared proteins are relevant to the kinetics of cell division, they could determine whether the daughter cells undergo self-renewal or differentiation. The kinetic function of the proteins of interest was investigated using a colony-replating assay and carboxyfluorescein succinimidyl ester (CFSE) staining. We used Notch/Numb as a model system, since they have a role in balancing symmetric/asymmetric divisions. Mitotic cells were examined microscopically and centrosomal markers γ-tubulin/pericentrin were used with activated Notch-1 and Numb. We monitored the first crucial divisions by CFSE staining and found an inverse relationship between activated Notch and Numb expression, suggesting a reciprocal regulation. We suggest that the subpopulations expressing activated Notch or Numb have different cell fates. To determine the influence of Notch signaling on progenitor cell self-renewal, we used the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl ester (DAPT). DAPT influences self-renewal/differentiation outcome by affecting the frequency of symmetric renewal divisions without affecting the rate of divisions. Overall, the purpose of this study was to establish a cellular system for predicting the symmetry/asymmetry of hematopoietic progenitor divisions at the level of centrosomes and protein distribution and to investigate the influence of these proteins on progenitor cell kinetics.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies(1). To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia(2-8). The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma(2,7,8). The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds(9). The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division(10). CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity(2). This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry(2). The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Slow-dividing satellite cells retain long-term self-renewal ability in adult muscle

Summary Satellite cells are muscle stem cells that have important roles in postnatal muscle growth and adult muscle regeneration. Although fastand slow-dividing populations in activated satellite cells have been observed, the functional differences between them remain unclear. Here we elucidated the relationship between proliferation behaviour and satellite cell function. To assess the frequency of cell division, satellite cells isolated from mouse EDL muscle were labelled with the fluorescent dye PKH26, stimulated to proliferate and then sorted by FACS. The vast majority of activated satellite cells were PKH26 low fast-dividing cells, whereas PKH26 high slow-dividing cells were observed as a minority population. The fast-dividing cells generated a higher number of differentiated and self-renewed cells compared with the slow-dividing cells. However, cells derived from the slow-dividing population formed secondary myogenic colonies when passaged, whereas those from the fast-dividing population rapidly underwent myogenic differentiation without producing self-renewing cells after a few rounds of cell division. Furthermore, slow-dividing cells transplanted into injured muscle extensively contributed to muscle regeneration in vivo. Id1, a HLH protein, was expressed by all activated satellite cells, but the expression level varied within the slow-dividing cell population. We show that the slow-dividing cells retaining long-term self-renewal ability are restricted to an undifferentiated population that express high levels of Id1 protein (PKH26 high Id1 high population). Finally, genome-wide gene expression analysis described the molecular characteristics of the PKH26 high Id1 high population. Taken together, our results indicate that undifferentiated slow-dividing satellite cells retain stemness for generating progeny capable of long-term self-renewal, and so might be essential for muscle homeostasis throughout life.

Slow-dividing satellite cells retain long-term self-renewal ability in adult muscle

Satellite cells are muscle stem cells that have important roles in postnatal muscle growth and adult muscle regeneration. Although fast- and slow-dividing populations in activated satellite cells have been observed, the functional differences between them remain unclear. Here we elucidated the relationship between proliferation behaviour and satellite cell function. To assess the frequency of cell division, satellite cells isolated from mouse EDL muscle were labelled with the fluorescent dye PKH26, stimulated to proliferate and then sorted by FACS. The vast majority of activated satellite cells were PKH26(low) fast-dividing cells, whereas PKH26(high) slow-dividing cells were observed as a minority population. The fast-dividing cells generated a higher number of differentiated and self-renewed cells compared with the slow-dividing cells. However, cells derived from the slow-dividing population formed secondary myogenic colonies when passaged, whereas those from the fast-dividing population rapidly underwent myogenic differentiation without producing self-renewing cells after a few rounds of cell division. Furthermore, slow-dividing cells transplanted into injured muscle extensively contributed to muscle regeneration in vivo. Id1, a HLH protein, was expressed by all activated satellite cells, but the expression level varied within the slow-dividing cell population. We show that the slow-dividing cells retaining long-term self-renewal ability are restricted to an undifferentiated population that express high levels of Id1 protein (PKH26(high)Id1(high) population). Finally, genome-wide gene expression analysis described the molecular characteristics of the PKH26(high)Id1(high) population. Taken together, our results indicate that undifferentiated slow-dividing satellite cells retain stemness for generating progeny capable of long-term self-renewal, and so might be essential for muscle homeostasis throughout life.