2 Department of Surgery, University of Toronto, Toronto, M5R 1A8, Canada
3 Department of Medical Genetics and Microbiology, University of Toronto, Toronto, M5R 1A8, Canada
4 Department of Chemical Engineering, University of Waterloo, Waterloo, N2L 3G1, Canada
Correspondence to P. Karpowicz: phillip.karpowicz{at}utoronto.ca
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Introduction |
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Intriguingly, it has been suggested that DNA itself is segregated unevenly between recipient daughter cells. Such a separation would not be a reversible one, like unevenness in protein or mRNA distribution, both of which could theoretically be regulated after division so that dissimilar daughter cells might eventually establish an equivalence in certain biochemical pathways. Asymmetric DNA distribution would be an immutable physical discrepancy between daughter cells that would define a division as asymmetric by virtue of an inherent, and measurable, physical difference in cells containing original templates and cells containing newly synthesized DNA. Such a separation was first interpreted from the uneven distribution of 3[H]thymidine in proliferating in vitro mouse embryonic fibroblasts (Lark et al., 1966), and later experiments suggested that stem cells (SC) in the intestinal epithelium of mice also segregated their chromosomes asymmetrically (Potten et al., 1978, 2002). Recent evidence continues to support chromosome cosegregation in mutated fibroblasts (Merok et al., 2002). This asymmetric distribution of chromosomes in dividing SCs was originally dubbed the immortal strand hypothesis (ISH; Cairns, 1975). Such a mechanism was envisioned to reduce the incidence of mutations arising from errors in DNA synthesis and repair in future progenitor cells derived from the SCs. An asymmetry in DNA inheritance between daughter cells might also retain sequence fidelity for genes conferring pluripotency to SCs. It has been suggested that SCs in somatic tissues actively suppress chromosome recombination events (Potten et al., 1978, 2002), and are exceptionally sensitive to DNA damage as demonstrated by the high incidence of apoptosis in irradiated SC populations. SCs are thus defined partially by their function to transmit a faithful copy of DNA template to future cell generations.
Many studies have failed to support the ISH in S. cerevisiae (Neff and Burke, 1991), mouse epidermal basal cells (Kuroki and Murakami, 1989), the proliferating cells of Caenorhabditis elegans, as well as murine embryos (Ito and McGhee, 1987; Ito et al., 1988). These positive and negative findings are equivocal as supporting and dissenting studies used distinct and contrasting cell types, at distinct and contrasting periods of an organism's development. Moreover, if such a mechanism manifests itself only in SCs, it may easily be overlooked as these comprise a minority in the cell population. Evidence of chromosome segregation in most studies to date has been undertaken retrospectively at the population level. Thus after three decades of research, it is still an open question if actively dividing SCs cosegregate older and newer DNA asymmetrically during mitosis.
According to the ISH, SCs cosegregate chromosomes to retain older DNA templates in one daughter SC but not the non-SC daughter (Fig. 1). Given that DNA replication is semiconservative, cosegregated chromosomes are distinguished because they contain one older strand, albeit one that is associated with a newer strand from one preceding round of DNA synthesis. We predicted that symmetric SC divisions would randomize segregation of chromosomes between daughter cells. The ISH was investigated in neural stem cells (NSCs) using a clonal cell culture system in which brain-derived colonies, arising from a single SC, are both self renewing and multipotent (Reynolds and Weiss, 1992; Morshead et al., 1994). The halogenated thymidine analogue, 5-bromo-2-deoxyuridine (BrdU) was used to label DNA strands. We asked: (a) would SCs retain BrdU(+) DNA strands in the absence of BrdU, if they divided symmetrically many times in the presence of BrdU (see Fig. 2 A); and (b) would SCs retain their original BrdU() strands, in the absence of BrdU, if they divided asymmetrically once and only once in the presence of BrdU (see Fig. 7).
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Here we present in vitro evidence that old and new DNA templates are distributed asymmetrically in NSC divisions in clonal population studies and at the single cell level.
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Results |
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Treatment of cells with BrdU did not alter SC phenotype. BrdU(+) cells retained the ability to self-renew, as demonstrated by subcloning secondary, tertiary, and quaternary BrdU-treated cell clones after BrdU exposure (unpublished data). Untreated clones grew to an average diameter of 144 ± 8 microns at 7 d, similarly to their BrdU-treated counterparts, which were 149 ± 5 microns in diameter. We determined that a clonally derived 149 micron BrdU-pulsed colony represented 3,075 ± 91 cells in total.
When primary SC colonies were passaged twice in the absence of BrdU, tertiary colonies generally had one or a few BrdU(+) cells 10 d after BrdU exposure (Fig. 2 C). In all cases, such colonies arose clonally from single BrdU(+) cells, that had not diluted out BrdU label over 10 d. This suggested either that: the proliferating founder BrdU(+) cells were cycling at a slow rate relative to their progeny; or, were postmitotic cells that had arisen in the first division of an actively proliferating BrdU(+) founder cell, which itself kept dividing to dilute out BrdU label; or, alternatively, were a result of heterogeneity in chromosome segregation.
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We assayed a second group of cells: the STO/SNL fibroblast cell line, transformed cells derived from embryonic mice that were passaged >40 times. Fibroblasts are not thought to be SCs. Like R1's, STO fibroblasts did not retain BrdU (Fig. 3 C). By five to six population doublings, at day 10, only 18.6 ± 0.7% of the cells retained any BrdU signal, and this was completely eradicated at day 12, which corresponds to less than seven doublings.
In both ESCs and fibroblasts, BrdU extinction was noted when cells were expanded over seven population doublings. A mouse cell, which contains 40 BrdU(+) chromosomes and which halved chromosomes containing BrdU label in each division symmetrically would indeed dilute this number to one single chromosome after five to six division events. On average, seven cell divisions in the absence of BrdU are sufficient to extinguish the label if cells partition BrdU-labeled chromosomes randomly. In contrast to fibroblasts and ESCs, which are thought to divide only symmetrically, NSC colonies contained cells able to retain the analogue well past this seven-division dilution threshold.
We attempted to see if NSCs would eventually dilute out all BrdU through symmetric divisions. BrdU-exposed SC colonies were passaged four times in the absence of BrdU. Overall this represents >25 doublings, and indeed in only a few cases were we able to find BrdU-labeled cells in colonies maintained past 14 such doublings (unpublished data). NSCs do not divide asymmetrically exclusively, but can certainly divide symmetrically, as evidenced by the formation of multiple secondary colonies arising from a single subcloned NSC colony plated at clonal density (Morshead et al., 1994). Only cells that have not divided symmetrically more than seven times retain BrdU at a detectable level. Symmetric divisions in neurosphere culture may account for the eventual loss of all BrdU signal in all cells, and asymmetric divisions may explain the retention of the BrdU signal in contrast to ESCs and fibroblasts.
BrdU-retaining cells are not quiescent
If BrdU-retaining cells were postmitotic or relatively mitotically stagnant, such heterogeneity in cell cycle within the neurosphere cell population would explain the contrast between these cells and embryonic cells or fibroblasts.
1-Dioctadecyl-3-tetramethylindocarbocyanine perchlorate (DiI) is a lipophilic fluorescent dye that associates with cell membranes and carboxyfluorescein diacetate succinimidyl ester (CFSE) is a cytosolic dye that renders cells fluorescent upon uptake. We reasoned that cells initially positive for either such dyes would subsequently halve their fluorescent dye intensity after each division, as the dye was redistributed among the daughter cells. This would enable the separation of fractions of cells that were dividing quickly from their quiescent counterparts, before such cells were examined for the presence of BrdU. DiI has already been proven amenable to FACS analysis (Malatesta et al., 2000) and there is no evidence that DiI can be passed between adjacent cells. Nonetheless, we cocultured neurosphere cells that had been exposed to DiI, with GFP(+)/DiI() neurosphere cells in high cell density aggregated colonies for 1 wk. We confirmed that none of the GFP(+) cells took up DiI label, confirming that the dye cannot be shared between adjacent cells (unpublished data).
Neurosphere cells were exposed to BrdU and then immediately pulsed with DiI. We sorted cells to confirm that these cells were also DiI(+). 97.6 ± 0.7% of cells emitted a high DiI signal by FACS. These results confirmed our starting population was positive for both indicators of cell proliferation.
The BrdU(+)/DiI(+) cells were proliferated for 1 wk in the absence of BrdU and then sorted into DiI(HI+) and DiI(LOW+) fractions. In 1 wk, the DiI signal was diminished in most of the cells as shown by the shift in DiI intensity (Fig. 4 A). We collected 9.7 ± 2.2% of the cells as a DiI(HI+) fraction and 63.1 ± 7.4% of all cells as the DiI(LOW+) fraction, leaving a buffer fraction of 30% cells between the two groups to reduce contamination between them. DiI signal was assessed by visual inspection to confirm that DiI(HI+) cells were indeed strongly positive for the membrane dye (Fig. 4 B), whereas DiI(LOW+) cells displayed no signal (Fig. 4 C).
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Within the DiI(LOW+) group we observed many BrdU() cells (75.7 ± 3.0%; Fig. 4 E), which was expected as the neurosphere cells had already demonstrated a loss of BrdU label through symmetric divisions, and the extinction of both cell division indicators was predicted. Intriguingly, this actively dividing fraction also contained 23.1 ± 2.7% weakly or moderately BrdU(+) cells, and 1.2 ± 0.2% heavily BrdU(+) cells. Altogether these occupied 15% of the total cell population. Together with the 7.1% of BrdU(+)/DiI(HI+) cells above, this is lower than the 33.6 ± 2.0% of cells we originally gathered during our analysis of BrdU retention at day 7 of neurosphere culture (Fig. 3 A). Nonetheless, 1.2% of these cells had BrdU(+) signals at a strength that was qualitatively equivalent with that of cells immediately after BrdU withdrawal. The retention of DNA label in fast proliferating cells during 1 wk of culture was suggestive of the cosegregation of BrdU(+) chromosomes during asymmetric cell divisions.
We reproduced these results using CFSE instead of DiI (unpublished data). In addition, we quantified the intensity of fluorescence emitted by cells immediately after CFSE exposure whose average was found to be >4,000 higher than that diluted by cells proliferated for 7 d. Indeed we calculated that the average level of intensity emitted by even the highest CFSE fluorescent cells, at 7 d culture, reflected at least 12 population doublings. This is the number of divisions one would expect in a single neurosphere clone of >3,000 cells at this time point. Moreover, when we diluted the concentration of initial CFSE dye applied to cells to approximate seven population doublings (seven halvings of that concentration), we found that cells treated with this concentration were still, on average, >250 times more fluorescent than those exposed to undiluted dye and allowed 7 d to dilute it. We thus confirm that in 7 d proliferation conditions, most neurosphere cells are proliferating and undergo over seven population doublings, at which BrdU fluorescence is diluted past the threshold of detection in ESCs and fibroblasts. However, a subset of proliferating neurosphere cells retain BrdU.
In vivo, SCs are thought to divide slowly (Morshead et al., 1994), but based on our evidence we predicted this was not the case in vitro. If SCs divided slowly, it would follow that they would be enriched in the DiI(HI+) fraction. We assessed each fraction for secondary colony forming ability (Fig. 4 F), which is indicative of SC presence via self renewal. The DiI(LOW+) population gave rise to 7.4 (±1.5) times as many spheres as the DiI(HI+) population at clonal density. This suggested that this fast-dividing DiI(LOW+) fraction contained most if not all of the SCs. Subcloning the DiI(LOW+) and DiI(HI+) fractions revealed that not one secondary sphere arose from the DiI(HI+) sphere cells though many secondary spheres arose from the DiI(LOW+) sphere cell population. This suggests that the DiI(HI+) spheres arose from progenitor cells that were unable to self renew whereas self-renewing SCs were fully restricted to the DiI(LOW+) fraction. What is more, 24% of the DiI(LOW+) population contained BrdU(+) cells.
Some BrdU-retaining cells express markers of proliferating, multipotent precursors
In vivo neural precursor cells are positive for Nestin, a filament protein that is present in proliferating neural precursors (Lendahl et al., 1990). We observed that all cells in proliferating clones were Nestin(+) at 4 d after BrdU removal, and that every clone contained one or more BrdU(+) cells (Fig. 5 A). At 4, 7, and 10 d under proliferation conditions, all colonies derived from BrdU-exposed cells were composed entirely of Nestin(+) cells, and contained no glial fibrillary acidic protein (GFAP)(+) cells (a marker of astrocytes), or ß-3-tubulin(+) cells (a marker of neuronal cells; unpublished data). We then stained cells at 10 d after BrdU removal for Ki67, a cell proliferation marker, and found that 79.1 ± 7.5% were Ki67(+). At both these time points we confirmed that every single cell colony contained Ki67(+) cells and that BrdU(+) cells also displayed Ki67 positivity (Fig. 5 B). We found similar results using proliferating cell nuclear antigen (PCNA; also known as DNA polymerase clamp), another marker of proliferation (unpublished data). This data suggests that under proliferation conditions all colonies are composed of cycling Nestin(+) cells.
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We took differentiated neurospheres from differentiation conditions and replated them at clonal density in proliferation conditions, to see if these undifferentiated cells would subclone. Most cells subcloned, remained differentiated as assessed by their obvious glial, or neuronal morphology. Such cells did not divide and displayed low, if any, Nestin positivity. Interestingly, a proportion of cells did not appear to be differentiated neurons or glia by morphology, displayed high Nestin(+), and divided rapidly (Fig. 5 E). Of 2,000 cells removed from the differentiation substratum and examined at 3 d in vitro (DIV), approximately eight secondary clones arose, and of these six had at least one BrdU(+) cell present despite a total of 17 d culture having elapsed since BrdU exposure (Fig. 5 F). Hence, we suggest that secondary clones obtained after 1 wk of differentiation arise from BrdU(+) cells, which themselves persist as undifferentiated BrdU(+)/Nestin(+) precursors under differentiation conditions. On average 11 neurospheres, arising from differentiated colonies, were produced for every 2,000 cells plated. We suggest that these neurospheres at 7 d are the same cell clones examined at 3 d.
This data reveals that self-renewing NSCs persist in differentiation conditions, and that it is likely that some of these retain BrdU. It is extraordinary that undifferentiated and cycling Nestin(+)/BrdU(+) cells would persist in clones composed of an average of 3,000 cells, but which in some cases grew to large 300 micron clones numbering up to 15,000 cells. All clones contained BrdU(+) cells. Such a phenomenon is strongly suggestive of chromosome cosegregation in NSCs, because such cells must have already achieved more than seven cell doublings, a time point at which we have shown BrdU to be no longer detectable in symmetrically dividing STO or R1 cells.
Cell cycle arrest and real time imaging confirm asymmetric segregation of older and newly synthesized DNA
We asked whether in vitro neural precursors could distribute DNA asymmetrically using cytokinetic and karyokinetic inhibitors and immunofluorescence. 10 d after BrdU cells were exposed to an actin binding protein, cytochalasin D, to arrest them during cytokinesis, although karyokinesis had already occurred. Such treatment resulted in the recovery of many binucleate cells, composing approximately half of the total cell population. The complete dissociation of cells, including binucleate cells, into a single cell suspension was verified on a hemocytometer. Though most BrdU(+) binucleate cells displayed equivalent BrdU signal in Fig. 6 A we found instances of cells that had one labeled nucleus and one unlabeled nucleus (Fig. 6 B). Such cells had been arrested by mitotic inhibition over a period of 24 h, meaning that it is likely that many of these cells had a cell cycle of <24 h. Thus, it is likely that at least 10 divisions occurred in these cells over 10 d in neurosphere culture. Notwithstanding 10 consecutive divisions, a subset of cells cosegregated BrdU-labeled chromosomes into one nucleus and remained positively labeled in contrast to the majority of cells examined at this time point. Quantification revealed that 78.3 ± 4.5 binucleate cells had two unlabeled nuclei, 11.4 ± 2.7% had equally labeled nuclei, and 10.3 ± 1.9% exhibited BrdU signal in only one of the daughter nuclei (Fig. 6 C). No evidence of uneven BrdU(+) signal in fibroblast cells treated with cytochalasin D was found (unpublished data), in contrast to neural cells.
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We continued the above experiment, but allowed single dissociated cells 2 h to complete division upon removal of nocodazole. Consequently, single mitotic cells became cell doublets. We again found unevenly labeled daughter cells similar to the results obtained with cytochalasin Dtreated cells (Fig. 6 E), as well as cell doublets, which were BrdU() or evenly BrdU(+) (Fig. 6 F).
Thus far our results did not examine asymmetric DNA partitioning within living individual mitotic cells. We made use of a real time imaging system to track cell division within clones arising from single neurosphere cells. Clones were filmed and then fixed at varying time points, and cell lineages were reconstructed. Unlike our studies so far, we did not expose cells for an extended time to BrdU. Instead, cells were plated in BrdU-containing medium and allowed to undergo DNA synthesis to divide exactly once. We reasoned that, just before mitosis, each mouse cell would contain 40 pairs (4N) of BrdU(+) chromosomes to be distributed to both daughters. After division in BrdU, both daughter cells would contain 40 chromosomes, half unlabeled with the original unlabeled DNA template strand and half labeled with the new and BrdU(+) synthesized strand (Fig. 7 A). Therefore each cell daughter would be positive for BrdU signal at the two cell stage, when BrdU was removed. Whereas in our previous work we inferred immortal strand retention in SCs by the presence of analogue, here we examined the loss of newly synthesized BrdU(+) DNA and retention of older, unlabeled DNA in SCs. Two asymmetric divisions, the first in the presence of BrdU, the second in the absence of BrdU, would result in a dissociation of labeled and unlabeled DNA strands. Daughter SCs would retain the original unlabeled strands.
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Using the same strategy, we plated cells in the microwells of Terasaki plates in the presence of BrdU. We confirmed that single cells were initially present in each well, and after overnight incubation, wells containing cell doublets were scored before the removal of BrdU. Cells were then allowed to proliferate for 4 d before the removal of mitogens and addition of serum and substrate to initiate cell differentiation. Colonies were then assessed for BrdU, ß-3-tubulin, and Nestin. We examined 29 clones and found that 9 of these showed asymmetric BrdU partitioning, whereas 13 showed asymmetry in cell fate, containing a mixture of both Nestin and ß-3-tubulin(+) cells (unpublished data). Strikingly, eight of the nine clones with asymmetric BrdU partitioning also demonstrated concomitant asymmetric cell fate. Within asymmetric clones, all ß-3-tubulin(+) cells were BrdU(+), but only 37.8 ± 12.2% of the Nestin(+) cells colabeled with BrdU, suggesting that some progenitors and/or SCs shed newly synthesized DNA preferentially. None of the clones in this experiment produced the number of offspring expected from seven population doublings, the number at which BrdU would be reaching its threshold of dilution. These results suggest that: (a) asymmetric DNA partitioning is correlated with asymmetric cell fate; and (b) only undifferentiated precursors cosegregate and retain their original unlabeled DNA, whereas labeled DNA is passed on to cells destined to differentiate.
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Discussion |
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The expectation that cells would retain a full complement of labeled chromosomes has been used to invalidate the ISH (Ito et al., 1988). This expectation is false, because cells dilute BrdU-containing chromosomes randomly through symmetric divisions, and may segregate varying ratios of BrdU-labeled and -unlabeled chromosomes as immortal strandbearing chromosomes. Indeed, BrdU heterogeneity among immortal DNA strands is entirely consistent with the cosegregation phenomenon. For example, if a single SC had one half of each chromosome strand labeled with BrdU, it would possess 40 one-halflabeled, and 40-nonlabeled chromosomes immediately after DNA synthesis had taken place. If such a cell was to divide symmetrically, it would randomly obtain between 0 to 40 of the labeled chromosomes to be consequently segregated as immortal strands. Thus, a cell will not be indelibly marked with analogue as a result of DNA synthesis according to the ISH. This is consistent with our results.
It has been suggested that chromosome cosegregation confers a means for SCs, that arise in the embryo and, which divide in the animal until senescence, to avoid passing potentially deleterious mutations, occurring as a result of errors in DNA synthesis and DNA repair to their progeny (Cairns, 2002). SCs may also need to avoid recombining chromosomes, as recombination might obviate or at least attenuate any benefits accrued through the segregation of older chromosomes. Suppression of recombination in SCs would itself provide a mechanism to avoid loss of heterozygosity events that could lead to cell transformation (Tischfield and Shao, 2003).
Studies on the mollusc (Tomasovic and Mix, 1974) demonstrated a surprising retention of incorporated DNA label in cells within continuously regenerating tissues of the adult animal. Cells from the adult mouse have been found to retain thymidine analogue (Potten et al., 1978, 2002). However, investigations of cosegregation during development have failed to observe DNA label retention in murine blastocysts or morula in vivo (Ito et al., 1988), nor in the embryos of C. elegans (Ito and McGhee, 1987). Similarly, our evidence does not support the ISH in ESCs derived from the 3.5-d blastocyst of early murine embryos. Further work needs to address this discrepancy explicitly.
Template strand retention might attenuate the end replication problem in telomeres during DNA synthesis and thus allow a SC greater divisions without the need for telomerase or other related mechanisms. The occurrence of symmetric divisions in SCs means that the end replication problem would still apply to SCs, although these may possess an overall greater potential number of total divisions, before becoming senescent, relative to non-SCs. Consequently, we hypothesize that SCs cosegregating older chromosomes possess a greater proliferative capacity than non-SCs in the absence of enzymatic telomere length maintenance.
Mechanisms involving microtubule/centrosomal asymmetry in both protein (Liakopoulos et al., 2003) and mRNA (Lambert and Nagy, 2002) localization have been shown to occur during cell division. It is possible that older chromosome strands could be associated with the mitotic spindle apparatus that would cosegregate immortal strandbearing chromosomes in an intrinsic fashion. A molecular basis for such an uneven chromosome segregation is unknown, but a theoretical model for such a system has been proposed, which would involve sequence recognition of either leading or lagging templates in dividing cells (Jablonka and Jablonka, 1982). It is possible that leading versus lagging DNA synthesis might prime chromosomes for separation during synthesis itself. The yeast, Schizosaccharomyces pombe, uses a DNA strandspecific imprinting mechanism to produce daughter cells, where only one of the two changes its mating cell type by the process of mating type switching (Dalgaard and Klar, 2001). Such an asymmetry is conferred at the template level, where an imprint is installed only during lagging strand replication, but not in that of the leading strand. The lagging strand imprint permits subsequent DNA recombination by a double strand repair mechanism, which differentiates daughter cell chromosomes. In this system it appears that inheritance of specific chain of the parental chromosome is crucial for cellular differentiation and asymmetry between daughter cell progeny. Without the imprint cells do not maintain the multipotent lineage and can only produce differentiated progeny, behaviors that bear intriguing similarity to the multipotent nature of self-renewing SCs in multicellular eukaryotes. We envision such a mechanism to be primarily epigenetic, though progeny arising asymmetrically from the SC lineage might carry sequence differences as a result of errors in DNA synthesis. The uneven segregation of DNA pattern in an endlessly cycling cell might be sufficient to define the epigenetic persistence of the SC itself.
The pioneering efforts of Meselson and Stahl (1958), demonstrating that the semiconservative replication of DNA resulted in equal partitioning of genetic material, overall have suggested a fundamentally random nature to the distribution of genetic copy between generations. It has been generally assumed that eukaryotic chromosomes are randomly distributed to daughter cells and that daughter cell asymmetry is not a result of DNA asymmetry, but a rather a result of genetic product differences. This may not apply to all mitotic inheritance; our results here support the hypothesis that a small population of neural cells retain their original DNA when dividing asymmetrically; and that these cells possess NSC characteristics.
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Materials and methods |
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Differentiation
Neurospheres were isolated and transferred to 24-well plates (Nunclon) coated with 15.1 mg/ml MATRIGEL basement membrane matrix (Becton Dickinson) diluted 1:25. Alternatively, 5-cm Petri dishes (Nunclon) were coated with MATRIGEL and clones were transferred in bulk. Cells were differentiated for 7 d in serum-free media containing 1% FBS (Hyclone). Cells were removed from MATRIGEL, using 0.25% porcine trypsinEDTA solution (Sigma-Aldrich) applied for 5 min at 37°C.
BrdU and dye labeling
0.6 µM BrdU (Sigma-Aldrich) was used to label synthesized DNA. To remove BrdU, cells were centrifuged, washed, and reconstituted in fresh media. BrdU was applied at the same concentration and time interval in ESCs and fibroblasts. Vybrant DiI (Molecular Probes) was administered to neurosphere cells after dissociation using 5 µl/ml of DiI stock for 5 min at 37°C. CFSE (Molecular Probes) was used according to manufacturer's instructions. Cells were washed three times using serum-free media to remove dyes.
Immunofluorescence and microscopy
Dissociated cells or colonies were coated with MATRIGEL for 30 min at 37°C. Cell attachment was assessed by gently tapping plates under microscope. Cells were also attached using CELL-TAK (Becton Dickinson) according to manufacturer's instructions. Cells were fixed using 4% paraformaldehyde (Sigma-Aldrich) dissolved in cold Stockholm's phosphate-buffered saline (pH 7.3) for 15 min. Neurospheres were equilibrated in 30% sucrose (Sigma-Aldrich) and StPBS overnight at 4°C, embedded in cryoprotectant (Thermo Electron Corporation), and sectioned on a Jencon's OTF5000 cryostat. To detect BrdU, cells were exposed to 4 N HCl for 30 min. Cells were blocked using 10% normal goat serum (Sigma-Aldrich) in StPBS, pH 7.3, 0.3% Triton (Sigma-Aldrich) for 45 min at room temperature. Primary antibodies were applied overnight in StPBS, 1.0% NGS, 0.3% Triton (Sigma-Aldrich). Anti-BrdU Bu1/75 (1:500; Abcam), anti-Nestin (1:1,0002,000; Chemicon), antiglial fibrillary acidic protein (1:400; Biomedical Technologies), antiß-tubulin isotype III (1:500; Sigma-Aldrich), anti-Ki67 (1:10; Becton Dickinson), proliferating cell nuclear antigen (1:10; Zymed Laboratories), and antipan-histone (1:500; Chemicon) were used. Secondary antibodies were applied at 37°C for 50 min in StPBS 1.0% normal goat serum. TRITC, FITC, and CY3-conjugated antibodies (1:250; Jackson ImmunoResearch Laboratories) or secondary 350 and 568 nm Alexa Fluor antibodies (1:300; Molecular Probes) were used. Nuclei were sometimes counterstained with 10 µg/ml Hoechst 33258 (Sigma-Aldrich). Cells were photographed in StPBS or Gel Mount (Biomeda Corp.). Cells were visualized at 40x/0.55 (dry lens) objective using a Nikon DIAPHOT 200 microscope, and a 40x/0.60 Olympus IX81 microscope with the Olympus Microsuite version 3.2 analysis imaging system software (Soft Imaging Systems Corp.). Cell nuclei were counted within a known area of 15 µm thickness to calculate cell density. For confocal microscopy, cells were visualized at 100x/0.30 (oil immersion lens) objective using a Zeiss Axiovert 100 LSM410 with LSM version 3.993 imaging software (Carl Zeiss MicroImaging Corp.). Photos were processed using Adobe Photoshop 6.0 software.
Cell division inhibition
Cells were exposed to 2 µM of cytochalasin D (Sigma-Aldrich) or 0.1 µg/ml Nocodazole (Sigma-Aldrich) for 24 h at 37°C. Nocodazole inhibition was removed by aspirating media containing mitotic inhibitor, washing with serum-free media, and resuspending cells in medium containing FGF2, heparin, and EGF. Cells were then fixed 2530 min later.
FACS
Cells were sorted on FACS DiVa (Becton-Dickinson) system. Cells were sorted at 9,000 events per second and fractions were kept on ice until plated. For each sample, freshly pulsed DiI or CFSE cells were used to confirm positivity at the outset of each sort.
Cell imaging
Cells were imaged at 40x/0.75 (dry lens) magnification using an Axiovert 200 inverted microscope (Carl Zeiss MicroImaging, Inc.). Samples were illuminated every 2 min during image acquisition and images were captured with Sony XCD-SX900 digital camera, using ImageJ software (National Institutes of Health). Cells were loaded in BrdU-containing media and filmed until one division had occurred. BrdU was then immediately removed and fresh media substituted.
Submitted: 11 February 2005
Accepted: 15 July 2005
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References |
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