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Support for the immortal strand hypothesis: neural stem cells partition DNA asymmetrically in vitro.

Karpowicz P, Morshead C, Kam A, Jervis E, Ramunas J, Ramuns J, Cheng V, van der Kooy D - J. Cell Biol. (2005)

Bottom Line: The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates.We investigated cosegregation in neural stem cells (NSCs).It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, University of Toronto, Toronto, M5R 1A8, Canada. phillip.karpowicz@utoronto.ca

ABSTRACT
The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.

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Neurosphere cells retain BrdU in contrast to ESCs and fibroblasts. (A) Proportion of BrdU-labeled cells (bars) and population expansion (line) in adult neurosphere culture. 98.6 ± 0.2% of 62,500 cells plated are BrdU(+). At day 3, cells have achieved two population doublings, and at 7 d, seven doublings. 8.7 ± 1.3% of cells retain BrdU signal at the 10 d time point of 10 population doublings. (B) Proportion of BrdU-labeled cells (bars) and population expansion (line) in the R1 ESC line. Between 2 and 4 d, embryonic cells have reached the threshold during which BrdU is lost, demonstrated by a dramatic decrease from 81.1 ± 3.2% to 13.0 ± 1.2% cells labeled. ESCs lose all BrdU signal after seven doublings evidenced by day 7 (six population doublings). (C) Proportion of BrdU-labeled cells (bars) and population expansion (line) in the STO fibroblast cell line. At day 6, cells achieve three populations doublings demonstrating that fibroblasts have 2× the cell cycle time as neural precursors. At day 12, and with less than seven doublings, fibroblasts' BrdU signal is abolished.
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fig3: Neurosphere cells retain BrdU in contrast to ESCs and fibroblasts. (A) Proportion of BrdU-labeled cells (bars) and population expansion (line) in adult neurosphere culture. 98.6 ± 0.2% of 62,500 cells plated are BrdU(+). At day 3, cells have achieved two population doublings, and at 7 d, seven doublings. 8.7 ± 1.3% of cells retain BrdU signal at the 10 d time point of 10 population doublings. (B) Proportion of BrdU-labeled cells (bars) and population expansion (line) in the R1 ESC line. Between 2 and 4 d, embryonic cells have reached the threshold during which BrdU is lost, demonstrated by a dramatic decrease from 81.1 ± 3.2% to 13.0 ± 1.2% cells labeled. ESCs lose all BrdU signal after seven doublings evidenced by day 7 (six population doublings). (C) Proportion of BrdU-labeled cells (bars) and population expansion (line) in the STO fibroblast cell line. At day 6, cells achieve three populations doublings demonstrating that fibroblasts have 2× the cell cycle time as neural precursors. At day 12, and with less than seven doublings, fibroblasts' BrdU signal is abolished.

Mentions: Neurosphere cells were exposed to BrdU and then proliferated in the absence of BrdU. Population expansion was assessed simultaneously with the presence of BrdU label in dissociated cells up to 10 d after BrdU withdrawal (Fig. 3 A). This period of time spanned an estimated 9 to 10 population doublings. However, this is likely to be an underestimate of the actual number of cell divisions as there is considerable cell death in clones that was not taken into account (unpublished data). Indeed, as the average colony contains over 3,000 cells after 1 wk of culture, this represents ∼12 population doublings in the absence of cell death. Over time we observed an attenuation of BrdU signal in many cells. At day 7, most cells (33.6 ± 2.0% BrdU[+]) ceased to possess any detectable BrdU signal. This is likely to be a result of the attenuation of BrdU signal via cell divisions to a threshold at which the presence of BrdU is so slight that it cannot be detected by immunocytochemistry using our detection protocol. Despite this severe loss, there was a striking perseverance of BrdU-labeled cells at 10 population doublings, day 10, with 8.7 ± 1.3% of cells exhibiting varying levels of BrdU(+) signal.


Support for the immortal strand hypothesis: neural stem cells partition DNA asymmetrically in vitro.

Karpowicz P, Morshead C, Kam A, Jervis E, Ramunas J, Ramuns J, Cheng V, van der Kooy D - J. Cell Biol. (2005)

Neurosphere cells retain BrdU in contrast to ESCs and fibroblasts. (A) Proportion of BrdU-labeled cells (bars) and population expansion (line) in adult neurosphere culture. 98.6 ± 0.2% of 62,500 cells plated are BrdU(+). At day 3, cells have achieved two population doublings, and at 7 d, seven doublings. 8.7 ± 1.3% of cells retain BrdU signal at the 10 d time point of 10 population doublings. (B) Proportion of BrdU-labeled cells (bars) and population expansion (line) in the R1 ESC line. Between 2 and 4 d, embryonic cells have reached the threshold during which BrdU is lost, demonstrated by a dramatic decrease from 81.1 ± 3.2% to 13.0 ± 1.2% cells labeled. ESCs lose all BrdU signal after seven doublings evidenced by day 7 (six population doublings). (C) Proportion of BrdU-labeled cells (bars) and population expansion (line) in the STO fibroblast cell line. At day 6, cells achieve three populations doublings demonstrating that fibroblasts have 2× the cell cycle time as neural precursors. At day 12, and with less than seven doublings, fibroblasts' BrdU signal is abolished.
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Related In: Results  -  Collection

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fig3: Neurosphere cells retain BrdU in contrast to ESCs and fibroblasts. (A) Proportion of BrdU-labeled cells (bars) and population expansion (line) in adult neurosphere culture. 98.6 ± 0.2% of 62,500 cells plated are BrdU(+). At day 3, cells have achieved two population doublings, and at 7 d, seven doublings. 8.7 ± 1.3% of cells retain BrdU signal at the 10 d time point of 10 population doublings. (B) Proportion of BrdU-labeled cells (bars) and population expansion (line) in the R1 ESC line. Between 2 and 4 d, embryonic cells have reached the threshold during which BrdU is lost, demonstrated by a dramatic decrease from 81.1 ± 3.2% to 13.0 ± 1.2% cells labeled. ESCs lose all BrdU signal after seven doublings evidenced by day 7 (six population doublings). (C) Proportion of BrdU-labeled cells (bars) and population expansion (line) in the STO fibroblast cell line. At day 6, cells achieve three populations doublings demonstrating that fibroblasts have 2× the cell cycle time as neural precursors. At day 12, and with less than seven doublings, fibroblasts' BrdU signal is abolished.
Mentions: Neurosphere cells were exposed to BrdU and then proliferated in the absence of BrdU. Population expansion was assessed simultaneously with the presence of BrdU label in dissociated cells up to 10 d after BrdU withdrawal (Fig. 3 A). This period of time spanned an estimated 9 to 10 population doublings. However, this is likely to be an underestimate of the actual number of cell divisions as there is considerable cell death in clones that was not taken into account (unpublished data). Indeed, as the average colony contains over 3,000 cells after 1 wk of culture, this represents ∼12 population doublings in the absence of cell death. Over time we observed an attenuation of BrdU signal in many cells. At day 7, most cells (33.6 ± 2.0% BrdU[+]) ceased to possess any detectable BrdU signal. This is likely to be a result of the attenuation of BrdU signal via cell divisions to a threshold at which the presence of BrdU is so slight that it cannot be detected by immunocytochemistry using our detection protocol. Despite this severe loss, there was a striking perseverance of BrdU-labeled cells at 10 population doublings, day 10, with 8.7 ± 1.3% of cells exhibiting varying levels of BrdU(+) signal.

Bottom Line: The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates.We investigated cosegregation in neural stem cells (NSCs).It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, University of Toronto, Toronto, M5R 1A8, Canada. phillip.karpowicz@utoronto.ca

ABSTRACT
The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.

Show MeSH
Related in: MedlinePlus