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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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A proportion of BrdU-retaining cells possess markers of proliferating and undifferentiated neural progenitors. (A) All clones at 4 d after BrdU exposure are Nestin(+). (i) Bright field shows 4 d clone; (ii) Nestin is blue; (iii) BrdU-labeled nuclei are green; (iv) merge shows Nestin in blue, histone-labeled nuclei in red, and BrdU-labeled cells in green. (B) All clones 10 d after BrdU exposure contain Ki67(+) cells. Note strong Ki67 positivity of BrdU(+) cell (arrow). (i) Bright field shows clone; (ii) Ki67 expression is red; (iii) BrdU-labeled nuclei are green; (iv) merge shows Ki67 in red, BrdU-labeled cells are green, Hoechst-labeled nuclei are blue. (C) 14 d after BrdU differentiated clone, with arrows indicating GFAP(+) cell nucleus. (i) Bright field; (ii) merge shows GFAP in red and BrdU in green. (D)14 d after BrdU differentiated clone, with arrows indicating Nestin(+) cell nucleus. (i) Bright field; (ii) Nestin is blue; (iii) merge shows Nestin in blue, histone-labeled nuclei in red, and BrdU-labeled cells in green. (E) Clones arising from 7 d differentiated spheres (total of 17 d after BrdU). Cells show high Nestin(+) and undifferentiated cell morphology. (i) Bright field shows clone; (ii) Nestin is blue; (iii) BrdU-labeled nuclei are green; (iv) merge shows Nestin in blue, histone-labeled nuclei are red, and BrdU-labeled cells are green. (F) Numbers of Nestin(+) clones arising, from neurospheres exposed to differentiation conditions after BrdU removal. 3 DIV refers to clones that have been 17 d without BrdU, and 7 DIV clones have been without BrdU for 21 d. Nearly all clones at 3 DIV arise from BrdU(+) cells. The total number of SC colonies is slightly higher than this number (some taking longer to start proliferating), suggesting that all clones at 3 DIV are SC colonies.
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fig5: A proportion of BrdU-retaining cells possess markers of proliferating and undifferentiated neural progenitors. (A) All clones at 4 d after BrdU exposure are Nestin(+). (i) Bright field shows 4 d clone; (ii) Nestin is blue; (iii) BrdU-labeled nuclei are green; (iv) merge shows Nestin in blue, histone-labeled nuclei in red, and BrdU-labeled cells in green. (B) All clones 10 d after BrdU exposure contain Ki67(+) cells. Note strong Ki67 positivity of BrdU(+) cell (arrow). (i) Bright field shows clone; (ii) Ki67 expression is red; (iii) BrdU-labeled nuclei are green; (iv) merge shows Ki67 in red, BrdU-labeled cells are green, Hoechst-labeled nuclei are blue. (C) 14 d after BrdU differentiated clone, with arrows indicating GFAP(+) cell nucleus. (i) Bright field; (ii) merge shows GFAP in red and BrdU in green. (D)14 d after BrdU differentiated clone, with arrows indicating Nestin(+) cell nucleus. (i) Bright field; (ii) Nestin is blue; (iii) merge shows Nestin in blue, histone-labeled nuclei in red, and BrdU-labeled cells in green. (E) Clones arising from 7 d differentiated spheres (total of 17 d after BrdU). Cells show high Nestin(+) and undifferentiated cell morphology. (i) Bright field shows clone; (ii) Nestin is blue; (iii) BrdU-labeled nuclei are green; (iv) merge shows Nestin in blue, histone-labeled nuclei are red, and BrdU-labeled cells are green. (F) Numbers of Nestin(+) clones arising, from neurospheres exposed to differentiation conditions after BrdU removal. 3 DIV refers to clones that have been 17 d without BrdU, and 7 DIV clones have been without BrdU for 21 d. Nearly all clones at 3 DIV arise from BrdU(+) cells. The total number of SC colonies is slightly higher than this number (some taking longer to start proliferating), suggesting that all clones at 3 DIV are SC colonies.

Mentions: 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 β-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.


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)

A proportion of BrdU-retaining cells possess markers of proliferating and undifferentiated neural progenitors. (A) All clones at 4 d after BrdU exposure are Nestin(+). (i) Bright field shows 4 d clone; (ii) Nestin is blue; (iii) BrdU-labeled nuclei are green; (iv) merge shows Nestin in blue, histone-labeled nuclei in red, and BrdU-labeled cells in green. (B) All clones 10 d after BrdU exposure contain Ki67(+) cells. Note strong Ki67 positivity of BrdU(+) cell (arrow). (i) Bright field shows clone; (ii) Ki67 expression is red; (iii) BrdU-labeled nuclei are green; (iv) merge shows Ki67 in red, BrdU-labeled cells are green, Hoechst-labeled nuclei are blue. (C) 14 d after BrdU differentiated clone, with arrows indicating GFAP(+) cell nucleus. (i) Bright field; (ii) merge shows GFAP in red and BrdU in green. (D)14 d after BrdU differentiated clone, with arrows indicating Nestin(+) cell nucleus. (i) Bright field; (ii) Nestin is blue; (iii) merge shows Nestin in blue, histone-labeled nuclei in red, and BrdU-labeled cells in green. (E) Clones arising from 7 d differentiated spheres (total of 17 d after BrdU). Cells show high Nestin(+) and undifferentiated cell morphology. (i) Bright field shows clone; (ii) Nestin is blue; (iii) BrdU-labeled nuclei are green; (iv) merge shows Nestin in blue, histone-labeled nuclei are red, and BrdU-labeled cells are green. (F) Numbers of Nestin(+) clones arising, from neurospheres exposed to differentiation conditions after BrdU removal. 3 DIV refers to clones that have been 17 d without BrdU, and 7 DIV clones have been without BrdU for 21 d. Nearly all clones at 3 DIV arise from BrdU(+) cells. The total number of SC colonies is slightly higher than this number (some taking longer to start proliferating), suggesting that all clones at 3 DIV are SC colonies.
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fig5: A proportion of BrdU-retaining cells possess markers of proliferating and undifferentiated neural progenitors. (A) All clones at 4 d after BrdU exposure are Nestin(+). (i) Bright field shows 4 d clone; (ii) Nestin is blue; (iii) BrdU-labeled nuclei are green; (iv) merge shows Nestin in blue, histone-labeled nuclei in red, and BrdU-labeled cells in green. (B) All clones 10 d after BrdU exposure contain Ki67(+) cells. Note strong Ki67 positivity of BrdU(+) cell (arrow). (i) Bright field shows clone; (ii) Ki67 expression is red; (iii) BrdU-labeled nuclei are green; (iv) merge shows Ki67 in red, BrdU-labeled cells are green, Hoechst-labeled nuclei are blue. (C) 14 d after BrdU differentiated clone, with arrows indicating GFAP(+) cell nucleus. (i) Bright field; (ii) merge shows GFAP in red and BrdU in green. (D)14 d after BrdU differentiated clone, with arrows indicating Nestin(+) cell nucleus. (i) Bright field; (ii) Nestin is blue; (iii) merge shows Nestin in blue, histone-labeled nuclei in red, and BrdU-labeled cells in green. (E) Clones arising from 7 d differentiated spheres (total of 17 d after BrdU). Cells show high Nestin(+) and undifferentiated cell morphology. (i) Bright field shows clone; (ii) Nestin is blue; (iii) BrdU-labeled nuclei are green; (iv) merge shows Nestin in blue, histone-labeled nuclei are red, and BrdU-labeled cells are green. (F) Numbers of Nestin(+) clones arising, from neurospheres exposed to differentiation conditions after BrdU removal. 3 DIV refers to clones that have been 17 d without BrdU, and 7 DIV clones have been without BrdU for 21 d. Nearly all clones at 3 DIV arise from BrdU(+) cells. The total number of SC colonies is slightly higher than this number (some taking longer to start proliferating), suggesting that all clones at 3 DIV are SC colonies.
Mentions: 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 β-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.

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