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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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NSCs are fast dividing cells in vitro, and show BrdU retention. (A) Distribution of DiI, initially and after 1 wk in vitro. DiI signal decreases as a result of DiI dilution via cell proliferation. (DiI pos) DiI(HI+) fraction; (DiI neg) DiI(LOW+) fraction. (B) DiI(HI+) fraction of slowly dividing neurosphere cells where DiI signal is vivid. (i) Dissociated cells in bright field; (ii) DiI signal in red. (C) DiI(LOW+) fraction of rapidly dividing neurosphere cells where DiI signal is noticeably lower than in DiI(HI+) fraction. (i) Dissociated cells in bright field; (ii) DiI signal in red. (D) Data shows DiI(HI+) population (10% of total). As expected, slowly cycling cells do not greatly attenuate BrdU or DiI. The BrdU(−) population may be the same 1% of cells that are BrdU(−) immediately after BrdU exposure. (E) Data shows DiI(LOW+) population (63% of total). A subset of BrdU(+) cells are DiI(LOW+) after extended cell proliferation in vitro. BrdU retention in rapidly cycling cells suggests these are cosegregating their DNA. (F) Comparison in clonal sphere formation between DiI(HI+) and DiI(LOW+) fractions. The majority of neurospheres arise from the fast-cycling DiI(LOW+) population (7.5-fold increase over DiI[HI+]). This suggests SCs are in this actively proliferating fraction.
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fig4: NSCs are fast dividing cells in vitro, and show BrdU retention. (A) Distribution of DiI, initially and after 1 wk in vitro. DiI signal decreases as a result of DiI dilution via cell proliferation. (DiI pos) DiI(HI+) fraction; (DiI neg) DiI(LOW+) fraction. (B) DiI(HI+) fraction of slowly dividing neurosphere cells where DiI signal is vivid. (i) Dissociated cells in bright field; (ii) DiI signal in red. (C) DiI(LOW+) fraction of rapidly dividing neurosphere cells where DiI signal is noticeably lower than in DiI(HI+) fraction. (i) Dissociated cells in bright field; (ii) DiI signal in red. (D) Data shows DiI(HI+) population (10% of total). As expected, slowly cycling cells do not greatly attenuate BrdU or DiI. The BrdU(−) population may be the same 1% of cells that are BrdU(−) immediately after BrdU exposure. (E) Data shows DiI(LOW+) population (63% of total). A subset of BrdU(+) cells are DiI(LOW+) after extended cell proliferation in vitro. BrdU retention in rapidly cycling cells suggests these are cosegregating their DNA. (F) Comparison in clonal sphere formation between DiI(HI+) and DiI(LOW+) fractions. The majority of neurospheres arise from the fast-cycling DiI(LOW+) population (7.5-fold increase over DiI[HI+]). This suggests SCs are in this actively proliferating fraction.

Mentions: 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).


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)

NSCs are fast dividing cells in vitro, and show BrdU retention. (A) Distribution of DiI, initially and after 1 wk in vitro. DiI signal decreases as a result of DiI dilution via cell proliferation. (DiI pos) DiI(HI+) fraction; (DiI neg) DiI(LOW+) fraction. (B) DiI(HI+) fraction of slowly dividing neurosphere cells where DiI signal is vivid. (i) Dissociated cells in bright field; (ii) DiI signal in red. (C) DiI(LOW+) fraction of rapidly dividing neurosphere cells where DiI signal is noticeably lower than in DiI(HI+) fraction. (i) Dissociated cells in bright field; (ii) DiI signal in red. (D) Data shows DiI(HI+) population (10% of total). As expected, slowly cycling cells do not greatly attenuate BrdU or DiI. The BrdU(−) population may be the same 1% of cells that are BrdU(−) immediately after BrdU exposure. (E) Data shows DiI(LOW+) population (63% of total). A subset of BrdU(+) cells are DiI(LOW+) after extended cell proliferation in vitro. BrdU retention in rapidly cycling cells suggests these are cosegregating their DNA. (F) Comparison in clonal sphere formation between DiI(HI+) and DiI(LOW+) fractions. The majority of neurospheres arise from the fast-cycling DiI(LOW+) population (7.5-fold increase over DiI[HI+]). This suggests SCs are in this actively proliferating fraction.
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Related In: Results  -  Collection

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fig4: NSCs are fast dividing cells in vitro, and show BrdU retention. (A) Distribution of DiI, initially and after 1 wk in vitro. DiI signal decreases as a result of DiI dilution via cell proliferation. (DiI pos) DiI(HI+) fraction; (DiI neg) DiI(LOW+) fraction. (B) DiI(HI+) fraction of slowly dividing neurosphere cells where DiI signal is vivid. (i) Dissociated cells in bright field; (ii) DiI signal in red. (C) DiI(LOW+) fraction of rapidly dividing neurosphere cells where DiI signal is noticeably lower than in DiI(HI+) fraction. (i) Dissociated cells in bright field; (ii) DiI signal in red. (D) Data shows DiI(HI+) population (10% of total). As expected, slowly cycling cells do not greatly attenuate BrdU or DiI. The BrdU(−) population may be the same 1% of cells that are BrdU(−) immediately after BrdU exposure. (E) Data shows DiI(LOW+) population (63% of total). A subset of BrdU(+) cells are DiI(LOW+) after extended cell proliferation in vitro. BrdU retention in rapidly cycling cells suggests these are cosegregating their DNA. (F) Comparison in clonal sphere formation between DiI(HI+) and DiI(LOW+) fractions. The majority of neurospheres arise from the fast-cycling DiI(LOW+) population (7.5-fold increase over DiI[HI+]). This suggests SCs are in this actively proliferating fraction.
Mentions: 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).

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