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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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After a 2-d exposure to BrdU, cells from adult murine forebrain ventricles demonstrate heterogenous BrdU labeling in vitro. (A) BrdU retention strategy: (1) Each double strand (1 chromosome) represents 10 chromosomes of a mouse cell. Cells are unlabeled for BrdU (black). (2) During multiple rounds of DNA synthesis, BrdU (green) is taken up and distributed in both symmetric and asymmetric divisions in the presence of BrdU. (3) BrdU is removed and the daughter cells now undergo DNA synthesis in the absence of BrdU. (4) BrdU should be retained if labeled chromosomes are cosegregated as immortal strands into SCs. (B) BrdU–neurosphere assay. (1) Cells from adult forebrain lateral ventricles are cultured for 7 d at clonal density. (2) After dissociation, cells are pulsed with BrdU for 2 d, at 3 DIV. (3) BrdU is removed and cells are passaged at clonal density for an additional 7 d. (4) Cells are examined (A), passaged (B), or differentiated (C). (C) 10 d after BrdU exposure, cell clones still contained heavily BrdU(+) cells (arrows) and BrdU(−) cells. The retention of BrdU(+), in cells seeded at clonal density, suggests that BrdU(+) cells give rise to both labeled and unlabeled progeny. (i) Bright field shows 3 d cell clumps; (ii) histone-labeled nuclei are red, BrdU-labeled cells are green; merge shows overlap as yellow.
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fig2: After a 2-d exposure to BrdU, cells from adult murine forebrain ventricles demonstrate heterogenous BrdU labeling in vitro. (A) BrdU retention strategy: (1) Each double strand (1 chromosome) represents 10 chromosomes of a mouse cell. Cells are unlabeled for BrdU (black). (2) During multiple rounds of DNA synthesis, BrdU (green) is taken up and distributed in both symmetric and asymmetric divisions in the presence of BrdU. (3) BrdU is removed and the daughter cells now undergo DNA synthesis in the absence of BrdU. (4) BrdU should be retained if labeled chromosomes are cosegregated as immortal strands into SCs. (B) BrdU–neurosphere assay. (1) Cells from adult forebrain lateral ventricles are cultured for 7 d at clonal density. (2) After dissociation, cells are pulsed with BrdU for 2 d, at 3 DIV. (3) BrdU is removed and cells are passaged at clonal density for an additional 7 d. (4) Cells are examined (A), passaged (B), or differentiated (C). (C) 10 d after BrdU exposure, cell clones still contained heavily BrdU(+) cells (arrows) and BrdU(−) cells. The retention of BrdU(+), in cells seeded at clonal density, suggests that BrdU(+) cells give rise to both labeled and unlabeled progeny. (i) Bright field shows 3 d cell clumps; (ii) histone-labeled nuclei are red, BrdU-labeled cells are green; merge shows overlap as yellow.

Mentions: 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).


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)

After a 2-d exposure to BrdU, cells from adult murine forebrain ventricles demonstrate heterogenous BrdU labeling in vitro. (A) BrdU retention strategy: (1) Each double strand (1 chromosome) represents 10 chromosomes of a mouse cell. Cells are unlabeled for BrdU (black). (2) During multiple rounds of DNA synthesis, BrdU (green) is taken up and distributed in both symmetric and asymmetric divisions in the presence of BrdU. (3) BrdU is removed and the daughter cells now undergo DNA synthesis in the absence of BrdU. (4) BrdU should be retained if labeled chromosomes are cosegregated as immortal strands into SCs. (B) BrdU–neurosphere assay. (1) Cells from adult forebrain lateral ventricles are cultured for 7 d at clonal density. (2) After dissociation, cells are pulsed with BrdU for 2 d, at 3 DIV. (3) BrdU is removed and cells are passaged at clonal density for an additional 7 d. (4) Cells are examined (A), passaged (B), or differentiated (C). (C) 10 d after BrdU exposure, cell clones still contained heavily BrdU(+) cells (arrows) and BrdU(−) cells. The retention of BrdU(+), in cells seeded at clonal density, suggests that BrdU(+) cells give rise to both labeled and unlabeled progeny. (i) Bright field shows 3 d cell clumps; (ii) histone-labeled nuclei are red, BrdU-labeled cells are green; merge shows overlap as yellow.
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Related In: Results  -  Collection

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fig2: After a 2-d exposure to BrdU, cells from adult murine forebrain ventricles demonstrate heterogenous BrdU labeling in vitro. (A) BrdU retention strategy: (1) Each double strand (1 chromosome) represents 10 chromosomes of a mouse cell. Cells are unlabeled for BrdU (black). (2) During multiple rounds of DNA synthesis, BrdU (green) is taken up and distributed in both symmetric and asymmetric divisions in the presence of BrdU. (3) BrdU is removed and the daughter cells now undergo DNA synthesis in the absence of BrdU. (4) BrdU should be retained if labeled chromosomes are cosegregated as immortal strands into SCs. (B) BrdU–neurosphere assay. (1) Cells from adult forebrain lateral ventricles are cultured for 7 d at clonal density. (2) After dissociation, cells are pulsed with BrdU for 2 d, at 3 DIV. (3) BrdU is removed and cells are passaged at clonal density for an additional 7 d. (4) Cells are examined (A), passaged (B), or differentiated (C). (C) 10 d after BrdU exposure, cell clones still contained heavily BrdU(+) cells (arrows) and BrdU(−) cells. The retention of BrdU(+), in cells seeded at clonal density, suggests that BrdU(+) cells give rise to both labeled and unlabeled progeny. (i) Bright field shows 3 d cell clumps; (ii) histone-labeled nuclei are red, BrdU-labeled cells are green; merge shows overlap as yellow.
Mentions: 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).

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