Limits...
CDK6 levels regulate quiescence exit in human hematopoietic stem cells.

Laurenti E, Frelin C, Xie S, Ferrari R, Dunant CF, Zandi S, Neumann A, Plumb I, Doulatov S, Chen J, April C, Fan JB, Iscove N, Dick JE - Cell Stem Cell (2015)

Bottom Line: Short-term (ST)-HSCs are also quiescent but contain high CDK6 protein levels that permit rapid cell cycle entry upon mitogenic stimulation.Enforced CDK6 expression in LT-HSCs shortens quiescence exit and confers competitive advantage without impacting function.Thus, differential expression of CDK6 underlies heterogeneity in stem cell quiescence states that functionally regulates this highly regenerative system.

View Article: PubMed Central - PubMed

Affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada. Electronic address: el422@cam.ac.uk.

Show MeSH

Related in: MedlinePlus

Human HSC Subsets in the Xenograft Divide with Distinct Frequencies and Display Distinct Transcriptional Profiles(A) Number of cells per mouse of indicated populations in the bone marrow of the mice at indicated time points post-transplantation of 70,000 Lin− CB (saturating number of LT-HSCs). Median and interquantile ranges are shown. ∗∗∗p < 0.01 by one-way ANOVA and Tukey test.(B) The number of repopulating LT-HSCs per mouse at indicated time points post-transplantation were calculated by multiplying the number of phenotypic LT-HSCs shown in (A) by the frequency of long-term repopulating cells indicated in Table S1.(C) BrdU incorporation kinetics over 12 days of LT-HSC (black) and ST-HSC (red) enriched populations isolated from pools of two to five mice engrafted with 70,000 Lin− CB cells. BrdU was started either at 4 (left panel, expanding phase) or 20 weeks post-transplantation (right panel, equilibrium phase). n = 1–4 pools of three to five mice from six (4 weeks) or one (20 weeks) independent CB samples. Curve is least-squares fit. Left panel: R2 > 0.96; right panel: R2 > 0.98. Doubling times (half times of fit) in hours are shown in the insert. ∗∗∗p < 0.01 by extra-sum of squares test.(D and E) Derivation of a 241-gene signature distinguishing LT- and ST-HSCs in unperturbed CB over 20 weeks in a xenotransplant. (D) Examples of five expression profiles of genes with known HSC function over the course of 20 weeks of xenotransplant (black: LT-HSCs, red: ST-HSCs), mean ± S.E.M shown, n = 3 per time point. (E) Selected gene ontology terms significantly enriched in the 241-gene LT-HSC/ST-HSC core signature. Shown is the −log10 of the Benjamini-Hochberg adjusted p value.See also Figure S1.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359055&req=5

fig1: Human HSC Subsets in the Xenograft Divide with Distinct Frequencies and Display Distinct Transcriptional Profiles(A) Number of cells per mouse of indicated populations in the bone marrow of the mice at indicated time points post-transplantation of 70,000 Lin− CB (saturating number of LT-HSCs). Median and interquantile ranges are shown. ∗∗∗p < 0.01 by one-way ANOVA and Tukey test.(B) The number of repopulating LT-HSCs per mouse at indicated time points post-transplantation were calculated by multiplying the number of phenotypic LT-HSCs shown in (A) by the frequency of long-term repopulating cells indicated in Table S1.(C) BrdU incorporation kinetics over 12 days of LT-HSC (black) and ST-HSC (red) enriched populations isolated from pools of two to five mice engrafted with 70,000 Lin− CB cells. BrdU was started either at 4 (left panel, expanding phase) or 20 weeks post-transplantation (right panel, equilibrium phase). n = 1–4 pools of three to five mice from six (4 weeks) or one (20 weeks) independent CB samples. Curve is least-squares fit. Left panel: R2 > 0.96; right panel: R2 > 0.98. Doubling times (half times of fit) in hours are shown in the insert. ∗∗∗p < 0.01 by extra-sum of squares test.(D and E) Derivation of a 241-gene signature distinguishing LT- and ST-HSCs in unperturbed CB over 20 weeks in a xenotransplant. (D) Examples of five expression profiles of genes with known HSC function over the course of 20 weeks of xenotransplant (black: LT-HSCs, red: ST-HSCs), mean ± S.E.M shown, n = 3 per time point. (E) Selected gene ontology terms significantly enriched in the 241-gene LT-HSC/ST-HSC core signature. Shown is the −log10 of the Benjamini-Hochberg adjusted p value.See also Figure S1.

Mentions: The cycling properties of mouse HSC subpopulations are described, but they have not been validated in the human HSC hierarchy. Human LT-HSCs, isolated from umbilical cord blood (CB) as Lin− CD34+ CD38− CD45RA− CD90+ CD49f+ (Notta et al., 2011), provide robust multilineage repopulation beyond 30 weeks in the NSG mouse xenograft assay with about 10% frequency (Notta et al., 2011) and efficiently engraft upon secondary transplantation (Table S1). In contrast, Lin− CD34+ CD38− CD45RA− CD90− CD49f− cells generate multilineage grafts over intermediate time periods (Notta et al., 2011), but they lack serial transplantation ability and thus have limited self-renewal (Table S1). According to the criteria used in mouse, this population corresponds to ST-HSCs. Importantly, LT- and ST-HSC-enriched populations can be purified with the cell surface markers indicated above from NSG mice repopulated with human cells (Table S1). Similar to transplantation models in mice, phenotypic human LT- and ST-HSCs expand in the first 4 weeks after xenotransplant (with >50% that actively cycle) then regain quiescence by 20 weeks when a transient equilibrium phase is reached (Figures 1A, S1A, and S1B). In our model, functionally repopulating LT-HSCs expanded ≈30-fold in 20 weeks (Figure 1B).


CDK6 levels regulate quiescence exit in human hematopoietic stem cells.

Laurenti E, Frelin C, Xie S, Ferrari R, Dunant CF, Zandi S, Neumann A, Plumb I, Doulatov S, Chen J, April C, Fan JB, Iscove N, Dick JE - Cell Stem Cell (2015)

Human HSC Subsets in the Xenograft Divide with Distinct Frequencies and Display Distinct Transcriptional Profiles(A) Number of cells per mouse of indicated populations in the bone marrow of the mice at indicated time points post-transplantation of 70,000 Lin− CB (saturating number of LT-HSCs). Median and interquantile ranges are shown. ∗∗∗p < 0.01 by one-way ANOVA and Tukey test.(B) The number of repopulating LT-HSCs per mouse at indicated time points post-transplantation were calculated by multiplying the number of phenotypic LT-HSCs shown in (A) by the frequency of long-term repopulating cells indicated in Table S1.(C) BrdU incorporation kinetics over 12 days of LT-HSC (black) and ST-HSC (red) enriched populations isolated from pools of two to five mice engrafted with 70,000 Lin− CB cells. BrdU was started either at 4 (left panel, expanding phase) or 20 weeks post-transplantation (right panel, equilibrium phase). n = 1–4 pools of three to five mice from six (4 weeks) or one (20 weeks) independent CB samples. Curve is least-squares fit. Left panel: R2 > 0.96; right panel: R2 > 0.98. Doubling times (half times of fit) in hours are shown in the insert. ∗∗∗p < 0.01 by extra-sum of squares test.(D and E) Derivation of a 241-gene signature distinguishing LT- and ST-HSCs in unperturbed CB over 20 weeks in a xenotransplant. (D) Examples of five expression profiles of genes with known HSC function over the course of 20 weeks of xenotransplant (black: LT-HSCs, red: ST-HSCs), mean ± S.E.M shown, n = 3 per time point. (E) Selected gene ontology terms significantly enriched in the 241-gene LT-HSC/ST-HSC core signature. Shown is the −log10 of the Benjamini-Hochberg adjusted p value.See also Figure S1.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359055&req=5

fig1: Human HSC Subsets in the Xenograft Divide with Distinct Frequencies and Display Distinct Transcriptional Profiles(A) Number of cells per mouse of indicated populations in the bone marrow of the mice at indicated time points post-transplantation of 70,000 Lin− CB (saturating number of LT-HSCs). Median and interquantile ranges are shown. ∗∗∗p < 0.01 by one-way ANOVA and Tukey test.(B) The number of repopulating LT-HSCs per mouse at indicated time points post-transplantation were calculated by multiplying the number of phenotypic LT-HSCs shown in (A) by the frequency of long-term repopulating cells indicated in Table S1.(C) BrdU incorporation kinetics over 12 days of LT-HSC (black) and ST-HSC (red) enriched populations isolated from pools of two to five mice engrafted with 70,000 Lin− CB cells. BrdU was started either at 4 (left panel, expanding phase) or 20 weeks post-transplantation (right panel, equilibrium phase). n = 1–4 pools of three to five mice from six (4 weeks) or one (20 weeks) independent CB samples. Curve is least-squares fit. Left panel: R2 > 0.96; right panel: R2 > 0.98. Doubling times (half times of fit) in hours are shown in the insert. ∗∗∗p < 0.01 by extra-sum of squares test.(D and E) Derivation of a 241-gene signature distinguishing LT- and ST-HSCs in unperturbed CB over 20 weeks in a xenotransplant. (D) Examples of five expression profiles of genes with known HSC function over the course of 20 weeks of xenotransplant (black: LT-HSCs, red: ST-HSCs), mean ± S.E.M shown, n = 3 per time point. (E) Selected gene ontology terms significantly enriched in the 241-gene LT-HSC/ST-HSC core signature. Shown is the −log10 of the Benjamini-Hochberg adjusted p value.See also Figure S1.
Mentions: The cycling properties of mouse HSC subpopulations are described, but they have not been validated in the human HSC hierarchy. Human LT-HSCs, isolated from umbilical cord blood (CB) as Lin− CD34+ CD38− CD45RA− CD90+ CD49f+ (Notta et al., 2011), provide robust multilineage repopulation beyond 30 weeks in the NSG mouse xenograft assay with about 10% frequency (Notta et al., 2011) and efficiently engraft upon secondary transplantation (Table S1). In contrast, Lin− CD34+ CD38− CD45RA− CD90− CD49f− cells generate multilineage grafts over intermediate time periods (Notta et al., 2011), but they lack serial transplantation ability and thus have limited self-renewal (Table S1). According to the criteria used in mouse, this population corresponds to ST-HSCs. Importantly, LT- and ST-HSC-enriched populations can be purified with the cell surface markers indicated above from NSG mice repopulated with human cells (Table S1). Similar to transplantation models in mice, phenotypic human LT- and ST-HSCs expand in the first 4 weeks after xenotransplant (with >50% that actively cycle) then regain quiescence by 20 weeks when a transient equilibrium phase is reached (Figures 1A, S1A, and S1B). In our model, functionally repopulating LT-HSCs expanded ≈30-fold in 20 weeks (Figure 1B).

Bottom Line: Short-term (ST)-HSCs are also quiescent but contain high CDK6 protein levels that permit rapid cell cycle entry upon mitogenic stimulation.Enforced CDK6 expression in LT-HSCs shortens quiescence exit and confers competitive advantage without impacting function.Thus, differential expression of CDK6 underlies heterogeneity in stem cell quiescence states that functionally regulates this highly regenerative system.

View Article: PubMed Central - PubMed

Affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada. Electronic address: el422@cam.ac.uk.

Show MeSH
Related in: MedlinePlus