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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.

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Distinct CDK6 Levels Govern the G0 Exit Kinetics of LT- and ST-HSCs(A) Log2 signal intensity for CDK6 mRNA probe. Shown are individual measures (black circles: LT-HSCs, red squares: ST-HSCs, green triangles: GMPs) and the median and interquantile ranges (horizontal bars); n = 3. All multiple comparisons have been tested.(B) Immunofluorescence for CDK6 protein in LT- and ST-HSCs sorted from CB (left panel) or cultured for 4 days (right panel). Representative pictures and histograms of CDK6 fluorescence density are normalized to the fluorescence density of the IgG control in the same population. Positivity threshold was set over the median + 1 SD of the IgG control distribution and the percetnage of positive cells is indicated. 100–570 cells are analyzed with n = 3 CB samples. Scale bar represents 10 μM.(C) Normalized median CDK6 fluorescence density. Mean ± SEM is shown; n = 3 CB samples. GMPs, granulocyte-monocyte progenitors.(D) Immunofluorescence for CyclinD3 protein in LT-HSCs, ST-HSCs, and GMPs from freshly isolated CB (Day 0, left panel) or after 4 days of culture (Day 4, right panel). Shown are histograms of CyclinD3 fluorescence density normalized to the fluorescence density of the IgG control in the same population. Positivity threshold (dotted line) was set over the median + 1 SD of the IgG control distribution. n = 135–315 cells analyzed for day 0 and n = 49–245 cells for day 4.(E) Normalized median CyclinD3 fluorescence density at the indicated time points. Mean ± SEM is shown; n = 3 CB samples. ∗∗p < 0.05 by paired t test.(F) Time course analysis of CDK6 and CyclinD3 upon stimulation by mitogenic signals. Percentages of CDK6+ (top panel) or CyclinD3+ (bottom panel) cells in each of the indicated populations at the indicated time points after isolation from CB are shown. Mean ± S.E.M shown. n = 3 CB samples, except for day 2, where n = 2 CB samples. ∗∗p < 0.05 by paired t test.See also Figure S3.
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fig3: Distinct CDK6 Levels Govern the G0 Exit Kinetics of LT- and ST-HSCs(A) Log2 signal intensity for CDK6 mRNA probe. Shown are individual measures (black circles: LT-HSCs, red squares: ST-HSCs, green triangles: GMPs) and the median and interquantile ranges (horizontal bars); n = 3. All multiple comparisons have been tested.(B) Immunofluorescence for CDK6 protein in LT- and ST-HSCs sorted from CB (left panel) or cultured for 4 days (right panel). Representative pictures and histograms of CDK6 fluorescence density are normalized to the fluorescence density of the IgG control in the same population. Positivity threshold was set over the median + 1 SD of the IgG control distribution and the percetnage of positive cells is indicated. 100–570 cells are analyzed with n = 3 CB samples. Scale bar represents 10 μM.(C) Normalized median CDK6 fluorescence density. Mean ± SEM is shown; n = 3 CB samples. GMPs, granulocyte-monocyte progenitors.(D) Immunofluorescence for CyclinD3 protein in LT-HSCs, ST-HSCs, and GMPs from freshly isolated CB (Day 0, left panel) or after 4 days of culture (Day 4, right panel). Shown are histograms of CyclinD3 fluorescence density normalized to the fluorescence density of the IgG control in the same population. Positivity threshold (dotted line) was set over the median + 1 SD of the IgG control distribution. n = 135–315 cells analyzed for day 0 and n = 49–245 cells for day 4.(E) Normalized median CyclinD3 fluorescence density at the indicated time points. Mean ± SEM is shown; n = 3 CB samples. ∗∗p < 0.05 by paired t test.(F) Time course analysis of CDK6 and CyclinD3 upon stimulation by mitogenic signals. Percentages of CDK6+ (top panel) or CyclinD3+ (bottom panel) cells in each of the indicated populations at the indicated time points after isolation from CB are shown. Mean ± S.E.M shown. n = 3 CB samples, except for day 2, where n = 2 CB samples. ∗∗p < 0.05 by paired t test.See also Figure S3.

Mentions: To identify the molecular determinants underlying differences in the duration of LT- and ST-HSC divisions, we screened the 241-gene core signature for genes known to be involved in either G0 exit or G1 progression. CDK6 was selected because its mRNA was consistently upregulated in ST-HSCs both in CB and upon xenotransplantation (Figures 3A, S3A, and S3B), and CDK6/CyclinD complexes regulate G0 exit and early G1 (Sherr and Roberts, 2004). Importantly, the CDK6 protein was undetectable in most of the quiescent CB LT-HSCs, but it was upregulated after 4 days of culture when all HSC subsets actively cycle (Figures 3B and 3C). In sharp contrast, before culture, ST-HSCs already expressed high levels of CDK6 protein, similar to that found in G1 GMPs, despite being quiescent (Figures 3B, 3C, and S3C). Similarly, adult ST-HSCs isolated from either bone marrow or mobilized peripheral blood also expressed high levels of CDK6, though it was undetectable in adult LT-HSCs (Figures S3D–S3F). To gain insight into how ST-HSCs can remain quiescent with high levels of CDK6 protein, and because CDK6 kinase activity depends on its association with CyclinD proteins, we examined the levels of CyclinD1 and CyclinD3 protein in sorted LT- and ST-HSC subsets from purified CB at day 0. We found that neither one expressed CyclinD1 nor CyclinD3; as expected, both were expressed in G1 GMPs (Figures S3G, S3H, 3D, and 3E). Therefore the CDK6 in ST-HSCs is not part of an active complex, which explains the absence of RB phosphorylation in these cells (Figure 2F) and their quiescence. To gain insight into how the CyclinD-CDK6 complexes integrate proliferative signals once HSCs are activated, we did a time course analysis of CDK6, CyclinD1, and CyclinD3 protein expression. After 2 days of culture, less than 5% of LT- and ST-HSCs divided (Figure 2G). Interestingly, about 54% of LT-HSCs expressed CDK6 and 44% and 25% express CyclinD1 and CyclinD3, respectively (Figures S3H and 3F). In contrast, almost all ST-HSCs had upregulated CyclinD3 protein by day 2, and 52% express CyclinD1. By day 4, when all LT- and ST-HSCs actively cycle (Figure 2G), each HSC subset expresses both CDK6 and CyclinD3 proteins (Figure 3F). These data indicate that, upon activation by mitogens, the assembly of the CDK/CyclinD complex is more rapid and more robust in ST-HSCs than in LT-HSCs. Overall, these data reveal two unexpected findings. First, ST-HSCs exist in a G0 state, yet they express a known driver of G1 progression (CDK6) while lacking the cognate partners (CyclinD1 and CyclinD3) of CDK6. Second, the hierarchical organization based on functional repopulation properties also exhibits a hierarchy of CDK6/CyclinD complex components reflecting distinct cycling properties: LT-HSCs are negative for both CDK6 and CyclinD; ST-HSCs express exclusively CDK6; and lineage-restricted progenitors, e.g., GMPs, express both.


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)

Distinct CDK6 Levels Govern the G0 Exit Kinetics of LT- and ST-HSCs(A) Log2 signal intensity for CDK6 mRNA probe. Shown are individual measures (black circles: LT-HSCs, red squares: ST-HSCs, green triangles: GMPs) and the median and interquantile ranges (horizontal bars); n = 3. All multiple comparisons have been tested.(B) Immunofluorescence for CDK6 protein in LT- and ST-HSCs sorted from CB (left panel) or cultured for 4 days (right panel). Representative pictures and histograms of CDK6 fluorescence density are normalized to the fluorescence density of the IgG control in the same population. Positivity threshold was set over the median + 1 SD of the IgG control distribution and the percetnage of positive cells is indicated. 100–570 cells are analyzed with n = 3 CB samples. Scale bar represents 10 μM.(C) Normalized median CDK6 fluorescence density. Mean ± SEM is shown; n = 3 CB samples. GMPs, granulocyte-monocyte progenitors.(D) Immunofluorescence for CyclinD3 protein in LT-HSCs, ST-HSCs, and GMPs from freshly isolated CB (Day 0, left panel) or after 4 days of culture (Day 4, right panel). Shown are histograms of CyclinD3 fluorescence density normalized to the fluorescence density of the IgG control in the same population. Positivity threshold (dotted line) was set over the median + 1 SD of the IgG control distribution. n = 135–315 cells analyzed for day 0 and n = 49–245 cells for day 4.(E) Normalized median CyclinD3 fluorescence density at the indicated time points. Mean ± SEM is shown; n = 3 CB samples. ∗∗p < 0.05 by paired t test.(F) Time course analysis of CDK6 and CyclinD3 upon stimulation by mitogenic signals. Percentages of CDK6+ (top panel) or CyclinD3+ (bottom panel) cells in each of the indicated populations at the indicated time points after isolation from CB are shown. Mean ± S.E.M shown. n = 3 CB samples, except for day 2, where n = 2 CB samples. ∗∗p < 0.05 by paired t test.See also Figure S3.
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fig3: Distinct CDK6 Levels Govern the G0 Exit Kinetics of LT- and ST-HSCs(A) Log2 signal intensity for CDK6 mRNA probe. Shown are individual measures (black circles: LT-HSCs, red squares: ST-HSCs, green triangles: GMPs) and the median and interquantile ranges (horizontal bars); n = 3. All multiple comparisons have been tested.(B) Immunofluorescence for CDK6 protein in LT- and ST-HSCs sorted from CB (left panel) or cultured for 4 days (right panel). Representative pictures and histograms of CDK6 fluorescence density are normalized to the fluorescence density of the IgG control in the same population. Positivity threshold was set over the median + 1 SD of the IgG control distribution and the percetnage of positive cells is indicated. 100–570 cells are analyzed with n = 3 CB samples. Scale bar represents 10 μM.(C) Normalized median CDK6 fluorescence density. Mean ± SEM is shown; n = 3 CB samples. GMPs, granulocyte-monocyte progenitors.(D) Immunofluorescence for CyclinD3 protein in LT-HSCs, ST-HSCs, and GMPs from freshly isolated CB (Day 0, left panel) or after 4 days of culture (Day 4, right panel). Shown are histograms of CyclinD3 fluorescence density normalized to the fluorescence density of the IgG control in the same population. Positivity threshold (dotted line) was set over the median + 1 SD of the IgG control distribution. n = 135–315 cells analyzed for day 0 and n = 49–245 cells for day 4.(E) Normalized median CyclinD3 fluorescence density at the indicated time points. Mean ± SEM is shown; n = 3 CB samples. ∗∗p < 0.05 by paired t test.(F) Time course analysis of CDK6 and CyclinD3 upon stimulation by mitogenic signals. Percentages of CDK6+ (top panel) or CyclinD3+ (bottom panel) cells in each of the indicated populations at the indicated time points after isolation from CB are shown. Mean ± S.E.M shown. n = 3 CB samples, except for day 2, where n = 2 CB samples. ∗∗p < 0.05 by paired t test.See also Figure S3.
Mentions: To identify the molecular determinants underlying differences in the duration of LT- and ST-HSC divisions, we screened the 241-gene core signature for genes known to be involved in either G0 exit or G1 progression. CDK6 was selected because its mRNA was consistently upregulated in ST-HSCs both in CB and upon xenotransplantation (Figures 3A, S3A, and S3B), and CDK6/CyclinD complexes regulate G0 exit and early G1 (Sherr and Roberts, 2004). Importantly, the CDK6 protein was undetectable in most of the quiescent CB LT-HSCs, but it was upregulated after 4 days of culture when all HSC subsets actively cycle (Figures 3B and 3C). In sharp contrast, before culture, ST-HSCs already expressed high levels of CDK6 protein, similar to that found in G1 GMPs, despite being quiescent (Figures 3B, 3C, and S3C). Similarly, adult ST-HSCs isolated from either bone marrow or mobilized peripheral blood also expressed high levels of CDK6, though it was undetectable in adult LT-HSCs (Figures S3D–S3F). To gain insight into how ST-HSCs can remain quiescent with high levels of CDK6 protein, and because CDK6 kinase activity depends on its association with CyclinD proteins, we examined the levels of CyclinD1 and CyclinD3 protein in sorted LT- and ST-HSC subsets from purified CB at day 0. We found that neither one expressed CyclinD1 nor CyclinD3; as expected, both were expressed in G1 GMPs (Figures S3G, S3H, 3D, and 3E). Therefore the CDK6 in ST-HSCs is not part of an active complex, which explains the absence of RB phosphorylation in these cells (Figure 2F) and their quiescence. To gain insight into how the CyclinD-CDK6 complexes integrate proliferative signals once HSCs are activated, we did a time course analysis of CDK6, CyclinD1, and CyclinD3 protein expression. After 2 days of culture, less than 5% of LT- and ST-HSCs divided (Figure 2G). Interestingly, about 54% of LT-HSCs expressed CDK6 and 44% and 25% express CyclinD1 and CyclinD3, respectively (Figures S3H and 3F). In contrast, almost all ST-HSCs had upregulated CyclinD3 protein by day 2, and 52% express CyclinD1. By day 4, when all LT- and ST-HSCs actively cycle (Figure 2G), each HSC subset expresses both CDK6 and CyclinD3 proteins (Figure 3F). These data indicate that, upon activation by mitogens, the assembly of the CDK/CyclinD complex is more rapid and more robust in ST-HSCs than in LT-HSCs. Overall, these data reveal two unexpected findings. First, ST-HSCs exist in a G0 state, yet they express a known driver of G1 progression (CDK6) while lacking the cognate partners (CyclinD1 and CyclinD3) of CDK6. Second, the hierarchical organization based on functional repopulation properties also exhibits a hierarchy of CDK6/CyclinD complex components reflecting distinct cycling properties: LT-HSCs are negative for both CDK6 and CyclinD; ST-HSCs express exclusively CDK6; and lineage-restricted progenitors, e.g., GMPs, express both.

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