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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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CDK6 EE LT-HSCs Outcompete Wild-Type HSCs without Exhaustion(A–D) NSG mice were injected with sorted Lin− CD34+ CD38− cells transduced with CDK6 EE or control (LUC) lentiviral vectors (GFP+ cells) and untransduced competitive cells (GFP−). Bone marrow was harvested at indicated time points post-transplantation and analyzed by flow cytometry. (A) Percentage of GFP+ cells among engrafted human hematopoietic cells (CD45+). Time 0 corresponds to percentage of GFP+ cells before injection in four independent CB samples. 4 weeks post-transplantation: n = 13 LUC and 14 CDK6 EE mice; 20 weeks post-transplantation: n = 25 LUC and 23 CDK6 EE mice. (B) Lymphoid to myeloid ratio (percentage of CD19+/CD33+) among GFP+ cells at 20 weeks post-transplantation. n = 25 LUC and 23 CDK6 EE mice. In (A) and (B), boxplots represent median, 25th, and 75th percentiles and whiskers represent min and max. Gray boxes, LUC; white boxes, CDK6 EE. (C) Percentage of GFP+ cells among LT-HSCs at 20 weeks post-transplantation. (D) Absolute number of LT-HSCs at 20 weeks post-transplantation. In (C) and (D), n = 6 mice from two CB samples. Individual mice, median, and interquantile range are shown. In (A)–(D), ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01 by Mann-Whitney test.(E and F) CDK6 EE LT-HSCs expand over serial transplantation. LUC, CDK6 EE (GFP+), or untransduced (GFP−) LT-HSCs were sorted from primary transplanted mice (n = 2 pools of three to five mice) and injected at four different doses into secondary NSG mice. (E) Engraftment levels (percentage of CD45+ cells) 12 weeks after secondary transplantation (>0.01% CD45+ GFP+ or CD45+ GFP−) at the two highest doses (200 and 400 cells/mouse). Individual mice, median, and interquantile range are shown. (F) Summary table of number of mice engrafted at each dose tested and estimation of LT-HSC frequencies in each group by the ELDA statistical method. See also Figure S5.
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fig5: CDK6 EE LT-HSCs Outcompete Wild-Type HSCs without Exhaustion(A–D) NSG mice were injected with sorted Lin− CD34+ CD38− cells transduced with CDK6 EE or control (LUC) lentiviral vectors (GFP+ cells) and untransduced competitive cells (GFP−). Bone marrow was harvested at indicated time points post-transplantation and analyzed by flow cytometry. (A) Percentage of GFP+ cells among engrafted human hematopoietic cells (CD45+). Time 0 corresponds to percentage of GFP+ cells before injection in four independent CB samples. 4 weeks post-transplantation: n = 13 LUC and 14 CDK6 EE mice; 20 weeks post-transplantation: n = 25 LUC and 23 CDK6 EE mice. (B) Lymphoid to myeloid ratio (percentage of CD19+/CD33+) among GFP+ cells at 20 weeks post-transplantation. n = 25 LUC and 23 CDK6 EE mice. In (A) and (B), boxplots represent median, 25th, and 75th percentiles and whiskers represent min and max. Gray boxes, LUC; white boxes, CDK6 EE. (C) Percentage of GFP+ cells among LT-HSCs at 20 weeks post-transplantation. (D) Absolute number of LT-HSCs at 20 weeks post-transplantation. In (C) and (D), n = 6 mice from two CB samples. Individual mice, median, and interquantile range are shown. In (A)–(D), ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01 by Mann-Whitney test.(E and F) CDK6 EE LT-HSCs expand over serial transplantation. LUC, CDK6 EE (GFP+), or untransduced (GFP−) LT-HSCs were sorted from primary transplanted mice (n = 2 pools of three to five mice) and injected at four different doses into secondary NSG mice. (E) Engraftment levels (percentage of CD45+ cells) 12 weeks after secondary transplantation (>0.01% CD45+ GFP+ or CD45+ GFP−) at the two highest doses (200 and 400 cells/mouse). Individual mice, median, and interquantile range are shown. (F) Summary table of number of mice engrafted at each dose tested and estimation of LT-HSC frequencies in each group by the ELDA statistical method. See also Figure S5.

Mentions: In mouse models, failure to maintain quiescence and/or increased cycling are mostly associated with decreased self-renewal and eventual HSC exhaustion (Orford and Scadden, 2008; Pietras et al., 2011; Rossi et al., 2012). To examine the long-term effect of exclusively accelerating the duration of exit from quiescence upon reception of mitogenic stimuli, we enforced CDK6 expression in LT-HSCs in vivo, where HSCs return to G0 after most divisions under homeostatic conditions (Wilson et al., 2008 and Figure S1B). Competitive xenotransplantation experiments showed that CDK6 EE does not confer a proliferative advantage within the first 4 weeks post-transplantation, during which HSCs are actively cycling (Figure 5A). However, by 20 weeks post-transplantation, most HSCs have regained quiescence. At this point, CDK6 EE cells, unlike LUC cells, significantly outcompete untransduced cells (median GFP+ percentage: LUC, 59.4%; CDK6 EE, 76.2%, p = 0.007, Figure 5A) without displaying any lineage bias (Figures 5B and S5A). This expansion originated from LT-HSCs (Figures 5C and 5D) and extended to all progenitor populations (Figure S5B). To determine whether the CDK6 EE in phenotypic LT-HSCs altered serial repopulating capacity, secondary transplantation was performed. There was no significant difference in the graft size at 12 weeks after secondary transplantation when high numbers of control (LUC/GFP−) and CDK6 EE LT-HSCs were transplanted (Figures 5E and S5C). However, limiting dilution analysis revealed a 4-fold increase in the frequency of repopulating LT-HSCs in the CDK6 EE group compared to controls (Figure 5F), confirming LT-HSC expansion over two rounds of transplantation. Importantly, like our in vitro results, CDK6 EE did not change the rate of cell cycle transit of LT- or ST-HSCs (Figure S5D) but accelerated the first division of LT-HSCs (Figure S5E). These data show that the unique shortening in the duration of G0 exit conferred by CDK6 EE gives LT-HSCs a competitive advantage without altering self-renewal or differentiation abilities.


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)

CDK6 EE LT-HSCs Outcompete Wild-Type HSCs without Exhaustion(A–D) NSG mice were injected with sorted Lin− CD34+ CD38− cells transduced with CDK6 EE or control (LUC) lentiviral vectors (GFP+ cells) and untransduced competitive cells (GFP−). Bone marrow was harvested at indicated time points post-transplantation and analyzed by flow cytometry. (A) Percentage of GFP+ cells among engrafted human hematopoietic cells (CD45+). Time 0 corresponds to percentage of GFP+ cells before injection in four independent CB samples. 4 weeks post-transplantation: n = 13 LUC and 14 CDK6 EE mice; 20 weeks post-transplantation: n = 25 LUC and 23 CDK6 EE mice. (B) Lymphoid to myeloid ratio (percentage of CD19+/CD33+) among GFP+ cells at 20 weeks post-transplantation. n = 25 LUC and 23 CDK6 EE mice. In (A) and (B), boxplots represent median, 25th, and 75th percentiles and whiskers represent min and max. Gray boxes, LUC; white boxes, CDK6 EE. (C) Percentage of GFP+ cells among LT-HSCs at 20 weeks post-transplantation. (D) Absolute number of LT-HSCs at 20 weeks post-transplantation. In (C) and (D), n = 6 mice from two CB samples. Individual mice, median, and interquantile range are shown. In (A)–(D), ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01 by Mann-Whitney test.(E and F) CDK6 EE LT-HSCs expand over serial transplantation. LUC, CDK6 EE (GFP+), or untransduced (GFP−) LT-HSCs were sorted from primary transplanted mice (n = 2 pools of three to five mice) and injected at four different doses into secondary NSG mice. (E) Engraftment levels (percentage of CD45+ cells) 12 weeks after secondary transplantation (>0.01% CD45+ GFP+ or CD45+ GFP−) at the two highest doses (200 and 400 cells/mouse). Individual mice, median, and interquantile range are shown. (F) Summary table of number of mice engrafted at each dose tested and estimation of LT-HSC frequencies in each group by the ELDA statistical method. See also Figure S5.
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fig5: CDK6 EE LT-HSCs Outcompete Wild-Type HSCs without Exhaustion(A–D) NSG mice were injected with sorted Lin− CD34+ CD38− cells transduced with CDK6 EE or control (LUC) lentiviral vectors (GFP+ cells) and untransduced competitive cells (GFP−). Bone marrow was harvested at indicated time points post-transplantation and analyzed by flow cytometry. (A) Percentage of GFP+ cells among engrafted human hematopoietic cells (CD45+). Time 0 corresponds to percentage of GFP+ cells before injection in four independent CB samples. 4 weeks post-transplantation: n = 13 LUC and 14 CDK6 EE mice; 20 weeks post-transplantation: n = 25 LUC and 23 CDK6 EE mice. (B) Lymphoid to myeloid ratio (percentage of CD19+/CD33+) among GFP+ cells at 20 weeks post-transplantation. n = 25 LUC and 23 CDK6 EE mice. In (A) and (B), boxplots represent median, 25th, and 75th percentiles and whiskers represent min and max. Gray boxes, LUC; white boxes, CDK6 EE. (C) Percentage of GFP+ cells among LT-HSCs at 20 weeks post-transplantation. (D) Absolute number of LT-HSCs at 20 weeks post-transplantation. In (C) and (D), n = 6 mice from two CB samples. Individual mice, median, and interquantile range are shown. In (A)–(D), ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01 by Mann-Whitney test.(E and F) CDK6 EE LT-HSCs expand over serial transplantation. LUC, CDK6 EE (GFP+), or untransduced (GFP−) LT-HSCs were sorted from primary transplanted mice (n = 2 pools of three to five mice) and injected at four different doses into secondary NSG mice. (E) Engraftment levels (percentage of CD45+ cells) 12 weeks after secondary transplantation (>0.01% CD45+ GFP+ or CD45+ GFP−) at the two highest doses (200 and 400 cells/mouse). Individual mice, median, and interquantile range are shown. (F) Summary table of number of mice engrafted at each dose tested and estimation of LT-HSC frequencies in each group by the ELDA statistical method. See also Figure S5.
Mentions: In mouse models, failure to maintain quiescence and/or increased cycling are mostly associated with decreased self-renewal and eventual HSC exhaustion (Orford and Scadden, 2008; Pietras et al., 2011; Rossi et al., 2012). To examine the long-term effect of exclusively accelerating the duration of exit from quiescence upon reception of mitogenic stimuli, we enforced CDK6 expression in LT-HSCs in vivo, where HSCs return to G0 after most divisions under homeostatic conditions (Wilson et al., 2008 and Figure S1B). Competitive xenotransplantation experiments showed that CDK6 EE does not confer a proliferative advantage within the first 4 weeks post-transplantation, during which HSCs are actively cycling (Figure 5A). However, by 20 weeks post-transplantation, most HSCs have regained quiescence. At this point, CDK6 EE cells, unlike LUC cells, significantly outcompete untransduced cells (median GFP+ percentage: LUC, 59.4%; CDK6 EE, 76.2%, p = 0.007, Figure 5A) without displaying any lineage bias (Figures 5B and S5A). This expansion originated from LT-HSCs (Figures 5C and 5D) and extended to all progenitor populations (Figure S5B). To determine whether the CDK6 EE in phenotypic LT-HSCs altered serial repopulating capacity, secondary transplantation was performed. There was no significant difference in the graft size at 12 weeks after secondary transplantation when high numbers of control (LUC/GFP−) and CDK6 EE LT-HSCs were transplanted (Figures 5E and S5C). However, limiting dilution analysis revealed a 4-fold increase in the frequency of repopulating LT-HSCs in the CDK6 EE group compared to controls (Figure 5F), confirming LT-HSC expansion over two rounds of transplantation. Importantly, like our in vitro results, CDK6 EE did not change the rate of cell cycle transit of LT- or ST-HSCs (Figure S5D) but accelerated the first division of LT-HSCs (Figure S5E). These data show that the unique shortening in the duration of G0 exit conferred by CDK6 EE gives LT-HSCs a competitive advantage without altering self-renewal or differentiation abilities.

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