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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 Levels Determine the Duration of Quiescence Exit in the HSC Pool(A–C) Cell division duration of single LT- and ST-HSCs after exposure to mitogenic signal in the presence or absence of PD033299 (50 nM). (A) Percentage of cells from the indicated populations that divided after 100 hr in culture. (B) Cumulative first division kinetics (excluding dead cells). Data from a representative CB example are shown. Curve is least-squares sigmoid fit. R2 > 0.99. (C) Mean time to first division (hours) (tfirstDiv = logEC50).(D–G) Cell division duration of single LT- and ST-HSCs after exposure to mitogenic signal with or without CDK6 EE. (D) Cumulative first division kinetics (excluding dead cells) of indicated populations transduced with indicated lentiviral vectors. Data from a representative CB are shown. Curve is least-squares sigmoid fit. R2 > 0.99. (E) Mean time to first division (hours) (tfirstDiv = logEC50). (F) Time of cell cycle transit of indicated populations in hours.(G) Expansion curves of LT- and ST-HSCs in culture. Shown is the average number of cells per single cell plated at the indicated time points after culture initiation. Data are from one representative experiment out of three. Time 0 represents the time of exposure to mitogenic stimulus.In (A), (C), (E), and (F), individual CB samples are shown; gray lines connect parameters from the same condition. ∗∗p < 0.05 by paired t test. See also Figure S4.
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fig4: CDK6 Levels Determine the Duration of Quiescence Exit in the HSC Pool(A–C) Cell division duration of single LT- and ST-HSCs after exposure to mitogenic signal in the presence or absence of PD033299 (50 nM). (A) Percentage of cells from the indicated populations that divided after 100 hr in culture. (B) Cumulative first division kinetics (excluding dead cells). Data from a representative CB example are shown. Curve is least-squares sigmoid fit. R2 > 0.99. (C) Mean time to first division (hours) (tfirstDiv = logEC50).(D–G) Cell division duration of single LT- and ST-HSCs after exposure to mitogenic signal with or without CDK6 EE. (D) Cumulative first division kinetics (excluding dead cells) of indicated populations transduced with indicated lentiviral vectors. Data from a representative CB are shown. Curve is least-squares sigmoid fit. R2 > 0.99. (E) Mean time to first division (hours) (tfirstDiv = logEC50). (F) Time of cell cycle transit of indicated populations in hours.(G) Expansion curves of LT- and ST-HSCs in culture. Shown is the average number of cells per single cell plated at the indicated time points after culture initiation. Data are from one representative experiment out of three. Time 0 represents the time of exposure to mitogenic stimulus.In (A), (C), (E), and (F), individual CB samples are shown; gray lines connect parameters from the same condition. ∗∗p < 0.05 by paired t test. See also Figure S4.

Mentions: To gain mechanistic insight into the correlation between CDK6 protein levels and cell division duration within HSC subsets, we altered CDK6 levels and investigated the effect on the kinetics of the first HSC division. To examine loss of function, we measured the duration of cell division when single LT- and ST-HSCs are exposed to a mitogenic stimulus in the presence of the highly specific CDK4-CDK6 inhibitor PD033299. The majority of LT-HSCs never divided in the presence of PD033299 (Figure 4A). Similarly there was a strong reduction in the number of ST-HSCs that could divide (Figure 4A). However, for those ST-HSCs that divided, inhibition of CDK6 brought the length of the first division to that of LT-HSCs (Figures 4B and 4C). Intriguingly, those 10% LT-HSCs dividing in the presence of PD033299 were not further delayed, potentially representing a subset of more “activated” cells within the LT-HSC phenotypic compartment. To examine the consequences of CDK6 gain of function, we enforced expression of CDK6 protein (CDK6 EE) with lentiviral vectors in LT- and ST-HSCs before their first division (Figures S4A and S4B). CDK6 EE did not change any ST-HSC cell cycle parameters (Figures 4D–4F, S4C, and S4E). By contrast, a division starting from G0 (first division) of CDK6 EE LT-HSCs was significantly shortened to values similar to those of ST-HSCs (Figures 4D and 4E); control transduced LT-HSCs (LUC) showed no such changes. CDK6 EE did not decrease the duration of a division starting from G1 that remained significantly longer than that of ST-HSCs (Figures 4F, S4C, and S4F). Also, CDK6 EE did not affect the long-term proliferative output of LT-HSCs in vitro in conditions where cells do not return to G0 (Figure 4G). These approaches show that CDK6 shortens divisions starting from G0, but not divisions starting from G1. Moreover, variation in CDK6 protein levels between HSC subsets results in active regulation of the duration of the latency that is unique to HSCs transiting out of G0, a process we define as G0 exit. Importantly, our data also establish that pre-existent CDK6 in ST-HSCs primes them for earlier cell division upon mitogenic stimuli.


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 Levels Determine the Duration of Quiescence Exit in the HSC Pool(A–C) Cell division duration of single LT- and ST-HSCs after exposure to mitogenic signal in the presence or absence of PD033299 (50 nM). (A) Percentage of cells from the indicated populations that divided after 100 hr in culture. (B) Cumulative first division kinetics (excluding dead cells). Data from a representative CB example are shown. Curve is least-squares sigmoid fit. R2 > 0.99. (C) Mean time to first division (hours) (tfirstDiv = logEC50).(D–G) Cell division duration of single LT- and ST-HSCs after exposure to mitogenic signal with or without CDK6 EE. (D) Cumulative first division kinetics (excluding dead cells) of indicated populations transduced with indicated lentiviral vectors. Data from a representative CB are shown. Curve is least-squares sigmoid fit. R2 > 0.99. (E) Mean time to first division (hours) (tfirstDiv = logEC50). (F) Time of cell cycle transit of indicated populations in hours.(G) Expansion curves of LT- and ST-HSCs in culture. Shown is the average number of cells per single cell plated at the indicated time points after culture initiation. Data are from one representative experiment out of three. Time 0 represents the time of exposure to mitogenic stimulus.In (A), (C), (E), and (F), individual CB samples are shown; gray lines connect parameters from the same condition. ∗∗p < 0.05 by paired t test. See also Figure S4.
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Related In: Results  -  Collection

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fig4: CDK6 Levels Determine the Duration of Quiescence Exit in the HSC Pool(A–C) Cell division duration of single LT- and ST-HSCs after exposure to mitogenic signal in the presence or absence of PD033299 (50 nM). (A) Percentage of cells from the indicated populations that divided after 100 hr in culture. (B) Cumulative first division kinetics (excluding dead cells). Data from a representative CB example are shown. Curve is least-squares sigmoid fit. R2 > 0.99. (C) Mean time to first division (hours) (tfirstDiv = logEC50).(D–G) Cell division duration of single LT- and ST-HSCs after exposure to mitogenic signal with or without CDK6 EE. (D) Cumulative first division kinetics (excluding dead cells) of indicated populations transduced with indicated lentiviral vectors. Data from a representative CB are shown. Curve is least-squares sigmoid fit. R2 > 0.99. (E) Mean time to first division (hours) (tfirstDiv = logEC50). (F) Time of cell cycle transit of indicated populations in hours.(G) Expansion curves of LT- and ST-HSCs in culture. Shown is the average number of cells per single cell plated at the indicated time points after culture initiation. Data are from one representative experiment out of three. Time 0 represents the time of exposure to mitogenic stimulus.In (A), (C), (E), and (F), individual CB samples are shown; gray lines connect parameters from the same condition. ∗∗p < 0.05 by paired t test. See also Figure S4.
Mentions: To gain mechanistic insight into the correlation between CDK6 protein levels and cell division duration within HSC subsets, we altered CDK6 levels and investigated the effect on the kinetics of the first HSC division. To examine loss of function, we measured the duration of cell division when single LT- and ST-HSCs are exposed to a mitogenic stimulus in the presence of the highly specific CDK4-CDK6 inhibitor PD033299. The majority of LT-HSCs never divided in the presence of PD033299 (Figure 4A). Similarly there was a strong reduction in the number of ST-HSCs that could divide (Figure 4A). However, for those ST-HSCs that divided, inhibition of CDK6 brought the length of the first division to that of LT-HSCs (Figures 4B and 4C). Intriguingly, those 10% LT-HSCs dividing in the presence of PD033299 were not further delayed, potentially representing a subset of more “activated” cells within the LT-HSC phenotypic compartment. To examine the consequences of CDK6 gain of function, we enforced expression of CDK6 protein (CDK6 EE) with lentiviral vectors in LT- and ST-HSCs before their first division (Figures S4A and S4B). CDK6 EE did not change any ST-HSC cell cycle parameters (Figures 4D–4F, S4C, and S4E). By contrast, a division starting from G0 (first division) of CDK6 EE LT-HSCs was significantly shortened to values similar to those of ST-HSCs (Figures 4D and 4E); control transduced LT-HSCs (LUC) showed no such changes. CDK6 EE did not decrease the duration of a division starting from G1 that remained significantly longer than that of ST-HSCs (Figures 4F, S4C, and S4F). Also, CDK6 EE did not affect the long-term proliferative output of LT-HSCs in vitro in conditions where cells do not return to G0 (Figure 4G). These approaches show that CDK6 shortens divisions starting from G0, but not divisions starting from G1. Moreover, variation in CDK6 protein levels between HSC subsets results in active regulation of the duration of the latency that is unique to HSCs transiting out of G0, a process we define as G0 exit. Importantly, our data also establish that pre-existent CDK6 in ST-HSCs primes them for earlier cell division upon mitogenic stimuli.

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