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Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion.

Kent DG, Li J, Tanna H, Fink J, Kirschner K, Pask DC, Silber Y, Hamilton TL, Sneade R, Simons BD, Green AR - PLoS Biol. (2013)

Bottom Line: Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact.This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells.Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F subclinical clonal expansions and the transition to overt MPNs will illuminate the earliest stages of tumor establishment and subclone competition, fundamentally shifting the way we treat and manage cancers.

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Related in: MedlinePlus

JAK2V617F E-SLAM HSCs, but not progenitors, are tilted toward differentiation.(A) The average clone size data for WT and JAK2V617F E-SLAM HSCs are approximately exponential over the 10-d time course. At early times, the data for both cell types show that the expansion is geometrical, with individual clones expanding from one to two to four to eight cells. After several rounds of division, the average cell division rate appears to accelerate significantly, while nearer to 10 d, there is a deceleration most likely due to cells exiting cycle. The dashed line shows what exponential growth would look like with the average doubling rate of the first 4–5 rounds of division (1.06 for WT and 1.26 for JAK2V617F), and the solid line represents the model fit to the actual data points for WT (left) and JAK2V617F (right) clones (for details, see Model S1). Note that, in both cases, division rates must increase to accommodate the expansion measured at day 10. (B) A schematic of the model dynamics. In the WT situation, cells move through a differentiation hierarchy with HSCs at the apex. In the model, the division of an HSC leads to symmetric duplication or differentiation with equal probability (i.e., x = 50%). Cells at the first generation of the differentiating hierarchy then have a capacity to duplicate or symmetrically differentiate into cells in the next tier of the hierarchy, and the model can be tuned to allow x to vary throughout (see Model S1 for further details). (C) The cumulative size distribution of clones 10 d postplating by cell type in WT clones—i.e., the KSL data point at (4k, 40%) in the WT graph—shows that 40% of the colonies have at least 4,000 KSL cells, etc. (D) Comparison of the balanced self-renewal model (i.e., with x = 50% within the entire stem and progenitor cell compartments) with parameters inferred from a fit to the colony growth curve (A) and cell type averages at 10 d postplating, against the experimental data (points) taken from (C) and (E). The vertical lines (color coded by cell type) represent the expected range of fluctuations of the cumulative size distribution due to small number statistics, and are inferred from the average and first standard deviation of the results of the model simulation with 1,000 trials each with a cohort of 68 (WT) and 125 (JAK2V617F) colonies, consistent with that used in experiment (for further details and model parameters, see Model S1). (E) The same data set as in 5C and 5D, but with model predictions when just 40% of the progenitor cell progeny (x = 40% for the non-HSCs tiers) remain at the same tier of the hierarchy. Note the departure of the line for the KSL population, which reflects the premature escape of cells from the top of the hierarchy. (F) The cumulative size distribution of colonies 10 d postplating by cell type in JAK2V617F clones. (G) The balanced self-renewal model (i.e., with x = 50% within the entire non-HSC progenitor compartment) overlaid onto the data from JAK2V617F clones with solid lines displaying the predictions of the model. The departure of the model from the observed data is visible in the total viable cells where the model predicts more viable cells in order to produce the observed number of KSL and Lin-/non-KSL cells. (H) Here the lines shown represent a model where HSC self-renewal has been set to 0 implying that every division of an HSC will result in differentiation to the next tier, but progenitor self-renewal remains intact within the rest of the non-HSC progenitor cell compartments. Note the strong overlap of the model with the data points from the JAK2V617F clones. In panels C–H, the total size colony is represented by black, Lin-/non-KSL cells are beige, and KSL cells are blue.
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pbio-1001576-g005: JAK2V617F E-SLAM HSCs, but not progenitors, are tilted toward differentiation.(A) The average clone size data for WT and JAK2V617F E-SLAM HSCs are approximately exponential over the 10-d time course. At early times, the data for both cell types show that the expansion is geometrical, with individual clones expanding from one to two to four to eight cells. After several rounds of division, the average cell division rate appears to accelerate significantly, while nearer to 10 d, there is a deceleration most likely due to cells exiting cycle. The dashed line shows what exponential growth would look like with the average doubling rate of the first 4–5 rounds of division (1.06 for WT and 1.26 for JAK2V617F), and the solid line represents the model fit to the actual data points for WT (left) and JAK2V617F (right) clones (for details, see Model S1). Note that, in both cases, division rates must increase to accommodate the expansion measured at day 10. (B) A schematic of the model dynamics. In the WT situation, cells move through a differentiation hierarchy with HSCs at the apex. In the model, the division of an HSC leads to symmetric duplication or differentiation with equal probability (i.e., x = 50%). Cells at the first generation of the differentiating hierarchy then have a capacity to duplicate or symmetrically differentiate into cells in the next tier of the hierarchy, and the model can be tuned to allow x to vary throughout (see Model S1 for further details). (C) The cumulative size distribution of clones 10 d postplating by cell type in WT clones—i.e., the KSL data point at (4k, 40%) in the WT graph—shows that 40% of the colonies have at least 4,000 KSL cells, etc. (D) Comparison of the balanced self-renewal model (i.e., with x = 50% within the entire stem and progenitor cell compartments) with parameters inferred from a fit to the colony growth curve (A) and cell type averages at 10 d postplating, against the experimental data (points) taken from (C) and (E). The vertical lines (color coded by cell type) represent the expected range of fluctuations of the cumulative size distribution due to small number statistics, and are inferred from the average and first standard deviation of the results of the model simulation with 1,000 trials each with a cohort of 68 (WT) and 125 (JAK2V617F) colonies, consistent with that used in experiment (for further details and model parameters, see Model S1). (E) The same data set as in 5C and 5D, but with model predictions when just 40% of the progenitor cell progeny (x = 40% for the non-HSCs tiers) remain at the same tier of the hierarchy. Note the departure of the line for the KSL population, which reflects the premature escape of cells from the top of the hierarchy. (F) The cumulative size distribution of colonies 10 d postplating by cell type in JAK2V617F clones. (G) The balanced self-renewal model (i.e., with x = 50% within the entire non-HSC progenitor compartment) overlaid onto the data from JAK2V617F clones with solid lines displaying the predictions of the model. The departure of the model from the observed data is visible in the total viable cells where the model predicts more viable cells in order to produce the observed number of KSL and Lin-/non-KSL cells. (H) Here the lines shown represent a model where HSC self-renewal has been set to 0 implying that every division of an HSC will result in differentiation to the next tier, but progenitor self-renewal remains intact within the rest of the non-HSC progenitor cell compartments. Note the strong overlap of the model with the data points from the JAK2V617F clones. In panels C–H, the total size colony is represented by black, Lin-/non-KSL cells are beige, and KSL cells are blue.

Mentions: To understand the self-renewal and differentiation capacity of individual HSCs and their progeny, we combined a quantitative analysis of short-term clone size data with a more detailed analysis of the colony size and cell type composition after 10 d in culture using the data presented in Figure 2. Consistent with previous reports [25],[29], HSCs exposed to SCF and IL-11 rarely entered the cell cycle before 24 h and had an average time to first division of approximately 40 h (Figure S1A). After this initial lag, HSC-derived clones underwent steady exponential expansion at a constant rate for the first 4–5 rounds of division, suggesting that few cells, if any, exited the cell cycle (Figure 5A). Clones then underwent a substantial increase in their average cell division rate. At the end of the time course, the average clone size increased less rapidly, consistent with cells committing to terminal differentiation (Model S1 and Figure S2).


Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion.

Kent DG, Li J, Tanna H, Fink J, Kirschner K, Pask DC, Silber Y, Hamilton TL, Sneade R, Simons BD, Green AR - PLoS Biol. (2013)

JAK2V617F E-SLAM HSCs, but not progenitors, are tilted toward differentiation.(A) The average clone size data for WT and JAK2V617F E-SLAM HSCs are approximately exponential over the 10-d time course. At early times, the data for both cell types show that the expansion is geometrical, with individual clones expanding from one to two to four to eight cells. After several rounds of division, the average cell division rate appears to accelerate significantly, while nearer to 10 d, there is a deceleration most likely due to cells exiting cycle. The dashed line shows what exponential growth would look like with the average doubling rate of the first 4–5 rounds of division (1.06 for WT and 1.26 for JAK2V617F), and the solid line represents the model fit to the actual data points for WT (left) and JAK2V617F (right) clones (for details, see Model S1). Note that, in both cases, division rates must increase to accommodate the expansion measured at day 10. (B) A schematic of the model dynamics. In the WT situation, cells move through a differentiation hierarchy with HSCs at the apex. In the model, the division of an HSC leads to symmetric duplication or differentiation with equal probability (i.e., x = 50%). Cells at the first generation of the differentiating hierarchy then have a capacity to duplicate or symmetrically differentiate into cells in the next tier of the hierarchy, and the model can be tuned to allow x to vary throughout (see Model S1 for further details). (C) The cumulative size distribution of clones 10 d postplating by cell type in WT clones—i.e., the KSL data point at (4k, 40%) in the WT graph—shows that 40% of the colonies have at least 4,000 KSL cells, etc. (D) Comparison of the balanced self-renewal model (i.e., with x = 50% within the entire stem and progenitor cell compartments) with parameters inferred from a fit to the colony growth curve (A) and cell type averages at 10 d postplating, against the experimental data (points) taken from (C) and (E). The vertical lines (color coded by cell type) represent the expected range of fluctuations of the cumulative size distribution due to small number statistics, and are inferred from the average and first standard deviation of the results of the model simulation with 1,000 trials each with a cohort of 68 (WT) and 125 (JAK2V617F) colonies, consistent with that used in experiment (for further details and model parameters, see Model S1). (E) The same data set as in 5C and 5D, but with model predictions when just 40% of the progenitor cell progeny (x = 40% for the non-HSCs tiers) remain at the same tier of the hierarchy. Note the departure of the line for the KSL population, which reflects the premature escape of cells from the top of the hierarchy. (F) The cumulative size distribution of colonies 10 d postplating by cell type in JAK2V617F clones. (G) The balanced self-renewal model (i.e., with x = 50% within the entire non-HSC progenitor compartment) overlaid onto the data from JAK2V617F clones with solid lines displaying the predictions of the model. The departure of the model from the observed data is visible in the total viable cells where the model predicts more viable cells in order to produce the observed number of KSL and Lin-/non-KSL cells. (H) Here the lines shown represent a model where HSC self-renewal has been set to 0 implying that every division of an HSC will result in differentiation to the next tier, but progenitor self-renewal remains intact within the rest of the non-HSC progenitor cell compartments. Note the strong overlap of the model with the data points from the JAK2V617F clones. In panels C–H, the total size colony is represented by black, Lin-/non-KSL cells are beige, and KSL cells are blue.
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pbio-1001576-g005: JAK2V617F E-SLAM HSCs, but not progenitors, are tilted toward differentiation.(A) The average clone size data for WT and JAK2V617F E-SLAM HSCs are approximately exponential over the 10-d time course. At early times, the data for both cell types show that the expansion is geometrical, with individual clones expanding from one to two to four to eight cells. After several rounds of division, the average cell division rate appears to accelerate significantly, while nearer to 10 d, there is a deceleration most likely due to cells exiting cycle. The dashed line shows what exponential growth would look like with the average doubling rate of the first 4–5 rounds of division (1.06 for WT and 1.26 for JAK2V617F), and the solid line represents the model fit to the actual data points for WT (left) and JAK2V617F (right) clones (for details, see Model S1). Note that, in both cases, division rates must increase to accommodate the expansion measured at day 10. (B) A schematic of the model dynamics. In the WT situation, cells move through a differentiation hierarchy with HSCs at the apex. In the model, the division of an HSC leads to symmetric duplication or differentiation with equal probability (i.e., x = 50%). Cells at the first generation of the differentiating hierarchy then have a capacity to duplicate or symmetrically differentiate into cells in the next tier of the hierarchy, and the model can be tuned to allow x to vary throughout (see Model S1 for further details). (C) The cumulative size distribution of clones 10 d postplating by cell type in WT clones—i.e., the KSL data point at (4k, 40%) in the WT graph—shows that 40% of the colonies have at least 4,000 KSL cells, etc. (D) Comparison of the balanced self-renewal model (i.e., with x = 50% within the entire stem and progenitor cell compartments) with parameters inferred from a fit to the colony growth curve (A) and cell type averages at 10 d postplating, against the experimental data (points) taken from (C) and (E). The vertical lines (color coded by cell type) represent the expected range of fluctuations of the cumulative size distribution due to small number statistics, and are inferred from the average and first standard deviation of the results of the model simulation with 1,000 trials each with a cohort of 68 (WT) and 125 (JAK2V617F) colonies, consistent with that used in experiment (for further details and model parameters, see Model S1). (E) The same data set as in 5C and 5D, but with model predictions when just 40% of the progenitor cell progeny (x = 40% for the non-HSCs tiers) remain at the same tier of the hierarchy. Note the departure of the line for the KSL population, which reflects the premature escape of cells from the top of the hierarchy. (F) The cumulative size distribution of colonies 10 d postplating by cell type in JAK2V617F clones. (G) The balanced self-renewal model (i.e., with x = 50% within the entire non-HSC progenitor compartment) overlaid onto the data from JAK2V617F clones with solid lines displaying the predictions of the model. The departure of the model from the observed data is visible in the total viable cells where the model predicts more viable cells in order to produce the observed number of KSL and Lin-/non-KSL cells. (H) Here the lines shown represent a model where HSC self-renewal has been set to 0 implying that every division of an HSC will result in differentiation to the next tier, but progenitor self-renewal remains intact within the rest of the non-HSC progenitor cell compartments. Note the strong overlap of the model with the data points from the JAK2V617F clones. In panels C–H, the total size colony is represented by black, Lin-/non-KSL cells are beige, and KSL cells are blue.
Mentions: To understand the self-renewal and differentiation capacity of individual HSCs and their progeny, we combined a quantitative analysis of short-term clone size data with a more detailed analysis of the colony size and cell type composition after 10 d in culture using the data presented in Figure 2. Consistent with previous reports [25],[29], HSCs exposed to SCF and IL-11 rarely entered the cell cycle before 24 h and had an average time to first division of approximately 40 h (Figure S1A). After this initial lag, HSC-derived clones underwent steady exponential expansion at a constant rate for the first 4–5 rounds of division, suggesting that few cells, if any, exited the cell cycle (Figure 5A). Clones then underwent a substantial increase in their average cell division rate. At the end of the time course, the average clone size increased less rapidly, consistent with cells committing to terminal differentiation (Model S1 and Figure S2).

Bottom Line: Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact.This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells.Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F subclinical clonal expansions and the transition to overt MPNs will illuminate the earliest stages of tumor establishment and subclone competition, fundamentally shifting the way we treat and manage cancers.

Show MeSH
Related in: MedlinePlus