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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

Cultured JAK2V617F E-SLAM HSCs produce more short-term progenitors, but lack long-term reconstitution ability.(A) Cells derived from cultures of 100–400 E-SLAM HSCs were harvested after 10 d of culture in SCF and IL-11 and then placed in a colony-forming cell (CFC) assay to determine the number and type of progenitor cells made or were transplanted into irradiated recipients to determine whether or not long-term reconstituting ability was retained. (B) Following 10–14 d of culture in the CFC assay, colonies were scored and enumerated. Colonies were scored as either Erythroid (E), Granulocyte/Macrophage (GM), or Granulocyte/Macrophage/Erythroid/Megakaryocyte (GEMM) progenitors and are represented by bar graphs showing the mean +/– SEM of four to six biological replicates from four independent experiments. A greater number of GM (p = 0.009) and E (p = 0.007) were observed in CFCs derived from JAK2V617F cultures. (C) Varying doses (40, 33, 4) of HSC starting equivalents (the proportion of the total culture that would have been made by that input number of HSCs) were transplanted to determine the frequency of cells that had retained long-term reconstituting ability in two independent experiments. This is followed by a limiting dilution analysis that estimates the frequency of HSCs retained in the culture. Cultures of JAK2V617F HSCs make 5–6-fold fewer HSCs in culture compared to WT littermate controls (p = 0.00469). * HSC dose is defined as the number of starting equivalents that were transplanted. In the case of “40,” this is representative of transplanting all of the cells that would be generated from a 10-d culture of 40 HSCs. ** A mouse was considered to be positive if it had >1% donor chimerism at 16–24 weeks and represented at least 0.5% of each lineage (GM, B, and T) at some point over the 16-wk period.
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pbio-1001576-g003: Cultured JAK2V617F E-SLAM HSCs produce more short-term progenitors, but lack long-term reconstitution ability.(A) Cells derived from cultures of 100–400 E-SLAM HSCs were harvested after 10 d of culture in SCF and IL-11 and then placed in a colony-forming cell (CFC) assay to determine the number and type of progenitor cells made or were transplanted into irradiated recipients to determine whether or not long-term reconstituting ability was retained. (B) Following 10–14 d of culture in the CFC assay, colonies were scored and enumerated. Colonies were scored as either Erythroid (E), Granulocyte/Macrophage (GM), or Granulocyte/Macrophage/Erythroid/Megakaryocyte (GEMM) progenitors and are represented by bar graphs showing the mean +/– SEM of four to six biological replicates from four independent experiments. A greater number of GM (p = 0.009) and E (p = 0.007) were observed in CFCs derived from JAK2V617F cultures. (C) Varying doses (40, 33, 4) of HSC starting equivalents (the proportion of the total culture that would have been made by that input number of HSCs) were transplanted to determine the frequency of cells that had retained long-term reconstituting ability in two independent experiments. This is followed by a limiting dilution analysis that estimates the frequency of HSCs retained in the culture. Cultures of JAK2V617F HSCs make 5–6-fold fewer HSCs in culture compared to WT littermate controls (p = 0.00469). * HSC dose is defined as the number of starting equivalents that were transplanted. In the case of “40,” this is representative of transplanting all of the cells that would be generated from a 10-d culture of 40 HSCs. ** A mouse was considered to be positive if it had >1% donor chimerism at 16–24 weeks and represented at least 0.5% of each lineage (GM, B, and T) at some point over the 16-wk period.

Mentions: The results described above show that JAK2V617F E-SLAM HSCs make larger, more differentiated clones compared to WT E-SLAM HSCs, but did not allow us to assess the production of functional progenitors. We therefore performed short-term progenitor assays and long-term transplantation assays on the progeny of cultured E-SLAM HSCs (Figure 3A). Individual pools each containing 100–400 E-SLAM HSCs were cultured for 10 d, and the progeny were assessed by CFC assays or transplanted at different doses into irradiated recipients. In CFC assays, JAK2V617F cells gave rise to significantly more BFU-E (p = 0.007) and CFU-GM (p = 0.009), but displayed no increase in the number of CFU-GEMM (Figure 3B). The CFC culture conditions do not detect lymphoid progenitors, and so these results do not exclude expansion of lymphoid-committed progenitors.


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)

Cultured JAK2V617F E-SLAM HSCs produce more short-term progenitors, but lack long-term reconstitution ability.(A) Cells derived from cultures of 100–400 E-SLAM HSCs were harvested after 10 d of culture in SCF and IL-11 and then placed in a colony-forming cell (CFC) assay to determine the number and type of progenitor cells made or were transplanted into irradiated recipients to determine whether or not long-term reconstituting ability was retained. (B) Following 10–14 d of culture in the CFC assay, colonies were scored and enumerated. Colonies were scored as either Erythroid (E), Granulocyte/Macrophage (GM), or Granulocyte/Macrophage/Erythroid/Megakaryocyte (GEMM) progenitors and are represented by bar graphs showing the mean +/– SEM of four to six biological replicates from four independent experiments. A greater number of GM (p = 0.009) and E (p = 0.007) were observed in CFCs derived from JAK2V617F cultures. (C) Varying doses (40, 33, 4) of HSC starting equivalents (the proportion of the total culture that would have been made by that input number of HSCs) were transplanted to determine the frequency of cells that had retained long-term reconstituting ability in two independent experiments. This is followed by a limiting dilution analysis that estimates the frequency of HSCs retained in the culture. Cultures of JAK2V617F HSCs make 5–6-fold fewer HSCs in culture compared to WT littermate controls (p = 0.00469). * HSC dose is defined as the number of starting equivalents that were transplanted. In the case of “40,” this is representative of transplanting all of the cells that would be generated from a 10-d culture of 40 HSCs. ** A mouse was considered to be positive if it had >1% donor chimerism at 16–24 weeks and represented at least 0.5% of each lineage (GM, B, and T) at some point over the 16-wk period.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672217&req=5

pbio-1001576-g003: Cultured JAK2V617F E-SLAM HSCs produce more short-term progenitors, but lack long-term reconstitution ability.(A) Cells derived from cultures of 100–400 E-SLAM HSCs were harvested after 10 d of culture in SCF and IL-11 and then placed in a colony-forming cell (CFC) assay to determine the number and type of progenitor cells made or were transplanted into irradiated recipients to determine whether or not long-term reconstituting ability was retained. (B) Following 10–14 d of culture in the CFC assay, colonies were scored and enumerated. Colonies were scored as either Erythroid (E), Granulocyte/Macrophage (GM), or Granulocyte/Macrophage/Erythroid/Megakaryocyte (GEMM) progenitors and are represented by bar graphs showing the mean +/– SEM of four to six biological replicates from four independent experiments. A greater number of GM (p = 0.009) and E (p = 0.007) were observed in CFCs derived from JAK2V617F cultures. (C) Varying doses (40, 33, 4) of HSC starting equivalents (the proportion of the total culture that would have been made by that input number of HSCs) were transplanted to determine the frequency of cells that had retained long-term reconstituting ability in two independent experiments. This is followed by a limiting dilution analysis that estimates the frequency of HSCs retained in the culture. Cultures of JAK2V617F HSCs make 5–6-fold fewer HSCs in culture compared to WT littermate controls (p = 0.00469). * HSC dose is defined as the number of starting equivalents that were transplanted. In the case of “40,” this is representative of transplanting all of the cells that would be generated from a 10-d culture of 40 HSCs. ** A mouse was considered to be positive if it had >1% donor chimerism at 16–24 weeks and represented at least 0.5% of each lineage (GM, B, and T) at some point over the 16-wk period.
Mentions: The results described above show that JAK2V617F E-SLAM HSCs make larger, more differentiated clones compared to WT E-SLAM HSCs, but did not allow us to assess the production of functional progenitors. We therefore performed short-term progenitor assays and long-term transplantation assays on the progeny of cultured E-SLAM HSCs (Figure 3A). Individual pools each containing 100–400 E-SLAM HSCs were cultured for 10 d, and the progeny were assessed by CFC assays or transplanted at different doses into irradiated recipients. In CFC assays, JAK2V617F cells gave rise to significantly more BFU-E (p = 0.007) and CFU-GM (p = 0.009), but displayed no increase in the number of CFU-GEMM (Figure 3B). The CFC culture conditions do not detect lymphoid progenitors, and so these results do not exclude expansion of lymphoid-committed progenitors.

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