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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 HSCs have an initial survival advantage and make larger, more differentiated clones.(A) Schematic for single cell in vitro cultures. Individual CD45+/EPCR+/CD48−/CD150+ (E-SLAM) cells, obtained from mice 6–10 mo following pIpC injection, were sorted into single wells and cultured for 10 d in 300 ng/mL SCF and 20 ng/mL IL-11 in four independent experiments. (B) The average cloning efficiency was higher (p = 0.05) for JAK2V617F (red bars) versus wild type (blue bars) cells and was measured by counting the number of sorted events that give rise to a colony after 10 d. (C) The average number of cells per clone was higher (p = 0.016) in JAK2V617F cells. JAK2V617F HSCs give rise to more differentiated cells (p = 0.006) as measured by the expression of one or more of a panel of lineage markers (CD5, Mac1, CD19, B220, Ly6g, 7-4, or Ter119, panel D) and expression of c-Kit and Sca1 as a surrogate for stem/progenitor cell number (E). Fourteen-day cultures of 100–400 E-SLAM HSCs in SCF+IL-11 followed by flow cytometric analysis of the cells show that, by proportion, JAK2V617F HSCs make more CD41+ (p = 0.003, F), and less Ly6g/Mac1+ cells (p = 0.008, G) than wild-type controls in three independent experiments. The proportion of CD71+ cells generated was not changed (H). (I) The absolute numbers of Ly6g/Mac1+ and CD71+ cells generated were not different, but the number of CD41+ cells produced was increased approximately 2-fold (p = 0.023).
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pbio-1001576-g002: JAK2V617F HSCs have an initial survival advantage and make larger, more differentiated clones.(A) Schematic for single cell in vitro cultures. Individual CD45+/EPCR+/CD48−/CD150+ (E-SLAM) cells, obtained from mice 6–10 mo following pIpC injection, were sorted into single wells and cultured for 10 d in 300 ng/mL SCF and 20 ng/mL IL-11 in four independent experiments. (B) The average cloning efficiency was higher (p = 0.05) for JAK2V617F (red bars) versus wild type (blue bars) cells and was measured by counting the number of sorted events that give rise to a colony after 10 d. (C) The average number of cells per clone was higher (p = 0.016) in JAK2V617F cells. JAK2V617F HSCs give rise to more differentiated cells (p = 0.006) as measured by the expression of one or more of a panel of lineage markers (CD5, Mac1, CD19, B220, Ly6g, 7-4, or Ter119, panel D) and expression of c-Kit and Sca1 as a surrogate for stem/progenitor cell number (E). Fourteen-day cultures of 100–400 E-SLAM HSCs in SCF+IL-11 followed by flow cytometric analysis of the cells show that, by proportion, JAK2V617F HSCs make more CD41+ (p = 0.003, F), and less Ly6g/Mac1+ cells (p = 0.008, G) than wild-type controls in three independent experiments. The proportion of CD71+ cells generated was not changed (H). (I) The absolute numbers of Ly6g/Mac1+ and CD71+ cells generated were not different, but the number of CD41+ cells produced was increased approximately 2-fold (p = 0.023).

Mentions: To study the stem cell defect in individual HSCs, we used a single-cell in vitro culture system previously reported to maintain numbers of long-term repopulating cells [20],[25]. Single E-SLAM HSCs (n = 720), obtained from JAK2V617F mice or WT littermate controls, were assessed for survival, early kinetics of cell division, proliferation, and differentiation state (Figure 2). Compared to WT E-SLAM HSCs, the number of wells giving rise to a 10-d clone from JAK2V617F E-SLAM HSCs was increased by approximately 50% (p = 0.05, Figure 2B) and the average clone size was also increased (p = 0.016, Figure 2C). Clones derived from JAK2V617F HSCs contained more differentiated cells (Lin+) (p = 0.006, Figure 2D), but did not show a significant increase in KSL cells (Figure 2E). Compared to WT equivalents, JAK2V617F E-SLAM HSCs displayed similar cell cycle kinetics during their first two rounds of cell division (Figure S1A) and gave rise to similar levels of apoptotic cells after 10 d of culture (Figure S1B). These results demonstrate that JAK2V617F E-SLAM HSCs are more clonogenic and give rise to more progeny expressing differentiation markers under conditions that normally maintain HSC numbers.


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 HSCs have an initial survival advantage and make larger, more differentiated clones.(A) Schematic for single cell in vitro cultures. Individual CD45+/EPCR+/CD48−/CD150+ (E-SLAM) cells, obtained from mice 6–10 mo following pIpC injection, were sorted into single wells and cultured for 10 d in 300 ng/mL SCF and 20 ng/mL IL-11 in four independent experiments. (B) The average cloning efficiency was higher (p = 0.05) for JAK2V617F (red bars) versus wild type (blue bars) cells and was measured by counting the number of sorted events that give rise to a colony after 10 d. (C) The average number of cells per clone was higher (p = 0.016) in JAK2V617F cells. JAK2V617F HSCs give rise to more differentiated cells (p = 0.006) as measured by the expression of one or more of a panel of lineage markers (CD5, Mac1, CD19, B220, Ly6g, 7-4, or Ter119, panel D) and expression of c-Kit and Sca1 as a surrogate for stem/progenitor cell number (E). Fourteen-day cultures of 100–400 E-SLAM HSCs in SCF+IL-11 followed by flow cytometric analysis of the cells show that, by proportion, JAK2V617F HSCs make more CD41+ (p = 0.003, F), and less Ly6g/Mac1+ cells (p = 0.008, G) than wild-type controls in three independent experiments. The proportion of CD71+ cells generated was not changed (H). (I) The absolute numbers of Ly6g/Mac1+ and CD71+ cells generated were not different, but the number of CD41+ cells produced was increased approximately 2-fold (p = 0.023).
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1001576-g002: JAK2V617F HSCs have an initial survival advantage and make larger, more differentiated clones.(A) Schematic for single cell in vitro cultures. Individual CD45+/EPCR+/CD48−/CD150+ (E-SLAM) cells, obtained from mice 6–10 mo following pIpC injection, were sorted into single wells and cultured for 10 d in 300 ng/mL SCF and 20 ng/mL IL-11 in four independent experiments. (B) The average cloning efficiency was higher (p = 0.05) for JAK2V617F (red bars) versus wild type (blue bars) cells and was measured by counting the number of sorted events that give rise to a colony after 10 d. (C) The average number of cells per clone was higher (p = 0.016) in JAK2V617F cells. JAK2V617F HSCs give rise to more differentiated cells (p = 0.006) as measured by the expression of one or more of a panel of lineage markers (CD5, Mac1, CD19, B220, Ly6g, 7-4, or Ter119, panel D) and expression of c-Kit and Sca1 as a surrogate for stem/progenitor cell number (E). Fourteen-day cultures of 100–400 E-SLAM HSCs in SCF+IL-11 followed by flow cytometric analysis of the cells show that, by proportion, JAK2V617F HSCs make more CD41+ (p = 0.003, F), and less Ly6g/Mac1+ cells (p = 0.008, G) than wild-type controls in three independent experiments. The proportion of CD71+ cells generated was not changed (H). (I) The absolute numbers of Ly6g/Mac1+ and CD71+ cells generated were not different, but the number of CD41+ cells produced was increased approximately 2-fold (p = 0.023).
Mentions: To study the stem cell defect in individual HSCs, we used a single-cell in vitro culture system previously reported to maintain numbers of long-term repopulating cells [20],[25]. Single E-SLAM HSCs (n = 720), obtained from JAK2V617F mice or WT littermate controls, were assessed for survival, early kinetics of cell division, proliferation, and differentiation state (Figure 2). Compared to WT E-SLAM HSCs, the number of wells giving rise to a 10-d clone from JAK2V617F E-SLAM HSCs was increased by approximately 50% (p = 0.05, Figure 2B) and the average clone size was also increased (p = 0.016, Figure 2C). Clones derived from JAK2V617F HSCs contained more differentiated cells (Lin+) (p = 0.006, Figure 2D), but did not show a significant increase in KSL cells (Figure 2E). Compared to WT equivalents, JAK2V617F E-SLAM HSCs displayed similar cell cycle kinetics during their first two rounds of cell division (Figure S1A) and gave rise to similar levels of apoptotic cells after 10 d of culture (Figure S1B). These results demonstrate that JAK2V617F E-SLAM HSCs are more clonogenic and give rise to more progeny expressing differentiation markers under conditions that normally maintain HSC numbers.

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