13 research outputs found
Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion
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
Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage.
Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALRdel/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALRdel/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALRdel/+ HSCs were more proliferative in vitro, but neither CALRdel/+ nor CALRdel/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF
Cultured JAK2<sup>V617F</sup> E-SLAM HSCs produce more short-term progenitors, but lack long-term reconstitution ability.
<p>(A) Cells derived from cultures of 100β400 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 10β14 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 +/β SEM of four to six biological replicates from four independent experiments. A greater number of GM (<i>p</i>β=β0.009) and E (<i>p</i>β=β0.007) were observed in CFCs derived from JAK2<sup>V617F</sup> 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 JAK2<sup>V617F</sup> HSCs make 5β6-fold fewer HSCs in culture compared to WT littermate controls (<i>p</i>β=β0.00469). * HSC dose is defined as the number of starting equivalents that were transplanted. In the case of β40,β 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 16β24 weeks and represented at least 0.5% of each lineage (GM, B, and T) at some point over the 16-wk period.</p
JAK2<sup>V617F</sup> alters the balance of HSC fate choices.
<p>(A) A paired daughter cell analysis of WT and JAK2<sup>V617F</sup> HSCs shows both daughters differentiate more often from JAK2<sup>V617F</sup> parent HSCs than from WT HSCs as shown by measuring the percentage of KSL cells remaining after 10 d. Each paired daughter set is connected by a line and the pairs are categorized into symmetric SR (both daughters above the WT average %KSL), asymmetric division (one daughter above and one below the average %KSL), and symmetric differentiation (both daughters below the average %KSL). Note the relative increase in symmetric differentiation at the expense of asymmetric divisions. (B) The same paired daughter pairs are displayed here by the absolute number of KSL cells produced. Here it is clear that some of the JAK2<sup>V617F</sup> pairs produce very few KSL cells (less than 100 per clone in some of the asymmetric divisions and symmetric differentiation divisions compared to WT HSCs, which are all above 100 KSL cells). (C) The pie graph on the left represents the outcome from 78 WT paired daughters (39 pairs), and the pie on the left represents the outcome from 76 mutant paired daughters (38 pairs). (D) Normally, HSCs will execute one of several programs in concert with the other HSCs to provide the requisite numbers of stem cells, progenitors, and differentiated cells for the organism. JAK2<sup>V617F</sup> disturbs this balance and increases the likelihood of differentiation. As HSCs with the V617F mutation age, they have both an increased chance of fully exhausting as well as an increased chance of progressing to a more severe disease state, likely due to the acquisition of additional genetic or epigenetic perturbations.</p
E-SLAM HSCs do not expand in old JAK2<sup>V617F</sup> knock-in mice and show reduced functional ability as well as a delayed entry into the cell cycle.
<p>(A) E-SLAM HSCs were increased in frequency in wild type (βΌ2-fold, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001576#pbio-1001576-g001" target="_blank">Figure 1E</a>) but not JAK2<sup>V617F</sup> marrow in 18β24-mo-old mice (<i>n</i>β=β10) compared to 6β10-mo-old mice resulting in a 3-fold overall reduction in E-SLAM HSCs compared to wild-type (<i>p</i>β=β0.002) in three independent experiments. (B) Individual HSCs were cultured and cell counts were recorded on day 1 and day 2 to determine whether or not they had undergone a division in three independent experiments. At day 2, significantly fewer (<i>p</i>β=β0.039) old JAK2<sup>V617F</sup> HSCs had divided. The cloning efficiency (C), number of cells per clone (D), and number of KSL cells per clone (F) were not different, but the JAK2<sup>V617F</sup> cells still produced more differentiated cell types after 10 d of culture (<i>p</i>β=β0.039, E). (G) Competitive transplantation of whole bone marrow from old JAK2<sup>V617F</sup> mice, transformed JAK2<sup>V617F</sup> mice, and their respective WT littermate controls. Relative chimerism is calculated by measuring donor chimerism as a percentage of donor+competitor chimerism and normalized to the average of the WT contribution (set to 1). The old JAK2<sup>V617F</sup> BM displays reduced chimerism (<i>p</i><0.01), whereas transformed JAK2<sup>V617F</sup> mice that have undergone transformation reacquire their self-renewal capacity.</p
JAK2<sup>V617F</sup> HSCs have an initial survival advantage and make larger, more differentiated clones.
<p>(A) Schematic for single cell in vitro cultures. Individual CD45<sup>+</sup>/EPCR<sup>+</sup>/CD48<sup>β</sup>/CD150<sup>+</sup> (E-SLAM) cells, obtained from mice 6β10 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 (<i>p</i>β=β0.05) for JAK2<sup>V617F</sup> (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 (<i>p</i>β=β0.016) in JAK2<sup>V617F</sup> cells. JAK2<sup>V617F</sup> HSCs give rise to more differentiated cells (<i>p</i>β=β0.006) 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 100β400 E-SLAM HSCs in SCF+IL-11 followed by flow cytometric analysis of the cells show that, by proportion, JAK2<sup>V617F</sup> HSCs make more CD41<sup>+</sup> (<i>p</i>β=β0.003, F), and less Ly6g/Mac1<sup>+</sup> cells (<i>p</i>β=β0.008, G) than wild-type controls in three independent experiments. The proportion of CD71<sup>+</sup> cells generated was not changed (H). (I) The absolute numbers of Ly6g/Mac1<sup>+</sup> and CD71<sup>+</sup> cells generated were not different, but the number of CD41<sup>+</sup> cells produced was increased approximately 2-fold (<i>p</i>β=β0.023).</p
JAK2V617F induces a loss of self-renewal activity and HSC numbers and leads to a lineage bias when limited HSCs are transplanted.
<p>(A) Relative chimerism following transplantation of 10<sup>6</sup> or 10<sup>5</sup> JAK2<sup>V617F</sup> or wild type (WT) whole bone marrow (BM) 6β10 months post-pIpC along with 5Γ10<sup>5</sup> whole BM competitor cells into eight recipient mice. Average chimerism was lower in mice receiving JAK2<sup>V617F</sup> cells (<i>p</i>β=β0.03). (B) The relative myeloid (purple) versus lymphoid (green) contribution in each of the recipient animals were determined by calculating a ratio between the contribution to the myeloid compartment [Donor GM/(Donor GM+Competitor GM)] and lymphoid compartment [Donor BT/(Donor BT+Competitor BT)]. (C) Relative chimerism in primary peripheral blood (PB) (white bars) in the eight animals receiving 10<sup>5</sup> cells compared to levels of PB chimerism in the 16 secondary recipients (two per primary animal, grey bars) in two independent transplantation experiments. An βXβ represents a recipient that showed less than 1% chimerism at 24 wk posttransplantation, and a β§ represents a recipient that only had contribution to the lymphoid lineages. (D) The relative myeloid (purple) versus lymphoid (green) contribution in each of the secondary recipient animals were determined by calculating a ratio between the contribution to the myeloid and lymphoid compartments as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001576#pbio-1001576-g001" target="_blank">Figure 1B</a>. (E) The FACS isolation strategy for CD45<sup>+</sup>/EPCR<sup>+</sup>/CD48<sup>β</sup>/CD150<sup>+</sup> (E-SLAM) cells. The panels are gated on viable white blood cells and show E-SLAM gates for WT (top) and JAK2<sup>V617F</sup> (bottom). (F) The frequency of E-SLAM HSCs per 10<sup>5</sup> viable bone marrow (BM) cells in four WT and four JAK2<sup>V617F</sup> mice 6β10 mo following pIpC injection from four independent experiments. The frequency is reduced in JAK2<sup>V617F</sup> animals (<i>p</i>β=β0.0288).</p
JAK2<sup>V617F</sup> E-SLAM HSCs, but not progenitors, are tilted toward differentiation.
<p>(A) The average clone size data for WT and JAK2<sup>V617F</sup> 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 4β5 rounds of division (1.06 for WT and 1.26 for JAK2<sup>V617F</sup>), and the solid line represents the model fit to the actual data points for WT (left) and JAK2<sup>V617F</sup> (right) clones (for details, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001576#pbio.1001576.s009" target="_blank">Model S1</a>). 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., xβ=β50%). 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001576#pbio.1001576.s009" target="_blank">Model S1</a> for further details). (C) The cumulative size distribution of clones 10 d postplating by cell type in WT clonesβi.e., the KSL data point at (4k, 40%) in the WT graphβshows that 40% of the colonies have at least 4,000 KSL cells, etc. (D) Comparison of the balanced self-renewal model (i.e., with xβ=β50% 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 (JAK2<sup>V617F</sup>) colonies, consistent with that used in experiment (for further details and model parameters, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001576#pbio.1001576.s009" target="_blank">Model S1</a>). (E) The same data set as in 5C and 5D, but with model predictions when just 40% of the progenitor cell progeny (xβ=β40% 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 JAK2<sup>V617F</sup> clones. (G) The balanced self-renewal model (i.e., with xβ=β50% within the entire non-HSC progenitor compartment) overlaid onto the data from JAK2<sup>V617F</sup> 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 JAK2<sup>V617F</sup> clones. In panels CβH, the total size colony is represented by black, Lin-/non-KSL cells are beige, and KSL cells are blue.</p
STAT1 is essential for HSC function and maintains MHCIIhi stem cells that resists myeloablation and neoplastic expansion
Adult hematopoietic stem cells (HSCs) are predominantly quiescent and can be activated in response to acute stress such as infection or cytotoxic insults. STAT1 is a pivotal downstream mediator of interferon (IFN) signaling and is required for IFN-induced HSC proliferation, but little is known about the role of STAT1 in regulating homeostatic hematopoietic stem/progenitor cells (HSPCs). Here we show that loss of STAT1 altered the steady state HSPC landscape, impaired HSC function in transplantation assays, delayed blood cell regeneration following myeloablation, and disrupted molecular programs which protect HSCs including control of quiescence. Our results also reveal STAT1-dependent functional HSC heterogeneity. A previously unrecognized subset of homeostatic HSCs with elevated MHCII expression (MHCIIhi) displayed molecular features of reduced cycling and apoptosis, and was refractory to 5-FU induced myeloablation. Conversely, MHCIIlo HSCs displayed increased megakaryocytic potential and were preferentially expanded in CALR mutant mice with thrombocytosis. Similar to mice, high MHCII expression is a feature of human HSCs residing in a deeper quiescent state. Our results therefore position STAT1 at the interface of stem cell heterogeneity and the interplay between stem cells and the adaptive immune system, areas of broad interest in the wider stem cell field