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JAK2V617F-bearing vascular niche in MPNs
colony-forming units; CFU-ECs or Hill) and, in some reports, in the true endothelial colony-forming cells (ECFC) based on in vitro assays.13-17 JAK2V617F mutation is also present in isolated liver or spleen ECs from patients with MPNs.15,18 Previously, we and others have shown that JAK2V617F-bearing ECs are critical in the development of the bleeding abnormalities in a murine model of JAK2V617F-positive MPNs in which JAK2V617F is expressed in all hematopoietic cells and endothelial cells.19 In addition, we have reported that the JAK2V617F -bear- ing vascular niche promotes the expansion of the JAK2V617F HSPCs in preference to JAK2WT HSPCs.20,21 All of these observations suggest that ECs are involved in the pathogenesis of MPNs.
In the present study, using the hematopoietic and endothelial specific Tie2-Cre system and different marrow transplantation models, we demonstrate that JAK2V617F-mutant HSPCs are relatively protected by the JAK2V617F-bearing vascular niche from the otherwise lethal irradiation administered during conditioning for marrow transplantation. Taken together, our studies indi- cate that the mutant vascular niche could contribute to the poor donor cell engraftment and the high incidence of dis- ease relapse well known to occur in patients with MPNs after allogeneic SCT. Therefore, targeting the altered hematopoietic vascular niche could provide more effective therapies for patients with MPNs.
Methods
Experimental mice
JAK2V617F Flip-Flop (FF1) mice22 were provided by Radek Skoda (University Hospital, Basal, Switzerland) and Tie2-Cre mice23 by Mark Ginsberg (University of California, San Diego, USA). The FF1 mice were crossed with Tie2-Cre mice to express JAK2V617F specifically in hematopoietic cells and ECs (Tie2/FF1 mice). All mice used were crossed onto a C57BL/6 background and were bred in a pathogen-free mouse facility at Stony Brook University. CD45.1+ congenic mice (SJL) were purchased from Taconic Inc. (Albany, NY, USA). Animal experiments were per- formed in accordance with the guidelines provided by the Institutional Animal Care and Use Committee.
Stem cell transplantation assays
The effects of the JAK2V617F-bearing vascular niche on MPN hematopoiesis were studied in vivo using marrow transplantation assays. First, we transplanted wild-type (WT) CD45.1 marrow cells into lethally irradiated (950cGy)24,25 8-14-week old Tie2/FF1 mice or WT controls (CD45.2). Peripheral blood was obtained every four weeks after transplantation, and CD45.1 donor chimerism and complete blood counts were measured.
To study the effects of HSPC JAK2V617F mutation on HSPC radioprotection, we generated a chimeric murine model with JAK2V617F-mutant HSPCs and WT vascular niche by transplant- ing JAK2V617F marrow cells (CD45.2) into lethally irradiated (950cGy) WT recipients (CD45.1). The transplantation of CD45.2 WT marrow cells into CD45.1 WT recipients served as a control. Following hematopoietic recovery and full donor cell engraftment, each set of mice were irradiated with 300cGy to create a radiation injury. Two hours later, marrow Lineageneg (Lin-) HSPCs were isolated using Lineage Cell Depletion Kit (Miltenyi Biotec, San Diego, CA, USA) for evaluation of cellular apoptosis and cell cycle status. For competitive marrow transplantation experiments, 5x105 post-irradiated marrow cells (CD45.2) were injected intra-
venously together with 1x105 competitor CD45.1 WT marrow cells into lethally irradiated (950 cGy) CD45.1 recipients. Peripheral blood was obtained every four weeks after transplanta- tion, and CD45.2 chimerism was measured.
To study the effects of EC JAK2V617F mutation on HSPC radio- protection, we generated a chimeric murine model with WT HSPCs and JAK2V617F-bearing vascular niche by transplanting WT marrow cells (CD45.1) into lethally irradiated (950cGy) Tie2/FF1 recipients (CD45.2). The transplantation of CD45.1 WT marrow cells into CD45.2 WT recipients served as a control. Following hematopoietic recovery and full donor cell engraftment, each set of mice were irradiated with 300cGy to create a radiation injury. Two hours later, marrow Lineageneg (Lin-) HSPCs were isolated for evaluation of cellular apoptosis.
Additional details of the methods used can be found in the
Online Supplementary Methods.
Results
Expression of JAK2V617F in Tie2+ cells protects marrow HSPCs from lethal irradiation
Mice expressing Cre under the control of the Tie2 pro- moter (Tie2-Cre) were crossed with JAK2V617F Flip-Flop (FF1) mice to generate mice bearing human JAK2V617F expression specifically in endothelial and hematopoietic cells (Tie2/FF1). The Tie2/FF1 mice develop an MPN-like phenotype with neutrophilia, thrombocytosis, significant splenomegaly, and greatly increased marrow vascular den- sity, megakaryopoiesis, and numbers of HSPCs.19,20
To investigate the effects of the JAK2V617F-bearing vas- cular niche on MPN hematopoiesis in vivo, WT CD45.1 marrow cells were transplanted directly into lethally irra- diated (950cGy) Tie2/FF1 mice or age-matched littermate control mice (CD45.2) (n=12 in each group) (Figure 1A). During a 3-month follow up, while all WT control recipi- ents displayed full donor engraftment, 7 of 12 (approx. 60%) Tie2/FF1 recipient mice displayed recovery of JAK2V617F-mutant hematopoiesis (mixed donor/recipi- ent chimerism) ten weeks after transplantation (Figure 1B).
We followed some of the Tie2/FF1 and WT control recipients (n=7 in each group) for more than eight months. In contrast to the Tie2/FF1 recipients with full donor engraftment, the mixed chimeric mice developed neu- trophilia, thrombocytosis, and splenomegaly (Figure 1C and D), similar to what has been observed in the primary Tie2/FF1 mice.19,26 Flow cytometry analysis revealed that JAK2V617F-mutant CD45.2+EPCR+CD48–CD150+ (E- SLAM) cells, which is a highly purified long-term repopu- lating HSPC population in normal and in MPN marrow,27,28 are significantly expanded in the mixed chimeric mice compared to Tie2/FF1 recipients with full donor engraft- ment or WT recipients (Figure 1E).
In virtually all our transplantation experiments per- formed over the past four years, we have used 950cGy radiation24 and have seen virtually 100% donor engraft- ment in every recipient. In contrast, 7 of 12 mice in our Tie2/FF1 recipients of normal marrow demonstrated mixed chimerism with an average of 23% recipient cells in peripheral blood at fourteen weeks following 950cGy irra- diation and marrow transplantation, and developed an MPN phenotype resembling the primary Tie2/FF1 mice during more than eight months of follow up. These find- ings suggest that the JAK2V617F-mutant HSPCs in Tie2/FF1 mice are relatively protected from the otherwise
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