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Figure 3. The JAK2V617F-bearing endothelial cells (ECs) protect JAK2V617F hematopoietic stem/progenitor cells (HSPCs) from lethal irradiation. (A and B) After 300cGy irradiation, there were higher total cell numbers (1.6-fold; P=0.026) (A) and hematopoietic progenitors (1.3-fold; P=0.010) (B) from JAK2V617F Lin– HSPCs cultured on JAK2V617F-bearing ECs compared to their being cultured on JAK2WT ECs. Cell numbers were shown as the relative ratio compared to JAK2WT Lin– HSPCs cultured on JAK2WT ECs under the same conditions. Data are from 3 independent experiments. (C) Outline of experiment design to generate a chimeric murine model with JAK2WT HSPCs and mutant vascular niche which is then subjected to sublethal irradiation. (D) CD45.1 WT Lin– HSPC cell apoptosis in WT or JAK2V617F-mutant vascular niche after 300cGy irradiation (n=4 in each group).
tured on primary EC feeder layers derived from WT or Tie2/FF1 (JAK2V617F) murine lungs. The Lin- HSPC-EC co-cultures were irradiated with 300cGy ex vivo and cell number was counted within 24 hours of irradiation. We observed higher total cell numbers (1.6-fold; P=0.026) and hematopoietic progenitors (1.3-fold; P=0.010) from JAK2V617F HSPCs cultured on JAK2V617F-bearing ECs compared to their being cultured on JAK2WT ECs, sug- gesting that the JAK2V617F-bearing vascular niche con- tributes directly to JAK2V617F-mutant HSPC radioprotec- tion (Figure 3A and B). No significant difference was observed in cell numbers or hematopoietic progenitors between JAK2WT HSPCs cultured on JAK2V617F-bearing ECs and their being cultured on JAK2WT ECs in vitro after irradiation.
To further investigate the effects of JAK2V617F-bearing vascular niche on the response of HSPCs to radiation injury in vivo, we generated a chimeric murine model with WT HSPCs and JAK2V617F-bearing vascular niche by transplanting WT marrow cells (CD45.1) into lethally irra- diated (950cGy) Tie2/FF1 recipients (CD45.2). The trans- plantation of CD45.1 WT marrow cells into CD45.2 WT recipients served as a control (Figure 3C). Based on our observation that Tie2/FF1 recipients did not develop any significant recovery of the JAK2V617F-mutant hematopoiesis until ten weeks after transplantation (Figure 1B), each set of chimeric mice were irradiated with 300cGy at 6-10 weeks following transplantation to create
a radiation injury. Two hours later, marrow Lin– HSPCs were isolated for evaluation of cellular apoptosis. We found that the WT Lin- HSPC cell apoptosis was decreased in the JAK2V617F-mutant vascular niche com- pared to WT vascular niche (12.7% vs. 19.7%; P=0.034) (Figure 3D). Taken together, these data suggest that the JAK2V617F-bearing vascular niche contributes directly to HSPC radioprotection.
In contrast to our observations in vivo, where JAK2V617F-mutant HSPCs had increased apoptosis com- pared to JAK2WT HSPCs in the WT vascular niche (Figure 2A and B), cell numbers or progenitor numbers from the JAK2V617F HSPCs and JAK2WT HSPCs were similar when cultured on JAK2WT ECs (Figure 3A and B). Similarly, while the JAK2V617F-bearing vascular niche is protective for WT HSPCs in vivo (Figure 3C and D), JAK2V617F EC did not significantly protect WT HSPC in vitro (Figure 3A and B). These results are likely due to the different cell-cell interactions and niche factors between the in vitro culture condition and in vivo microenvironment.
The JAK2V617F mutation alters vascular niche function to contribute to HSPC radioprotection
Next, we investigated how the JAK2V617F mutation alters EC function in the vascular niche to protect HSPCs from radiation injury. In our previous studies, we found that JAK2V617F-bearing ECs proliferate to a greater extent than JAK2WT ECs and display significantly increased
haematologica | 2018; 103(7)