The role of P-selectin in hematopoietic stem cell aging
Hematopoietic stem cells (HSCs) contribute to blood formation while simultaneously sustaining the stem cell pool. Aged HSCs increase in number while the functional activity of aged HSCs decreases substantially compared to their young counterparts, which is associated with myeloid malignancies, immune dysfunction and anemia. In previous studies we have reported that P-selectin (Selp) is the most consistently upregulated gene in murine aged HSCs. However, the role of Selp in HSCs aging is not clear.
In vitro: Colony forming unit assay was used to test clonogenic potential. Liquid culture was used to test HSC differentiation and expansion.
In vivo: The TetOP-H2B-GFP mouse model was used to determine whether Selphigh HSCs are more active or not by measuring GFP level after pulsing. We used competitive bone marrow transplantation models, in which the effects of Selp high-expression on HSCs were tested. We also administered young mice with TNF-α and Il1β to test the expression of Selp by flow cytometry.
We confirmed that Selp is up-regulated with age using flow cytometry on HSCs. We in vivo pulsed HSCs using the TetOP-H2B-GFP mouse model in which quiescent HSCs retain the GFP label after an extended chase period. We found that label-retaining GFPhigh HSCs are Selplow, while more active GFPlow HSCs are Selphigh, suggesting Selplow HSCs are more quiescent. Colony forming unit assays suggested Selphigh HSCs have decrease proliferative potential. Furthermore, Selphigh HSCs showed impaired differentiation when exposed to inflammatory stimuli in liquid culture.
Interestingly, HSC differentiation and expansion were repressed when Selphigh HSCs were treated with Selp ligand (Selplg), indicating that Selp-Selplg signaling is contributing to HSC activity. To further investigate the function of Selp upon aging, we separated Selphigh and Selplow HSCs from aged donor mice and transplanted these into lethally irradiated recipients. Selphigh HSCs showed myeloid bias and reduced secondary transplantation potential compared to Selplow HSCs. To assess whether premature HSC aging could also be induced, we overexpressed Selp in young HSCs and transplanted cells in young recipients. Interestingly, Selp overexpressing HSCs failed to produce erythrocytes in the peripheral blood.
Finally, TNF-α and Il1β in vivo administration induced Selp expression on HSCs, suggesting that elevated inflammatory responses in aged bone marrow is likely the cause of Selp up-regulation and may induce HSCs aging.
In conclusion, our data suggests that differential Selp expression affects HSC behavior and may contribute to HSC aging.