Dipeptidyl Peptidase 4 (DPP4) Is Altered By, and Modulates Sensitivity to, Extra-Physiologic Oxygen Shock Stress (EPHOSS): Implications for Hematopoietic Stem Cell Transplantation

Hematopoietic stem cells (HSC) reside in hypoxic niches (~1-4% O₂), however, HSC studies are consistently performed using cells isolated in ambient air (~20% O₂), regardless of subsequent processing in low oxygen tension. We recently published that by collecting/processing stem cells in physiologically native conditions of hypoxia, with all procedures performed inside a hypoxic chamber (3% O₂), we enhance the recovery of phenotypic, and functional, self-renewing long-term repopulating HSC (LT-HSC) with concomitantly decreasing numbers of progenitor cells. This occurs by inhibiting damage due to brief exposure of mouse bone marrow (BM) or human cord blood (CB) cells to ambient oxygen (a phenomenon we term Extra Physiologic Oxygen Shock/Stress (EPHOSS)) which we, in part, mechanistically linked to mitochondrial permeability transition pore (MPTP), Reactive Oxygen Species (ROS) and cyclophilin D. This data suggests that true numbers of HSCs, and the transplantation potency of BM and CB, have been consistently underestimated due to rapid differentiation of LT-HSCs in ambient air (EPHOSS), but the broad effects of EPHOSS on stem cell phenotype are unknown. We hypothesized that Dipeptidyl Peptidase 4 (DPP4) may be altered by EPHOSS and involved in the effects of EPHOSS on HSC.  We showed that DPP4, a serine peptidase whose enzymatic activity leads to the N terminal cleavage of select penultimate amino acids of proteins, alters homing and engraftment of HSC and the number of cytokines, chemokines and growth factors that have putative DPP4 truncation sites have been dramatically underestimated. Functional and mechanistic roles of full length (FL) versus DPP4 truncated (T) factors, the ability of DPP4 T proteins to induce signaling that FL factors cannot, and the effects of EPHOSS on DPP4 expression/activity, and vice versa, have not been investigated and may have yet unappreciated clinical application. Here we present novel data demonstrating that mouse bone marrow harvested in air in the presence of Diprotin A, a DPP4 inhibitor, or from DPP4 K/O mice, results in a significant increase in the number of phenotypic LT-HSC (p=.017), suggesting that inhibition of DPP4 can diminish the loss of phenotypic LT-HSC due to EPHOSS. Further, the percentage of DPP4+ cells is significantly increased in primitive fractions of mouse bone marrow and human cord blood (LSK ~15% DPP4+, LSKCD150+ ~40%DPP4+, CD34+CD38- of CB ~10% DPP4+, CD34+CD38-CD45RA-CD90+CD49F+ ~40% DPP4+, p=.007), the numbers of DPP4+ cells are additionally enhanced 15- 20% when cells (BM and hCB) are isolated in hypoxia, especially in the LT-HSC fraction (Air 40% DPP4+ Hypoxia 60% DPP4+, p=.005). However, DPP4 activity on lineage- bone marrow harvested in hypoxia showed a 2 fold decrease (p=.005) compared to lineage- cells harvested in air. Interestingly, this increase in the number of DPP4+ cells in hypoxia is not recapitulated when mouse BM is harvested in the presence a Cyclosporin A, a cylophilin D inhibitor, (even though the increase in numbers of LT-HSC is preserved similarly to that in hypoxia) suggesting an alternative mechanism for modulation of DPP4 other than inhibition of mitochondrial ROS/MPTP.  Unexpectedly, LT-HSC ROS levels (both mitochondrial and total) were not diminished in groups with decreased DPP4 activity (DPA or DPP4 K/O) harvested in air despite the blunting of EPHOSS leading to maintenance of the phenotypic LT-HSC increase over air harvest alone. These data suggest that pathways in addition to ROS, such as DPP4 expression/activity, may be influencing LT-HSC function after, and sensitivity to, EPHOSS as well as being modulated by EPHOSS. Further investigation of these collaborative pathways may facilitate increased HSC collections to enhance HSC transplantation.