Enhanced Engraftment of Human CD34+ Stem Cells in NOD/SCID Mice by Diprotin A Occurs by Inhibition of Recipient CD26/Dipeptidyl Peptidase-IV (DPP-IV).
Authors: Kawai Toshinao , Choi Uimook , Liu Po-Ching , Malech Harry L. 
Published: October 2019

Abstract
CD26/DPP-IV (CD26) is a membrane-anchored ectoenzyme with N terminus exopeptidase activity that cleaves X-Pro-dipeptides. Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 play a central role in trafficking of hematopoietic stem cells in the bone marrow. SDF-1 has a proline second from the N-terminus and is cleaved by CD26. Mouse hematopoietic progenitors express CD26 and a brief treatment of these cells with Diprotin A (Ile-Pro-Ile), a specific inhibitor of CD26, enhances engraftment. We examined the effect of Diprotin A treatment of peripheral blood human CD34+ stem cells (PBSC) with respect to subsequent responses to SDF-1 and with respect to engraftment in the NOD/SCID mouse xenograft model. We found that human CD34+ PBSC with colony forming potential are unlike mouse hematopoietic stem cells in that they lack the equivalent of CD26; and also are unlike mouse cells in that their response to SDF-1 and their engraftment in the NOD/SCID xenograft model are not affected by pre-treatment with Diprotin A. However, administration of Diprotin A intravenously to the NOD/SCID mouse at the time of transplant of human PBSC greatly enhances engraftment of the human PBSC, suggesting an effect primarily on the mouse stroma. Previous reports suggested that 70% of mouse lineage negative/sca-1 positive hematopoietic stem cells express CD26. However, freshly selected mobilized human CD34+ PBSC do not express detectable CD26, though after 4 days and 7 days of ex vivo culture in growth factors (SCF, flt3-ligand, TPO, IL3) 8.9% and 26.6% of cells express CD26, indicating that CD26 may only appear in later progenitors. At day 4 the cultured human PBSC were sorted by flow cytometry into CD26 positive and negative fractions. Only the CD26 negative fraction contained colony forming cells. 4 day-cultured human PBSC were exposed to Diprotin A 5mM for 15 minutes, washed and used in a filter transwell migration assay in response to SDF-1 at concentrations from 0.5 to 10 nM. There was no statistical difference between migration of Diprotin A treated and control PBSC, even in experiments with longer treatment with Diprotin A. When these human PBSC were transplanted into NOD/SCID mice there was no difference of engraftment between the Diprotin A treated group and control group. However, when 1x106 of 4 day-cultured PBSC were injected into NOD/SCID mice without or together with 2μmol of Diprotin A, there was a profound enhancement on subsequent engraftment in the group of mice injected with Diprotin A at time of transplant. At 6 weeks after transplantation the CD45+ human cell engraftment of the Diprotin A group was 6-fold increased compared to control group (49.6±8.2% vs. 8.1 ± 3.4%, p<.0001). Taken together with the colony assay, the in vitro migration studies, and lack of effect on engraftment when only the human PBSC are treated with Diprotin A, this result suggests that the enhanced engraftment of human PBSC in NOD/SCID mice is due to an action of Diprotin A on endogenous mouse CD26/DPP-IV (where the target is unknown, but possibly stromal cells). Although, further work is required to determine levels of expression of CD26/DPP-IV in human marrow stromal cells, it is possible to speculate that inhibitors of CD26/DPP-IV activity may provide a novel approach to improve stem cell engraftment in humans.