Inhibiting CD26 activity improves the migration of Flk2⁻CD34⁻ LT-HSCs and Flk2⁻CD34⁺ ST-HSCs in vitro. (A) Representative flow cytometric contour plots (n = 3 independent experiments) illustrating CD26 expression on Flk2⁻CD34⁻ LT-HSCs and Flk2⁻CD34⁺ ST-HSCs isolated from the BM of C57BL/6. (B) Data from (A) is presented as percent (%) positive cells ± SEM as compared with FMO control. The statistical analysis was performed with the unpaired t test (**P = .001). (C) RT-PCR was used to determine the expression of CD26 in the Flk2⁻CD34⁻ LT-HSCs and Flk2⁻CD34⁺ ST-HSCs. Results were obtained from n = 3 independent experiments and analyzed using the unpaired t test (*P = .03) and presented as mean ± SEM. (D) The activity of CD26 peptidase in both HSC populations before and after CD26 inhibition with Dip A was assessed from n = 3 independent experiments (*P = .03 and ***P < .001). (E) A transwell migration assay was used to assess the migration of both HSC populations toward SDF-1α with (+) and without (−) the presence of Dip A. Data were normalized by subtracting the percent migration of untreated cells without SDF-1α. Each sample was performed in triplicate, and n = 3 independent experiments were performed.