Transient Ex Vivo Inhibition of Heme Oxygenase 1 (HO-1) in Hematopoietic Stem/Progenitor Cells (HSPCs) By Small-Molecule Inhibitors Enhances Their Migratory Responsiveness to Bone Marrow (BM)-Secreted Chemoattractants a Novel and Simple Strategy to Improve Homing of HSPCs

Background. Heme oxygenase 1 (HO-1) is an inducible stress-response enzyme that not only catalyzes the degradation of heme (e.g., released from erythrocytes) but also has an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury. HO-1 has a well-documented anti-inflammatory potential and inhibits complement cascade (ComC)-mediated inflammatory responses. Moreover, HO-1 has been reported to have a negative effect on adhesion and migration of neutrophils in acute inflammation in a model of peritonitis. Radiation chimeras created after transplantation with HSPCs having a mutation in one of the alleles of HO-1 engrafted much faster; however, a persistent decrease in HO-1 activity in these animals resulted in their enhanced sensitivity to stress and susceptibility to irradiation (Blood 2008, 112, 4494-4502). Moreover, we recently demonstrated that HO-1-deficient HSPCs show enhanced in vitro migration up an SDF-1 gradient (Stem Cell Rev & Rep. 2015, 11, 110-118).

Hypothesis. Based on these findings, we hypothesized that transient inhibition of HO-1 by non-toxic, small-molecule inhibitors would enhance in vivo migration of HSPCs to bone marrow (BM)-derived chemoattractants and thus would facilitate their homing and accelerate hematopoietic recovery

Materials and Methods. To address this issue, we first generated several human hematopoietic cell lines in which HO-1 was upregulated or downregulated. We also exposed murine and human BM-derived cells to small-molecule inhibitors or activators of HO-1 and performed dose and timing toxicity studies. Next, murine BM mononuclear cells (MNCs) and human umbilical cord blood (UCB) MNCs were exposed to the small-molecule HO-1 inhibitor Sn(IV) protoporphyrin IX dichloride (SnPP) and tested for their chemotactic response in Transwell migration assays to all currently known HSPC chemoattractants, including stromal-derived factor 1 (SDF-1), sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and the extracellular nucleotides ATP and UTP. For in vivo assays, lethally irradiated mice were transplanted with BM MNCs exposed or not exposed to SnPP, and in recipient animals we evaluated i) the number of day-12 colony-forming units in spleen (CFU-S) and colony-forming units for granulocyte/macrophage (CFU-GM) progenitors in BM and ii) the kinetics of peripheral blood (PB) count recovery by measuring the number of leucocytes, lymphocytes, and platelets. We also performed competitive repopulation studies with a limited number of transplanted BM MNCs using the CD45.1 and CD45.2 congenic mouse models.

Results and Conclusions. We demonstrate here that HO-1 is a negative regulator of HSPC migration, and thus, by transiently inhibiting its activity in HSPCs with the non-toxic small-molecule inhibitor (SnPP), it is possible to accelerate homing and subsequent engraftment of HSPCs. We propose that this simple and inexpensive strategy could be employed in the clinical setting to improve seeding efficiency of transplanted HSPCs and their engraftment, particularly in those situations in which the number of HSPCs available for transplant is limited (e.g., from UCB or grafts harvested from poor mobilizers).