EHA Library - The official digital education library of European Hematology Association (EHA)

Abstract: S125

Type: Oral Presentation

Presentation during EHA21: On Friday, June 10, 2016 from 12:00 - 12:15

Location: Hall C13

Background
Poor graft function is an important, often fatal complication following allogeneic hematopoietic stem cell transplantation. However, the underlying mechanism is unclear. In murine study, effective cross-talk between hematopoietic stem cells (HSCs) and the bone marrow microenvironment is important for normal hematopoiesis. Normal HSCs reside in a hypoxic bone marrow microenvironment protecting them from oxidative stress which would otherwise inhibit self-renewal resulting in bone marrow failure. We recently reported an impaired bone marrow endosteal and vascular microenvironment in subjects with poor graft function posttransplant. However, whether an increased level of reactive oxygen species (ROS) causes poor graft function following allotransplant remains to be elucidated.

Aims
The aim of our study is to determine whether quantitative and/or functional abnormalities of donor CD34⁺ bone marrow cells result in poor graft function post-allotransplant.

Methods
 In the current prospective case-pair study,apoptosis, cell-cycle state and colony forming capacity of CD34⁺ bone marrow cells were quantified in subjects with poor or good graft function post-allotransplant. Moreover, expression of intracellular proteins including ROS, γ-H2AX, p53, phospho-p53, p21, phospho-p38, caspase-3 and caspase-9 were analyzed by flow cytometry. To study the effect of oxidative stress on post-allotransplant hematopoiesis, CD34⁺ cells from subjects with good graft function were treated of H₂O₂ with and without N-acetyl-L-cysteine. Subsequently, the hematopoietic reconstituting activity of the donor CD34⁺ bone marrow was evaluated using a NOD-Prkdc^scidIL2rg^null xenograft assay by intra-bone marrow injection.

Results
Increased levels of ROS were identified in CD34⁺ bone marrow cells in transplant recipients with poor graft function. This increase in ROS levels was associated with an elevated frequency of DNA double-strand breaks, apoptosis, exhaustion of quiescent CD34⁺ cells and defective colony-forming unit plating efficiency, particularly in the CD34⁺CD38^- fraction. Up-regulated intracellular p53, phospho-p53, p21, caspase-3 and caspase-9 levels (but not phospho-p38) were detected in CD34⁺ cells, particularly in the CD34⁺CD38^- fraction. To further study the potential role of ROS levels in post-transplant hematopoiesis, CD34⁺ bone marrow cells from subjects with good graft function and normals were treated with H₂O_2. Treatment with H₂O₂ increased ROS levels, resulting in defective CD34⁺ cells, an effect partially reversed by N-acetyl-L-cysteine. Moreover, CD34⁺ bone marrow cells from the donors to subjects with poor or good graft function exhibited comparable hematopoietic reconstitution capacities in the xeno-transplanted NOD-Prkdc^scidIL2rg^null mice.

Conclusion
Even if the transplanted donor’s bone marrow CD34⁺ cells are functionally normal pre-transplant, ROS-induced apoptosis may contribute to the exhaustion of CD34⁺ bone marrow cells in subjects with poor graft function following allotransplant. This effect could be partially reversed by N-acetyl-L-cystine. Thus, our data suggest a potential approach for treating poor graft function post-allotransplant.

Session topic: Stem cell transplantation - Experimental

Keyword(s): Allogeneic hematopoietic stem cell transplant, Hematopoietic stem and progenitor cells, Reactive oxygen species