N-Acetyl-L-Cysteine Improves Mesenchymal Stem Cell Function in Prolonged Isolated Thrombocytopenia Post-Allotransplant

Background: Prolonged isolated thrombocytopenia (PT), is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and defined as the engraftment of all peripheral blood cell lines other than a platelet (PLT) count ≤20×10E+9/L or dependence on PLT transfusions for more than 60 days after allo-HSCT. Several clinical risk factors have been proposed to be associated with PT after allo-HSCT. However, the underlying mechanisms remain to be elucidated. The processes of megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, cytokines, and bone marrow (BM) stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. However, little is known about the quantity and function of BM MSCs and how to improve the impaired BM MSCs in PT patients.

Aims

To evaluated the number and function of BM MSCs in patients with PT post-allotransplant. Moreover, to investigate the approach to enhance the number and function of BM MSCs derived from patients with PT and its underlying molecular mechanisms in vitro.

Methods

Three cohorts were included: patients with PT (N=83), patients with good graft function (GGF, N=32), defined as persistent successful engraftment after allotransplant, and transplant donors as normal controls (N=15). The number and functions of BM MSCs were evaluated by fibroblasts colony-forming unit (CFU-F) assay, cell proliferation assay and senescence-associated β-galactosidase (SAβ-gal) assay. Reactive oxygen species (ROS) levels and protein expression for p-p38, p38, p-p53, p53 was measured by flow cytometry. The number of megakaryocyte (MK), MK apoptosis, MK ploidy distribution and platelet release values after coculture were assessed by flow cytometry. To further investigate the potential effect for repairing the dysfunctional BM MSCs, N-Acetyl-L-cysteine (NAC,a ROS scavenger), SB203580 (p38 inhibitor) and PFT-a (p53 inhibitor) were administrated to the BM MSCs from PT patients. After 2 days in vitro culture, the percentage of SAβ-positive cells, the intracellular levels of ROS, p-p38, p-p53, the number and the function of MK were evaluated. For evaluation of the efficacy and safety of NAC in PT patients, 20 patients with PT were subsequently enrolled to a single-center pilot study.

Results

Human BM MSCs were demonstrated as spindle shape and typical immunophenotype of MSCs at day 21 of cultivation among subjects with PT, GGF and normal controls. 7 of the 32 GGF BM and 58 of the 83 PT BM failed to produce any adherent layers within 3 weeks of culture. BM MSCs derived from PT patients expanded more slowly and appeared flattened and larger. Proliferative capacity and CFU-F counts of BM MSCs from PT patients were significantly reduced compared to those of GGF patients and normal controls. Moreover, increased levels of ROS, which was associated with increased number of SAβ-positive cells, were identified in BM MSCs from PT patients. Intracellular p-p38 and p-p53 levels were significantly elevated in PT patients compared to those in GGF patients. Moreover, the percentages of MK were lower in CD34⁺ cells cocultured with PT-MSCs compared to those observed with GGF-MSCs. CFU-MKs counts were also lower in the PT-MSCs group. After NAC treatment in vitro, the proliferative capacity was increased significantly, whereas the number of senescent cells, the intracellular levels of ROS, p-p38 and p-p53 were reduced markedly in BM MSCs from PT patients. NAC exposure also improved the ability of BM MSCs from PT patients to support megakaryocytopoiesis. The preliminary clinical data of a single-center pilot study showed the complete response, response, and overall response rates at 2 months after NAC treatment (400 mg three times a day) were 10% (2/20), 55% (11/20) and 65% (13/20), respectively, and no apparent drug-associated side effects were observed.

Summary/Conclusion

In summary, the current study demonstrated the abnormalities of BM MSCs contribute to the pathogenesis of PT following allo-HSCT. Moreover, BM MSCs derived from PT patients exhibited quantitative and functional improvement following in vitro treatment with NAC. Although these findings require further validation, our in vitro data and preliminary clinical data suggested NAC is an effective and safe treatment option for PT patients after allo-HSCT.