Systmetic Study Of Biological Properties And Functions Of Bone Marrow Mesenchymal Stem Cells From Patients Of Aplastic Anemia

Background:Aplastic anaemia (AA) is mostly considered as an immune-mediated bone marrow failure syndrome, characterized by hypoplasia and pancytopenia with fatty bone marrow and reduced angiogenesis. Previous studies have demonstrated that hematopoietic stem cells (HSCs) from AA patients are defective in multiple biological properties and functions. However, too little is known about bone marrow mesenchymal stem cells (BM-MSCs) from patients with aplastic anemia.Objective:This study aims to assess systematically and thoroughly the biological properties and functions of BM-MSCs from patients with aplastic anemia.Methods:(1) BM-MSCs were isolated and purified from bone marrow by adherent culture and passaging in the culture medium (DMEM/DF12 and 2% fetal bovine serum) in vitro. (2) The morphology of BM-MSCs was stained with (3-tubulin and observed by confocal microscopy. (3) The immunophenotype markers of BM-MSCs were detected using flow cytometry. (4) BM-MSCs were induced to differentiate into adipocytes, osteoblasts and endothelial cells, which were identified with corresponding staining and detection of specific genes. (5) The capacity of forming CFU-F and proliferation of BM-MSCs were detected. (6) The cell cycle and apoptosis of BM-MSCs were detected using flow cytometry. (7) The gene profile of BM-MSCs was identified and compared using Affymetrix method. (8) CD34+ cells were purified from umbilical cord by miniMACS immunomagenetic method and co-cultured with BM-MSCs to assess the hematopoiesis-supportive function of BM-MSCs in the amplification of hematopoietic cells and clonogenic potential of CFUs (CFU-G, CFU-M, CFU-GM, CFU-Mix, BFU-E and CFU-MK). The expression of hematopoietic growth factors such as SCF, IL-3, EPO, TPO, IL-11, G-CSF, M-CSF and GM-CSF mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) method. (9) CD4+ cells were purified from peripheral blood by miniMACS immunomagenetic method and CD4- cells were further purified by adherent culture to remove monocytes, macrophages and dentritic cells. CD4+ cells and CD4- cells were then co-cultured with BM-MSCs to assess the suppression capacity of BM-MSCs in the clonogenic potential and proliferation of lymphocytes and the production of TNF-a and IFN-y by lymphocytes. CD4+ cells were also co-cultured with BM-MSCs to assess the contribution of BM-MSCs in promoting the proliferation of Tregs.Results:(1) We successively isolated and purified BM-MSCs from patients with aplastic anemia. (2) We showed abnormal morphology with irregular appearance using P-tubulin staining, reduced proliferation and clonogenic potential and increased apoptosis of BM-MSCs from AA patients. (3) We demonstrated that BM-MSCs from AA patients were susceptible to be induced to adipocytes but more difficult to osteoblasts and endothelial cells than healthy controls. (4) We also compared the gene expression profile in BM-MSCs from AA patients and healthy controls. A large number of genes associated with hematopoiesis, immune regulation, adipogenesis, apoptosis and cell cycle showed markedly differences between AA patients and healthy controls. (5) Furthermore, we showed that both BM-MSCs from AA patients and healthy controls had the potential of supporting hematopoiesis. But, BM-MSCs from AA patients had reduced potential to amplify CD34+ cells, maintain long-term hematopoiesis. It is more prominent that MSCs from AA patients were markedly defective in supporting the development of megakaryocytes. In addition, we showed that BM-MSCs from AA patients expressed lower levels SCF, TPO and IL-11 mRNA, but higher levels G-CSF and GM-CSF than healthy controls. (6) Finally, we found BM-MSCs from AA were defective in the suppression of proliferation of and clonogenic potential of T cells, TNF-αand IFN-γproduction by CD4+ and CD4- cells.Conclusion:(1) BM-MSCs from AA patients were multiply defective both in biological properties and functions, which might aggravate the bone marrow failure. (2) BM-MSCs from AA were abnormal in morphology and gene profile, defective in clonogenic potential, proliferation and differentiation with increased apoptosis. (3) BM-MSCs from AA were defective in the differentiation into endothelial cells and osteoblasts, which might be the precursor cells in modelling HSC niche. (4) BM-MSCs from AA were defective in maintaining hematopoiesis and supporting the development of megakaryocytes. (5) BM-MSCs from AA were defective in maintaining immune homeostasis. Background:Aplastic anaemia (AA) is considered as an immune-mediated bone marrow failure syndrome. The mechanism is involved with a variety of immune molecules including IFN-y, TNF-a and interleukins (ILs). IL-27 is a novel member of IL-12 family which mediates T cell response and enhances the production of IFN-y. However, little is known about the role of IL-27 in the development of AA.Objective:The present study was aimed to detect the expression of IL-27/IL-27R in bone marrow of patients with aplastic anemia and evaluate the role of IL-27/IL-27R in the pathogenesis of AA.Methods:(1) Bone marrow mononuclear cells (BMMNCs) were isolated from bone marrow using Ficoll-Hypaque gradients centrifugation and cultured in the culture medium (RPMI-1640 and 2% fetal bovine serum) in vitro. (2) The mRNA expression of IL-27 (p28 and EBI3) and IL-27 receptor components (WSX-1/TCCR and gp130) in BMMNCs were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). (3) The intracellular levels of IFN-y and TNF-a in bone marrow T lymphocytes of patients with AA and healthy controls were measured by flow cytometry. (4) The concentrations of IFN-y and TNF-a in the bone marrow plasma and supernatants of BMMNCs from patients with AA were measured by ELISA.Results:(1) We demonstrated that both the mRNA expression of IL-27/IL-27R subunits in the bone marrow mononuclear cells (BMMNCs) and the levels of IL-27 in the marrow plasma in AA were higher than that observed in controls. (2) Increased IL-27 correlated with the disease severity of AA. (3) Subsequently, we stimulated marrow T lymphocytes with rhIL-27 and found that IL-27 dramatically enhanced the production of TNF-a and IFN-y in both CD4+ and CD8+ T lymphocytes from AA patients. (4) We also detected increased TNF-a and IFN-y in the supernatants of BMMNCs from AA patients after IL-27 stimulation.Conclusion:In conclusion, our data suggest that IL-27 might play an important role in the pathogenesis of AA through enhancing the overproduction of IFN-y and TNF-a.