The Biological Characteristics Of Osteoblasts Derived From Myelodysplastic Syndrome

Objective To study the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro.Methods (1) PartⅠIn order to investigate the abnormal hematopoietic microenvironment in MDS, ELISA was used to detect levels of SDF-1αin bone marrow plasma from 12 MDS patients. (2) PartⅡUsing human fetal osteoblastic cell line 1.19 as a model to study the biological characteristics of osteoblasts, and their capacity in support of hematopoiesis. Flow cytometry was used to identify their immunophenotype,and RT-PCR was used to detect the expression of hematopoietic cytokines.(3) PartⅢTo study the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro. MSCs isolated from bone marrow of MDS patients and normal donors were cultured. Morphology, immunophenotype and colony forming unit fibroblast (CFU-F) of MSC were measured and analyzed. The third generation of cultured MSCs were induced to osteoblasts. After treated with mitomycin C , they became a feeder layer. Ficoll-isolated bone marrow mononuclear cell from normal donors were then seeded on the feeder layer to culture in vitro without exogenous cytokines. RT-PCR was used to determine the expression of hematopoietic cytokines in osteoblasts induced from human marrow mesenchymal stem cells.Results (1) PartⅠThe level of SDF-1αin refractory anemia with excess blasts (RAEB) patients was significant lower than that in refractory anemia (RA) patients , refractory anemia with ring sideroblasts(RAS) patients and controls (P < 0.05). The SDF-1αlevel was negatively correlated with the percentage of BM myeloblasts(r = -0.585, P=0.046).The correlation of SDF-1αlevels with outcomes of International Prognostic Scoring System (IPSS) was also observed(r = -0.657, P=0.02). Whereas, the SDF-1αlevels of MDS patients had no correlation with peripheral white blood cells counts, platelet counts and hemoglobin level (P>0.05).(2) PartⅡFCM revealed that hFOB cells were positive for CD44, CD73(SH3), CD105(SH2) and CD90 (Thy1), but negative for CD34, CD45, HLA-DR. RT-PCR found that hFOB cells expressed the ESC pluripotency marker of Oct-4, Rex-1, except hTERT. Meanwhile, it has also been proved that hFOB cells'capacity of adipogenic differentiation. Moreover, hFOB cells expressed mRNA of SCF , IL-6 , IL-11, SDF-1, GM-CSF and G-CSF. (3) PartⅢMSCs obtained from MDS patients and normal donors were displaying fibroblastoid morphology. Their growth pattern, immunophenotype and colony forming unit fibroblast (CFU-F) number were similar (P >0.05). Without exogenous cytokines, the osteoblasts derived from MDS can sustain GM–CFC survival for at least 3 weeks. The CFU-GM yield in supernatants after co-culture was not different from those of control in hematopoiesis supportive experiments in vitro ( P>0.05). RT-PCR clearly showed that the cultured BM-MSC from normal donor expressed mRNA of SCF, SDF-1, IL-6, and IL-11. As the MSC differentiated toward osteoblasts , the expression of G-CSF could be detected,whereas GM-CSF remained undetectable. The same expression pattern of above cytokines were also seen from osteoblasts induced from BM-MSC of MDS patients.Conclusions (1) The SDF-1/CXCR4 system may play a potential role in the pathogenesis of MDS. Detection of SDF-1αmay be helpful for diagnosis and predicting its prognosis of MDS. In addition, it may provide new clues for the treatment of MDS. (2) Human fetal osteoblastic cell line 1.19 possess the capacity of multilinage differentiation and express many hematopoietic cytokines. In conclusion , osteoblasts may play a pivotal role in hematopoiesis.(3) The biological characteristics of osteoblasts from bone marrow of MDS patients were generally not different from those of osteoblasts in bone marrow of normal controls. Both of them could support GM -CFC hematopoietic progenitor cells survival in vitro, according to their expression of multiple cytokines. In conclusion, the osteoblasts derived from MDS patients may not be involved in the malignant process.