Investigation On The Abnormal Osteogenic Differentiation Of Bone Marrow Mesenchymal Stromal Cells In Myelodysplastic Syndromes

Objectives: The aim of this study was to investigate the role and mechanism of Notch signaling in the abnormal osteogenic differentiation of bone marrow derived mesenchymal stromal cells(MSC)in patients with myelodysplastic syndrome(MDS),and to investigate the levels of hyaluronan(HA)in the bone marrow of MDS patients and the role of HA in osteogenic differentiation of MDS-MSC.Methods: MSC was cultured and amplified in vitro,and the expression of Notch ligand(JAG1-2?DLL1,3,4),receptor(Notch1-4)and Notch signal downstream genes(Hes1,Hes5,Hey1,Hey2 and Hey L)in MDS-MSC was detected by realtime-PCR before osteogenic differentiation,and the expression of Notch signal downstream genes in MDS-MSC was detected by realtime-PCR at Day 7,14 and 21 of osteogenic differentiation.We used DAPT,a ?-secretase inhibitor,to treat MDS-MSC or transfected normal MSC with adenovirus carrying Notch intracellular domain(NICD).Then realtime PCR and Western Blot were used to detect Notch signal downstream genes expression,ALP activity was measured at Day3 of osteogenetic differentiation,Alizarin red staining was detected at Day21 of osteogenic differentiation,realtime PCR and Western Blot were used to measure the osteogenic differentiation related gene expression(RUNX2,ALP and OCN)at Day21 of osteogenic differentiation.The HA content in MDS bone marrow serum and MSC culture medium supernatant was detected by radioimmunoassay.Realtime PCR was used to detect the expression of hyaluronan synthetase(HAS1-3)in MDS-MSC.We added exogenous HA to treat MDS-MSC or added 4MU,a HA synthetase inhibitor,to treat normal MSC.ALP activity was measured at Day3 of osteogenic differentiation,Alizarin red staining was detected at Day21 of osteogenic differentiation,realtime PCR and Western Blot were used to measure the osteogenic differentiation related gene expression(RUNX2,ALP and OCN)at Day21 of osteogenic differentiation.Results: The expression of Notch ligand(JAG1,DLL1),Notch receptor(Notch1,2)and Notch signal downstream genes(Hes1,Hes5)in MDS-MSC was increased.During osteogenic differentiation,the expression of Notch signal downstream genes in normal and higher-risk MDS-MSC was decreased significantly,while the expression of Hes1 and Hes5 in lower-risk MDS-MSC was continued to be activated.DAPT could improve the osteogenic differentiation ability of lower-risk MDS-MSC,while overexpression of NICD could inhibit the osteogenic differentiation ability of normal MSC.In addition,the BM serum HA levels were higher in higher-risk MDS patients.Patients with high levels of BM serum HA had worse prognosis.The HA levels were elevated in culture medium supernatants from higher-risk MDS-MSC.The expression of HAS2 was increased in higher-risk MDS-MSC.Exogenous addition of HA could promote osteogenic differentiation of MSC.The addition of4 MU inhibited normal osteogenic differentiation of MSC.Conclusion: In summary,our datas demonstrated that activation of Notch signal pathway is involved in the impaired osteogenic differentiation of lower-risk MDS-MSC.Higher-risk MDS patients has increased BM serum levels of HA.HA could promote osteogenic differentiation of MSC.