Preliminary Study On Biological And Genetical Characteristics Of Bone Marrow Mesenchymal Stem Cells In Myeloid Neoplasms

Objective:To investigate the changes of the bone marrow microenvironment(BMM)of myeloid neoplasms by studying the biological and genetic characteristics of myeloid mesenchymal stem cells(MSC)in acute myeloid leukemia(AML),myelodysplastic syndrome(MDS)and chronic myeloid leukemia(CML).Methods:(1)Isolation and identification of MSC:First,mononuclear cells(MNC)were isolated by Ficoll lymphocyte separation fluid,then the suspension cells were removed and adherent cells were left.Then flow cytometry,induced differentiation and fibroblast colony-forming unit assay were used to identify MSC.(2)Analysis of biological characteristics of MSC:Cell proliferative ability of MSC was assessed by CCK8 assay and fibroblast colony-forming unit assay.The percentage of senescence MSC was evaluated by SA-β-gal staining assay.Long-term culture-initiating cell(LTC-IC)assay was performed to explore the capacity of hematopoietic support.(3)Analysis of genetic characteristics of MSC:Conventional cytogenetic analysis(CCA)and fluorescent in situ hybridization(FISH)were used to detect the cytogenetics of hematopoietic cells(HC)and MSC.MDS kit probe and bcr-abl probe were respectively used to detect HC and MSC of patients with MDS and CML.Second-generation sequencing technology was used to detect gene mutations of HC and MSC of patients with AML and MDS.Results:1.Isolation and identification of MSC:The adherent cells in passage 3 were used for identification.(1)The flow cytometry results showed these adherent cells were absent of specific antigens of HC,such as CD34 and CD45,but positive for CD73,CD90 and CD105.(2)Differentiation assay showed that these adherent cells were able to differentiate into osteoblasts,adipocytes and chondrocytes,which indicated these cells had the potential of multi-directional differentiation.(3)Fibroblast colony-forming unit assay showed that the single adherent cell were able to form a visible colony,which indicated that the adherent cells with self-renewing stem cell characteristics.The above results are all in accordance with the minimum identification criteria of MSC established by the International Society for Cellular Therapy(ISCT).Therefore,these adherent cells can be considered as MSC.2.The biological characteristics of MSC:The MSC of myeloid neoplasms group(composed of AML,MDS and CML patients)and healthy donor(HD)control group showed differences in proliferation ability,percentage of senescent cells and hematopoietic supporting ability.The results of CCK8 assay showed that the proliferation rate of myeloid neoplasms group was significantly lower than that of the HD control group from the 3rd to 4th day.However,no significant differences among the groups of myeloid neoplasms.Similarly,fibroblast colony-forming unit assay also showed that the proliferative capacity of myeloid neoplasms group was significantly lower than that of HD group,but no significant differences among the groups of myeloid neoplasms.The numbers of colony-forming unit of HD,AML,MDS and CML were 13.55±2.73,9.73±2.53,7.75±1.91 and 7.60±3.06.The results of SA-β-Gal staining showed that the percentage of senescent MSC of AML,MDS and CML were 56.38±6.23%,53.75±14.66%and 60.17±6.52%,which was significantly higher than that of HD group(23.14±4.18%).MSC of myeloid neoplasms group were more susceptible to aging than HD-MSC.LTC-IC assay was implemented to evaluate hematopoietic support capacity of MSC.Compared with the HD group,the number of colonies formed by HD-CD34~+HSC after long-term co-culture with MSC of myeloid neoplasms group was significantly decreased.The number of LTC-IC of myeloid neoplasms(the sum of CFU-GM,CFU-E and BFU-E colonies)was about 1/3 of that in HD group,which indicated that hematopoietic support ability of MSC was impaired in myeloid neoplasms group.No significant differences among the groups of myeloid neoplasms.3.The genetic characteristics of MSC:In order to explore the genetic characteristics of MSC and HC in myeloid neoplasms,cytogenetics and hotspot gene mutation of MSC and HC in patients with AML,MDS and CML were detected.Among29 patients with AML,abnormal karyotype was found in HC of 16 cases by CCA,but no cytogenetic abnormalities were found in MSC.Using combination of CCA and FISH,we detected MSC and HC of 37 MDS patients and 15 CML patients.Among them,34cases of MDS-HC were successfully analyzed,of which 16 cases were detected of abnormal karyotype,but no cytogenetic abnormalities of MSC were found.10 cases of MDS-HC were found 5q-,7q-and+8 and other sites of change by FISH,but the results of MSC were also negative.Philadelphia chromosome and bcr-abl fusion gene were found in all CML-HC,but in none of MSC.Next-generation sequencing(NGS)was used to detect the gene mutations in HC and MSC of patients with AML and MDS.Several gene mutations were found in HC of patients with AML and MDS,however,only one sample of AML-MSC were detected AXSL1 mutations.Conclusion:The method we used is reliable to isolate MSC.MSC in this study possessed stem cell characteristics which comply with the minimum identification criteria defined MSC by ISCT.MSC of myeloid neoplasms patients showed decreased cell growth and impaired hematopoietic support capacity,as well as more susceptible to aging.Genetic characteristics was no correlation between MSC and HC.