| Objective: Bone marrow mesenchymal stromal cells(BM-MSCs)are essential structural and functional components of hematopoietic microenviroment and play an important role in hematopoietic stem and progenitor cell(HSPCs)proliferation and differentiation.However,an increasing number of studies have reported that BM-MSCs can support leukemia cells(LCs)survival,induce LCs homing and chemoresistance.Recent researches revealed that MSCs isolated from acute myeloid leukemia(AML)patients(AML-MSCs)show distinct gene expression and biological signatures from healthy donor derived BM-MSCs(HD-MSCs).However,the exact abnormalities of AML-MSCs and the origin of these abnormalities are still unknown.To evaluate the influence of leukemia cells(LCs)on MSCs,we cultured the two cell types using an in vitro co-culture system and compared the gene expression profile as well as the biological functions of different MSCs.Methods: Part 1.The proliferative activity and its influence factors of bone marrow MSCs derived from AML patients.AML-MSCs(n=6)and HD-MSCs(n=6)were separated and expanded in vitro.Cell surface markers,such as CD105,CD29,CD44,CD45,CD34,and human leukocyte antigen(HLA)-DR were detected using flow cytometry.Proliferative acivity of each MSCs were envaluted by 80% confluence time and their correlation with patients age and primary blast counts were analyzed using Pearson correlative analysis.To further evaluate the influence of LCs on HD-MSC proliferation,AML cell lines THP-1 and UT-7 were co-cultured with HD-MSCs in three different ways:(1)direct-contact(DC): AML cell lines were seeded on the HD-MSC layer directly;(2)Transwell system(TS): AML cell lines were seeded to the upper chamber of the Transwell insert;(3)conditioned culture medium(CM): the culture supernatants of AML cell lines were added to the HD-MSC culture system.After co-culture for 3 days,the LCs were removed and the MSCs were digested for cell counting.Part 2.The influence of different MSCs on proliferation and percentage of ALDH+ cells in AML cell lines.AML cell line UT-7、NB4、THP-1、Dami and Kasumi-1 were co-cultured with AML-MSC or HD-MSCs in direct contact system.Their corresponding cultures alone were set as control.Cell counting were performed at day 0,2,4,6 respectively,and the percents of ALDH+ cells in each cell line before and after co-culture for 6 days were detected using flow cytometry.Part 3.Influence of LCs on gene expression profile and function of bone marrow MSCs.THP-1 were added to HD-MSCs culture system to get LCs treated HD-MSCs(LCtr HD-MSCs),which were used to envalute the influence of LCs on MSCs.HD-MSCs cultured alone or together with THP-1 were digested following washed thoroughly to remove THP-1 and collected for transcriptome sequencing.Total RNA was extracted and purified before fragmented into small pieces.After construct a c DNA library,sequencing was performed on a BGISEQ500 platform(BGI,Shenzhen,China).After filtered,the clean reads were then mapped to the genome database and Bowtie2 were used to align the clean reads to the reference sequence.RSEM was used to calculate the number of reads mapping to genes.Differentially expressed genes between the 2 groups were screened using the criteria that log2 fold change must be ≥ 1 or ≤-1,p value must be ≤ 0.001,and the false discovery rate(FDR)must be ≤ 0.001.GO analysis and KEGG pathway analysis were used to envaluate the function of differently expressed genes.Further more,the functions,including support LCs proliferation,anti-AML cell apoptosis,LCs chemotaxis,and promote LCs chemoresistance of different MSCs were envaluated using flow cytometry,chemotaxis assay and CCK-8(Cell Counting Kit 8)assay.Results: Part 1.The proliferative activity and its influence factors of bone marrow MSCs derived from AML patients.Both AML-MSCs and HD-MSCs displayed similar spindle-shaped fibroblast like morphologies,expressed typical MSC markers(CD105+CD29+CD44+)and were negative for hematopoietic markers(CD45-CD34-HLA-DR-)in a comparable way.During MSC expansion,we found that AML-MSCs showed decreased proliferative activity.The adherent cells in primary AML-MSC cultures were significantly less than those in HD-MSCs cultures at the same culture time.AML-MSCs needed more days to reach 80% confluency than HD-MSCs.This difference declined with prolonging of the in vitro culture time.When cultured at passage 3,no significant difference were found between the two groups.Pearson correlative analysis showed a significant negative correlation between the proliferative avtivity and primary leukemic blast cell count but not patients’ age.These results indicate that the decreased proliferative activity may be induced by AML cells but is not related to patients’ age.To further evaluate the influence of LCs on MSC proliferation,we co-cultured AML cell lines THP-1 and UT-7 with HD-MSCs in direct-contact(DC),Transwell system(TS),or conditioned culture medium(CM).After co-culture for 3 days,we found that cell numbers were comparable in HD-MSCs cultured alone,in TS,and in CM,but were significantly reduced in DC.Thus,neither TS nor CM inhibited the proliferation of HD-MSCs.Part 2.The influence of different MSCs on proliferation and percentage of ALDH+ cells in AML cell lines.Both AML-MSCs and HD-MSCs promote the proliferation and survival of THP-1、NB4、Dami and Kasumi-1,but the difference between these two cell types were not significant.In addiation,neither HD-MSCs nor AML-MSCs increased the ALDH+ cell percent of each cell lines.Part 3.Influence of LCs on gene expression profile and function of bone marrow MSCs.Although IL-6,IL-8 were found to elevated in co-culture supernatant,there are insufficient evidence to confirm the influence of LCs on MSCs.Further transcriptome sequencing analysis showed a significantly different gene expression profile between the HD-MSCs and LCtr HD-MSCs.There were totally 2808 differently expressed genes between the two groups,which were mainly enriched in immune response,inflammatory response,cytokine-mediated signaling pathway,cell-cell signaling,cell cycle,chemotaxis etc.KEGG pathway analysis revealed that the differentially expressed genes were mainly involved in pathways in cancer,calcium signaling pathway,transcriptional mis-regulation in cancer,chemokine signaling pathway,osteoclast differentiation,leukocyte trans endothelial migration and cell cycle etc..Function analysis on different MSCs indicated that LCtr HD-MSCs showed compared anti-LCs apoptosis,chemotaxis,and promote LCs chemoresistance capacity with AML-MSCs.Conclusion: 1.In comparison with HD-MSCs,AML-MSCs showed decreased proliferative activity in vitro,which recovered gradually with prolonging of the in vitro culture time;2.The MSCs proliferative activity is negatively correlated with the primary leukemia blast cell counting but not patients age;3.AML cell line UT-7 and THP-1 inhibit the HD-MSCs proliferation via direct contaction;4.Neither HD-MSC or AML-MSC co-culture promote the ALDH+ cell expansion;5.LCs induce a significantly different gene expression profile in HD-MSCs.Those inflammatory and chemotaxis related genes were elevated after LCs induction;6.LCtr HD-MSCs exhibit improved chemotaxis acitivity,LCs protect ability,and can induce LCs chemoresistance towards Ara-C;... |
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