The Effect Of SDF-1/HOXB4Fusion Gene-modified Mesenchymal Stem Cells On The Amplification And Stemness Maintenance Of Hematopoietic Stem Cells In Vitro

ObjectiveTransplantation of human bone mesenchymal stem cells （MSCs） combined withhematopoietic stem cells （HSCs） can promote the hemopoietic reconstitution, but the lowrates of hematopoietic stem cells homing and limited proliferation capacity are majorproblems of transplantation. MSCs, as supporting cells, promoted HSCs homing to the bonemarrow through secreting stromal cell derived factor. Stromal cell-derived factor-1（SDF–1）and its receptor CXCR4play significant roles in promotion of CD34cells homing andproliferation, and hematopoietic reconstitution. Human homeobox B4（HOXB4）, highlyexpressed in hematopoietic stem cells, exerts important effect on the balance betweenself-renewal and differentiation. HOXB4expression can effectively enhance self-renewal ofHSCs. In this study, the adenovirus vectors respectively expressing SDF-1, HOXB4andSDF-1/HOXB4fusion genes were successfully constructed. Virus particles were packaged in293A cells, and the multiplicity of infection （MOI） of the virus supernatants was detected, andthen MSCs were transfected with the adenovirus vectors respectively in vitro. The expressionof these exogenous genes was detected at transcriptional and translational levels. After MSCswere co-cultured with umbilical cord blood CD34⁺cells, the effect of MSCs on theamplification and the stemness maintenance of CD34⁺cells.Research Methods1. The full-length cDNAs of SDF-1, HOXB4and SDF-1/HOXB4genes weresynthesized, and those with correct sequences were used as template for PCR amplification.And then, after DNA fragments were recovered and purified from agarose gel electrophoresis,the PCR products were digested with the restriction enzymes to construct a plasmid vector forDNA sequencing. Then the three DNA fragments with correct sequences as in GenBank were inserted in the expression adenovirus vector to construct the adenovirus expression systemencoding SDF-1, HOXB4and SDF-1/HOXB4genes respectively.2. To amplify the3recombinant adenovirus vectors, they were used to infect293A cellsto generate stocks with higher titer viral. Immunostaining with3,3’-diaminobenzidine （DAB）as selenium organic reagent was used to detect the titer of our adenoviral vectors afterscreening of efficient and stable packaging cells,2times of freeze-thaw cycle, andapplication.3. MSCs were isolated from fresh human bone marrow by density gradientcentrifugation. And after a short time of culture, the obtained MSCs were sorted by flowcytometry with monoclonal antibodies of CD90-FITC, CD45-FITC, CD44-FITC and CD34-PE, which were the hallmarks on the surface of MSCs. Above3recombinant adenovirusvector were used to infect the MSCs to generate the SDF-1transfected MSCs, the HOXB4transfected MSCs, and the SDF-1/HOXB4fusion gene transfected MSCs. The cellstransfected by blank adenovirus vector were used as control. Fluorescence intensity of greenfluorescent protein （GFP） was observed in above4transfected MSCs, and the expression ofthe3exogenous genes were detected in MSCs at mRNA and protein levels.4. CD34⁺cells were sorted from fresh human umbilical cord blood samples by densitygradient centrifugation and magnetic cells sorting system, and identified by flow cytometryand methylcellulose colony assay. At the same time, the4groups of MSCs infected withdifferent adenovirus vectors were prepared as feeder cells after exposure to60Co γ-rayradiation. Then, the MSCs were co-cultured with the CD34⁺stem cells for7d, the cellproliferation and the percentage of CD34⁺cells were measured.Results1. The adenovirus vectors respectively expressing SDF-1, HOXB4and SDF-1/HOXB4genes were successfully constructed. After the viruses were packaged in infect293A cells,the Ad-SDF1-IRES2-EGFP was confirmed with a titer of1.05×10¹¹ifu/mL, theAd-HOXB4-IRES2-EGFP of1.2×10¹¹ifu/mL, and the Ad-SDF-1-（GlySer）₃-HOXB4-IRES-EGFP of1×10¹¹ifu/mL.2. Human MSCs were isolated and cultured successfully. Flow cytometric analysisindicated that the obtained MSCs were91.0%positive to CD90, and97.5%positive to CD44,while0.992%positive to CD34, and0.599%positive to CD45, in accordance with thecharacteristics of MSCs. 3.After the MSCs were transfected with the virus supernatants stably and highlyexpressing the exogenous genes, the3genes were expressed in MSCs successfully. RT-PCRand Western blot analysis indicated that the expression of these3genes was significantlyhigher in the transfected MSCs than in the control group.4. CD34⁺stem cells were sorted successfully from human umbilical cord blood, and thecell purity was increased from2.87%to99.2%after flow cytometric analysis. When thesorted CD34⁺cells were cultured in methylcellulose-based medium for14d, CFU-E, CFU-M,CFU-G and CFU-GEMM were observed one after another, suggesting that the CD34⁺cellshaving good stemness and differentiation capacity after sorting.5. After the CD34⁺stem cells were co-cultured with4groups of MSCs transfected withdifferent adenovirus vectors as feeder cells for7d, the rate of proliferation was significantlyhigher in the SDF-1transfected MSCs （9.52±2.24）, and SDF-1/HOXB4transfected MSCs（8.20±2.34） than in the blank vector transfected MSCs （4.85±2.53）（P<0.05）, but nosignificant difference was seen in that of HOXB4transfected MSCs （5.74±2.43） and theblank vector transfected MSCs （P>0.05）. There was no significant difference in the rate ofproliferation between the SDF-1and SDF-1/HOXB4transfected MSCs, and between theSDF-1/HOXB4and HOXB4transfected MSCs （P>0.05）, but significant difference was seenbetween the SDF-1and HOXB4transfected MSCs （P<0.05）. The percentage of CD34⁺cellswas1.85%in the coculture system with the SDF-1/HOXB4transfected MSCs as feeder cells,which was significantly higher than the system with the blank vector transfected MSCs（0.59%）, the SDF-1transfected MSCs （1.20%）, and the HOXB4transfected MSCs （1.28%）.The right line of fluorescence intensity curve was obviously shifted to right in the coculturesystem with the SDF-1/HOXB4transfected MSCs as feeder cells.ConclusionThree adenovirus expression vectors pAD-SDF-1-IRES-EGFP, pAD-HOXB4-IRES-EGFP,and pAD-SDF-1-（GlySer）₃-HOXB4-IRES-EGFP are constructed successful. The SDF-1/HOXB4fusion gene modified MSCs promote the proliferation and stemness maintenanceof CD34⁺cells in vitro.