Lentiviral-mediated ho-1 gene transduced bone mesenchymal stem cells protects against acute graft-Versus-host disease in vitro and vivo
Authors: Wang Jishi , Ma Dan , Li Yan , Fang Qin , Chen Shuya , Sun Jia 
Published: October 2019

Abstract
Abstract 4675

Objective:
Bone mesenchymal stem cells (BMSCs) possessing immunoregulatory activities have been evaluated in the treatment of graft-versus-host disease (GVHD). In this study, mice's heme oxygenase 1 (HO-1) was transduced into mice's bone marrow-derived mesenchymal stem cells (mBMSCs), we assessed the immuno-suppressive capacity of lentiviral vector transduced BMSCs expressing HO-1 in BALB/c mice aGVHD model, and the immuno-regulatory effect of mBMSCs on alleviating acute GVHD in vivo was measured to provide laboratory data for gene therapy for aGVHD which used mBMSCs as vehicles.

Methods:
We cloned mice's HO-1 cDNA from mice's bone marrow and constructed recombinant lentivirus vectors (Lentivirus-V5-D-TOPO-HO-1-EGFP/Lentivirus–V 5-D-TOPO-EGFP) which titer was 1×1011 pfu/mL. These mBMSCs were separated, cultured, purified, and detected by morphology, flow cytometry, osteogenic, adipogenic and chondrogenic induction, and the mRNA level of the neural ganglioside GD2 gene which is a surface marker for the identification of MSCs by RT-PCR. Then recombinant vectors were transferred into mBMSCs, and the expression of EGFP and HO-1 were detected by fluorescence microscope, RT-PCR and Western blot respectively. The immunsuppressive capacity of HO-1 over-expressing mBMSCs was investigated using transwell assay in vitro. And before infusion, the homing of MSC was detected by Flow cytometry. In addition, we established BALB/c mice's aGVHD model after Allo-HSCT, the recombinant vectors tranfected mBMSCs and primary mBMSCs were injected into the BALB/c mice aGVHD model in tail vein, respectively. Four groups were separated in vivo test (Group A: aGVHD control; Group B: aGVHD model injected in mBMSCs; Group C: aGVHD model injected in mBMSCs transfected with EGFP; Group D: aGVHD model injected in mBMSCs transfected with HO-1 gene.) The survival, body weight and clinical score of GVHD in transplanted mice were monitored to evaluate the severity of aGVHD. The aGVHD targeted organ, such as Liver, intestine and lung from mice in each group were obtained for histological examination and pathological score. Plasma concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, IFN-γ and TNF-γ were also determined using a Cytometric Bead Array.

Results:
We cloned mice HO-1 gene from mice's bone marrow and constructed the recombinant adenovirus vectors (Lentivirus-V5-D-TOPO-HO-1- EGFP/Lentivirus-V5-D-TOPO-EGFP) successfully. mBMSCs was separated and identified successfully. Fluorescence microscope detected the expression of EGFP, while both RT-PCR and Western blot detected high expression of HO-1 in gene-transfected group cells. It clearly showed that mBMSCs in Group D can migrate more through the polycarbonate filter toward bone marrow micro-environment in the lower chamber than Group A, B, C in vitro, while homing of Group D showed similar trend compared with others groups. In the mice GVHD model, treatment with HO-1 over-expressing BMSCs significantly decreased the mortality rate and attenuated clinical and pathological GVHD scores, and volume of spleen was not more obviously enlarged than others groups. Moreover, compared with control groups, the plasma IL-2, IL-6, IFN-γ and TNF-γ levels in recipients infused with HO-1 over-expressing BMSCs were significantly decreased, while those of IL-4 and IL-10 were increased.

Conclusion:
Lentiviral vectors carrying the HO-1 were successfully used to transduce mBMSCs. In our report, mBMSCs transferred with lentiviral vectors expressed strongerly immunoregulatory activities to alleviate aGVHD. Not only did HO-1 enhance that migration of mBMSCs, but also strengthen homing of MSCs. In vivo experiments, the evidence of survival rate, body weight, clinical score and pathological score fully proved that HO-1-transduced BMSCs effectively controlled the occurrence of mice's aGVHD following allogeneic BM transplantation, and HO-1 may be a potential target to overcome aGVHD in vivo.

Disclosures:
Ma: National Natural Science Foundation of China: Research Funding. Li:National Natural Science Foundation of China: Research Funding. Fang:National Natural Science Foundation of China: Research Funding. Chen:National Natural Science Foundation of China: Research Funding. Sun:National Natural Science Foundation of China: Research Funding.