Investigating The Effects Of Bone Marrow MSCs Modified By RAAV-mediated TPO Gene On Megakaryocytopoiesis

Objective Thrombocytopenia is a major clinical problem in the management of patients with cancer receiving chemotherapy or radiotherapy, and nonchemotherapy or nonradiotherapy patients with myelodysplastic syndrome (MDS), idiopathic thrombocytopenic purpura (ITP), chronic liver disease, et al. Platelet transfusion therapy is currently the only acute treatment for severe thrombocytopenia. Although temporarily effective in controlling severe thrombocytopenia, platelet transfusion therapy is associated with several problems, including alloimmunization, transmission of infectious agents, and transfusion reactions. Therefore, we try to modify bone marrow mesenchymal stem cells (MSCs) with recombinant adeno-associated virus (rAAV), and explore the effect of TPO modified-bone marrow MSCs on megakaryocytopoiesis.Methods and Results1. Isolation and culture of bone marrow mesenchymal stem cellsHuman bone marrow derived MSCs were isolated and purified by density gradient centrifugation and direct adherence separately, and included osteoblasts and adipocytes. Von Kossa and oil red O were used to stain osteoblasts and adipocytes. Our results suggested that MSCs that having proliferative activity could be obtained by primary and passage culture. The proliferation ability was decreased gradually after several passages. After 2 weeks of osteoblasts inducing, calcium deposition was demonstrated by Von Kossa staining. After 3 weeks of adipocyte inducing, intra-cellular lipid vacuoles were stained red with oil red O. 2. Effects of bone marrow MSCs condition medium (MSCs-CM) on differentiate of human megakaryoblastic cell line Meg-01Then, serum-free bone marrow MSCs condition medium (MSCs-CM) was added to culture system of human megakaryoblastic cell line Meg-01 to induce differentiation and maturation. NF-E2 mRNA expression, which is necessary for megakaryocytes to produce platelets, was enhanced in megakaryocytic cell lines Meg-01 when cultures were exposed to MSCs-CM, compared with control cultures exposed to IMDM. This result suggested that MSCs-CM has a potential to promote megakaryocyte differentiate and maturation. To explain the effects of MSCs-CM on megakaryoblastic cell line Meg-01, Altas cDNA expression array was used to analyse the differential mRNA expression between Meg-01 cells with MSCs-CM and that without MSCs-CM (control). Among gene expressions of Meg-01 cells with MSCs-CM, seven genes were up-regulated and five genes were down-regulated compared with control. The comparison study indicated that the expression of mitosis related genes such as microtubule-associated protein, CDC20 were down-regulated, while the expression of Ca²⁺ correlative intracellular signal transduction pathways related genes such as calcium/calmodulin -dependent protein kinaseâ…£were up-regulated, while the expression of JAK3 correlative signal transduction pathways related genes such as Janus kinase 3 were up-regulated, while the expression of apoptosis related genes such as tumor necrosis factor, adenosine Al receptor were up-regulated, while the expression of megakaryocytic maturation related genes such as PDGF-A were up-regulated. These results provide a basis for molecule studying the megakaryocytic regulation by MSCs-CM.3. Effects of bone marrow MSCs-CM combined with TPO or IL-11 on megakaryocytopoiesis in vitroSome researches demonstrated that thrombopoietin (TPO) or interleukin-11(IL-11)is an effective cellar factor on megakaryocytopoiesis. The effect of MSCs-CM or MSCs-CM combined with TPO or MSCs-CM combined with IL-11 on the expansion of mature megakaryocytes and megakaryocytic progenitors from bone marrow were observed. The results showed that MSCs-CM or MSCs-CM combined with TPO or MSCs-CM combined with IL-11 could promote the expansion of mature megakaryocytes and megakaryocytic progenitors. Compared with MSCs-CM alone or TPO alone, the effect of MSCs-CM combined with TPO on the expansion of megakaryocytes was further enhanced, and the synergistic effects were exist between MSCs-CM and TPO. The effect of MSCs-CM combined with IL-11 on the expansion of megakaryocytes was not difference statistically compared with MSCs-CM alone or IL-11 alone. These results suggested that MSCs-CM combined with TPO would be an effective method for promoting megakaryoeytopoiesis.4. The method of packaging and purifying rAAV-TPOIn our study, the 1059 bp fragment of TPO was produced from human fetal liver by RT-PCR. The human TPO cDNA fragment was cloned into the multiple cloning site of pAAV-IRES-GFP. The plasmid pAAV-TPO-IRES-GFP (pAAV-TPO) was constructed by digesting with restriction enzyme and sequencing analysis. The calcium phosphate precipitate is formed by mixing pAAV-RC, pHelper or pAAV-TPO. The recombinant AAV-TPO-IRES-GFP (rAAV-TPO) was purified and condensed as the method including chloroform treatment, PEG/NaCl precipitation and chloroform extraction. The titer of rAAV was 2×10¹¹vg/ml, which was determined using quantitative DNA dot blots. The high purity of rAAV-TPO was achieved, which was determined by SDS-PAGE. We measured the transduction efficiency of HEK 293 cells infected with different titers rAAV-TPO particles per cell, and 1×10⁵ rAAV-TPO particles per cell was optimal for transduction experiment by counting green fluorescent cells under the inverted fluorescent microscope. Then, MSCs were either mock infected or infected with the rAAV-TPO-IRES-GFP (rAAV-TPO) vector. TPO mRNA and protein were both strongly expressed in TPO-transduced hMSCs. The expression of GFP in the TPO-transduced MSCs reached to 23.0%±7.18%. 5. Effects of MSCs modified by rAAV-mediated TPO gene on megakaryocytopoiesis in vitroThe condition medium from TPO-transduced human MSCs (TPO/MSCs-CM) was collected, and the effect of MSCs-CM or TPO/MSCs-CM on megakaryocytopoiesis from bone marrow were observed. The results demonstrated that hMSC-CM and TPO/hMSC-CM could promote megakaryocytic progenitors and CD41-positive cells production, while the effect of TPO/hMSC-CM on the expansion of megakaryocytes was further enhanced. These results suggested that human TPO modified-bone marrow MSCs (TPO/MSCs) mediated by rAAV may be possible to become as a new safe and effective method for megakaryocytopoiesis.6. Co-transplantation of human mesenchymal stem cells transduced with TPO gene enhances megakaryocytopoiesis in NOD/SCID mice.This has been the basis of the study in vitro. In order to explore further the effect of TPO/MSCs on megakaryocytopoiesis, the animal experiment in vivo was performed. In our study, mononuclear cells (MNCs) were transplanted to the irradiated NOD/SCID mice after X liner radiation and cultured MSCs or mesenchymal stem cells transduced with TPO gene (TPO/MSCs) were transplanted at the same time. The effects of TPO/MSCs or MSCs on megakaryocytopoiesis in mice were compared. After co-transplantation human MNCs with MSCs or TPO/MSCs, human Alu sequence gene could be detected in different tissues including bone marrow, peripheral blood, spleen and liver. The percent of human CD45⁺ cells had no statistically difference between MNCs plus MSCs group and MNCs plus TPO/MSCs group. After co-transplantation MNCs plus MSCs and MNCs plus TPO/MSCs, the average percentage of human CD45⁺ and human CD41⁺(hCD45⁺hCD41⁺) cells were 0.98±0.65% and 3.46±1.73% respectively in bone marrow, and the number of CFU-MK were 6.20±3.70 and 20.40±7.16 respectively per 2×10⁵ bone marrow mononuclear cells of mice. Histology in bone marrow, spleen and liver sections stained with hematoxylin/eosin (HE) was analyzed after transplantation. The polyploidy megakaryocytes were counted in MNCs plus MSCs group and MNCs plus TPO/MSCs group respectively, 3.40±1.14 and 12.60±5.45 in bone marrow, 2.20±0.84 and 6.80±1.64 in spleen, 0 and 0.80±0.44 in liver. In addition, the number of platelets was counted at every 3 days in MNCs plus MSCs group and MNCs plus TPO/MSCs group. The results showed that the number of platelets was increased in mice co-transplanted human MNCs with MSCs or TPO/MSCs. TPO/MSCs had a dramatic protection from platelet levels compared with MSCs. The recovery was accelerated more rapidly in MNCs plus TPO/MSCs group than MNCs plus MSCs group.Conclusion MSCs that have the proliferative activity and potential to differentiate were cultured successfully in vitro. TPO were expressed successfully in TPO-transduced MSCs mediated by rAAV. The effect of TPO/MSC-CM on the expansion of megakaryocytes was further increased than TPO/MSC-CM. The effects of co-transplantation of TPO-transduced MSCs and MNCs on megakaryocytopoiesis in NOD/SCID mice were more potent than co-transplantation of primary MSCs and MNCs.