Human Mesenchymal Stem Cells Over-expressing Pigment Epithelium-derived Factor Inhibits Hepatocellular Carcinoma In Nude Mice

To construct pigment epithelium-derived factor (PEDF) lentiviral vector and study the recombinant expression and biological activity of PEDF produced by PEDF-transduced human mesenchymal stem cells (hMSCs) derived from bone marrow. MethodsThe human PEDF cDNA derived from the expression plasmid pBLAST49-hPEDF was subcloned into the MluI site of the plasmid pLV-GFP to generate pLV-PEDF-GFP. Packaging of lentiviruses was performed using a transient cotransfection system. Lentiviruses were concentrated using the Fast-Trap Lentivirus Purification and Concentration Kit and their functional titers were determined by fluorescence-activated cell sorting (FACS). hMSCs were isolated, cultured and infected with lentivirus LV-GFP or LV-PEDF-GFP. Both LV-PEDF-GFP transduced cells (hMSCs-PEDF) and LV-GFP transduced cells (hMSCs-GFP) were monitored under a fluorescent microscopy, and the number of GFP-positive cells determined by fluorescence-activated cell sorting analysis was used as a parameter to estimate the genetically modified cell fraction. The recombinant expression of PEDF was determined by RT-PCR, enzyme linked immunosorbent assay (ELISA) and Western blot, and the biological activity of PEDF was evaluated by endothelial tube assay.ResultsThe successful construction of PEDF lentiviral vector was cofirmed by digestion with MluI and sequencing. The DNA sequence of PEDF was identical to the report in Genebank and did not reveal any mutation. Forty-eight hours after transduction, 96.38% of hMSCs-PEDF were GFP positive. Typically, vector titers were between 5×107 and 1×108 TU/mL, and upon ultrafiltration, titers of 2×1010 TU/mL could be achieved. High level expression of PEDF by hMSCs-PEDF was confirmed by RT-PCR,ELISA and Western blot, and recombinant PEDF could inhibit the tube formation of HUVECs.ConclusionsIn this experiment, PEDF lentiviral vector was successfully constructed and biological PEDF could be produced by PEDF-transduced hMSCs. The successful isolation, culture and genetic modification of hMSCs provide a powful support for the next research on therapy for HCC. To study whether the conditioned media (CM) collected from heptocarcinoma cells stimulate the migration of human mesenchymal stem cells (hMSCs), and to study the localization of hMSCs following tail vein injection in an orthotopic nude mice model of hepatocellular carcinoma (HCC).MethodsIn vitro migration assay using Boyden Transwell chambers were performed to evaluate the tropism of hMSCs for CM derived from MHCC-97H or HepG2 cells. An orthotopic metastatic hepatocellular carcinoma model was established using LV-GFP-transduced MHCC-97H cells, and hMSCs were labeled with the fluorescent dye CM-DiI prior to in vivo administration. On days 7 and 14 following hMSCs injection, organs (liver, spleen, lungs, heart, brain, pancreas and kidneys) were harvested and cryosectioned. Distribution of CM-DiI-labeled hMSCs was assessed by direct visualization with a fluorescence microscope.ResultsThe number of hMSCs or hMSCs-PEDF that migrated toward CM collected from MHCC-97H or HepG2 cells was significantly higher compared with controls, which strongly suggested that human hepatocarcinoma cells were capable of stimulating the migration of hMSCs and that the migratory ability of hMSCs was not affected by lentiviral transduction. At day seven following tail vein injection, CM-DiI-labeled hMSCs were localized mainly around the edge of the primary liver tumors, though a few hMSCs were also observed in the lungs of tumor-bearing mice. By 14 days post-injection, hMSCs were distributed extensively throughout the tumor mass, whereas they were undetectable in the lungs. No other examined organs (spleen, heart, brain, pancreas or kidneys) showed signs of CM-DiI-labeled hMSCs engraftment at either 7 or 14 days after injection.ConclusionsIt was first confirmed that hMSCs have the tropism for HCC, which suggested a potential role of hMSCs as a targeted, therapeutic delivery vehicle for the treatment of HCC. To evaluate the therapeutic potential of hMSCs-PEDF for the treatment of HCC.MethodsTen days after the implantation of MHCC-97H xenografts into the left liver lobes of the mice, the mice were divided into four treatment groups (n=22 for each group). Mice in each group were administered one of the following by tail vein injection: PBS, hMSCs-GFP, hMSCs-PEDF, or recombinant LV-PEDF. After 35 days of treatment, twelve randomly selected animals from each group were sacrificed and autopsied. Livers were removed and the volume of each primary liver tumor was calculated. Pulmonary metastases were assessed by microscopic examination of H&E-stained serial paraffin sections. Concentrations of human PEDF in sera were determined using a commercial PEDF ELISA kit, and immunohistochemistry using an antibody against CD34 to analyze the intratumoral microvessel density (MVD).ResultsIn vivo efficacy experiments showed that intravenous (i.v.) injection of PEDF-expressing hMSCs significantly suppressed both the growth of primary liver tumors and the development of pulmonary metastases. Moreover, hMSCs-based PEDF gene delivery moderately elevated systemic levels of human PEDF. Immunohistochemistry of primary liver tumors demonstrated lower microvessel density (MVD) in mice treated with hMSCs-PEDF compared with control mice. ConclusionsThis is the first study to show the potential of hMSCs as an effective delivery vehicle for therapeutic genes in the treatment of HCC.