Discogenic differentiation of human bone marrow mesenchymal stem cell with adenovirus mediated glucose transporter-1 and hypoxia inducible factor-1α gene therapy

Abstract

With aging and degeneration, intervertebral discs (IVDs) undergo profound and substantial changes in morphology and biochemical composition. An ideal solution for managing disc degeneration would be to repair the IVDs, by producing discogenic matrix. Recently, various approaches to biological repair of the disc function are under investigation, which are gene therapy, growth factor injection, cell therapy and cell-based tissue engineering. Hypoxia inducible factor-1α (HIF-1α) is the key molecules regulating energy metabolism and survival in the nucleus pulposus (NP) cell. The expression of HIF was demonstrated in the NP cell of normal human IVDs and can be used as a phenotypic marker of NP cell. Hypoxia responsive glucose transporter-1(GLUT-1) is a facilitative glucose transporter in the NP cell and also can be used as a phenotypic marker of NP cell along with HIF-1αThe discogenic induction from human bone marrow mesenchymal stem cell(BMSC) using adenoviral transduction of NP specific phenotypic factors of HIF-1α and GLUT-1 was investigated via in vitro and in vivo experiments. The BMSC was obtained from patients during surgery for lumbar spinal stenosis. Each control and experimental groups, in vitro and in vivo experiments were performed simultaneously. In vivo study, total of 6 conditions including 1 positive control with NP cell and 1 negative control with BMSC alone were set by the combination of control, viral vector and transducted genes. All groups were consisted of BMSC, BMSC with Ad-mock, the NP cell, BMSC with Ad-HIF-1α, BMSC with Ad-GLUT-1 and BMSC with Ad-HIF-1α and Ad-GLUT-1 in order. The gene transduction using adenovirus and subsequent differentiation of BMSC into NP cell was confirmed by reverse-transcription polymerase chain reaction(RT-PCR) and histologic analyses. In vivo experiment, xenograft of alginate-BMSC complex on the back of mice (DVA/1J) was done. Mice were sacrificed at 2 weeks and 4 weeks after subcutaneous implantation of alginate-BMSC complexes. Analyses of each specimen including histologic test were done. The mRNA of matrix component including aggrecan and collagen type II in the differentiated NP cell was tested. In vitro result at 24 and 48 hours culture, the BMSC with Ad-HIF-1α or Ad-GLUT-1 or both with Ad-HIF-1α and Ad-GLUT-1 groups showed increased mRNA expression of GLUT-1, aggrecan, and type II collagen compared to control and the expression level was comparable level of NP cell group. In HIF-1α mRNA expression, BMSC with Ad-HIF-1α, and the BMSC with Ad-HIF-1α and Ad-GLUT-1 groups showed increased level of expression of HIF-1α mRNA compared to those of other groups. Based on the different oxygen conditioned culture for 96 hours, expression of HIF-1α and GLUT-1 mRNA of normoxic-hypoxic group showed analogous pattern with that of continuous normoxic group. In matrix component mRNA expression, expression pattern of aggrecan and collagen type II mRNA was comparable with that of continuous hypoxic group.For in vivo study, the BMSC with Ad-HIF-1α, BMSC with Ad-GLUT-1 and BMSC with Ad-HIF-1α and Ad-GLUT-1 groups showed positive stains for collagen type II and aggrecan, which were analogous to the NP cell group. In conclusion, transduction of each gene of the HIF-1α, GLUT-1 using adenovirus vector was proved to be effective to induce the differentiation of BMSC into discogenic phenotype in vitro and in vivo.