Abstract: SA-PO584
Established Human Mesenchymal Stem Cell Lines Stably Secreting Matrix Metallopeptidase 7 to Treat Glomerulosclerosis in Light Chain Deposition Disease
Session Information
- Glomerular Diseases: Fibrosis, Extracellular Matrix
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix
Authors
- Feng, Xiuyan, LSU Shreveport, Shreveport, Louisiana, United States
- Zeng, Chun, Louisiana State University Health Sciences Center, Shreveport, Louisiana, United States
- Teng, Jiamin, University of South Alabama, Mobile, Alabama, United States
- Cai, Hui, Emory University School of Medicine, Atlanta, Georgia, United States
- Herrera, Guillermo A., Louisiana State University Health Sciences Center, Shreveport, Louisiana, United States
Background
Light chain deposition disease (LCDD) is a rare systemic disorder, caused by the overproduction and extracellular deposition of monoclonal immunoglobin light chain. The clinical manifestation is dominated by renal disease. The affected glomeruli are enlarged. The deposition of PAS-positive material produces capillary wall thickening and nodular expansion of the mesangium. The extent of glomerular involvement can vary from minimal mesangial expansion to a fully developed nodular glomerulosclerosis. Although the pathogenesis of the glomerulosclerosis in LCDD is not entirely clear, experimental studies have shown that mesangial cells incubated with monoclonal light chains obtained from patients with LCDD produce transforming growth factor β, which triggers the production of extracellular matrix proteins. Tenascin is the most common protein in expanded mesangium in LCDD and is degraded mostly by Matrix Metallopeptidase 7 (MMP7). This finding suggests that inducing MMP7 secretion by stem cells might be a possible therapeutic intervention to reverse nodular glomerulosclerosis in LCDD. In this study, we aimed to establish stable stem cell lines to facilitate degradation of tenascin.
Methods
1) pcDNA3-GFP-MMP-7 plasmid was purified using QIAprep Spin Miniprep Kit. The products are confirmed by gel electrophoresis.
2) HMSC was cultured in 6-well plate with 5% FBS specific medium. When 80% confluences was reached, the HMSCs were transfected by purified GFP-MMP7 plasmid using lipofectinamine 2000. Immunofluorescence microscopy was used to test the transfection efficiency.
3) 48 hours after transfection, the HMSCs were transferred into 10cm dishes. 72 hours later, G418 600ug/ml was added in to medium. Cell clones formed in 3-4 weeks and then were picked up to amplify. Confocal microscopy and western blot analysis were used to identify MMP-7 expression and secretion.
Results
1) GFP-MMP-7 plasmid was successfully amplified and purified.
2) HMSC were successfully transfected by GFP-MMP7 plasmid using lipofectinamine 2000. The transfection efficiency was about 30%, including 10% highly expressed cells.
Conclusion
We successfully established HMSC cell lines which highly express and secret MMP7. In our future study, the cell line will be used to treat kidney diseases caused by LCDD in animal model.