Yonsei Med J.  2007 Feb;48(1):109-119. 10.3349/ymj.2007.48.1.109.

Generation of Insulin-Producing Human Mesenchymal Stem Cells Using Recombinant Adeno-Associated Virus

• Kim JH
• Park SN
• Suh H

• KMID: 1093531
• DOI: http://doi.org/10.3349/ymj.2007.48.1.109

Abstract

The purpose of current experiment is the generation of insulin-producing human mesenchymal stem cells as therapeutic source for the cure of type 1 diabetes. Type 1 diabetes is generally caused by insulin deficiency accompanied by the destruction of islet beta-cells. In various trials for the treatment of type 1 diabetes, cell-based gene therapy using stem cells is considered as one of the most useful candidate for the treatment. In this experiment, human mesenchymal stem cells were transduced with AAV which is containing furin-cleavable human preproinsulin gene to generate insulin-producing cells as surrogate beta-cells for the type 1 diabetes therapy. In the rAAV production procedure, rAAV was generated by transfection of AD293 cells. Human mesenchymal stems cells were transduced using rAAV with a various multiplicity of infection. Transduction of recombinant AAV was also tested using beta-galactosidse expression. Cell viability was determined by using MTT assay to evaluate the toxicity of the transduction procedure. Expression and production of Insulin were tested using reverse transcriptase-polymerase chain reaction and immunocytochemistry. Secretion of human insulin and C-peptide from the cells was assayed using enzyme-linked immunosorbent assay. Production of insulin and C-peptide from the test group represented a higher increase compared to the control group. In this study, we examined generation of insulin-producing cells from mesenchymal stem cells by genetic engineering for diabetes therapy. This work might be valuable to the field of tissue engineering for diabetes treatment.

Keyword

Cell-based gene therapy; mesenchymal stem cells; adeno-associated virus; type 1 diabetes

Figure

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  Fig. 1 Structure of plasmid used to generate recombinant AAV. Furin-cleavable human preproinsulin gene was inserted into MCS (multiple cloning site) of pAAV-MCS by restriction enzyme treatment.

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  Fig. 2 Cells used in the experiment. Human mesenchymal stem cells (a, magnification × 100) and hamster islet cells (b, magnification × 400) are observed with light microscope.

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  Fig. 3 Agarose-gel electrophoresis of plasmids. (A) pHelper (11.6kb), (B) pAAV-RC and pAAV-LacZ (7.3kb), (C) pAAV-hPPI (4.8kb).

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  Fig. 4 AAV production from the transfected AD-293 cells. AD-293 cells before transfection (A) and at 3 days post-transfection (B). Arrows indicate the floated cells by rAAV production. Magnification × 100.

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  Fig. 5 Scanning electron microscopy of rAAV-(fur)hPPI. Magnification × 10K (left panel) and × 20K (right panel).

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  Fig. 6 β-Galactosidase staining of the hMSCs infected with rAAV-LacZ. Magnification × 100.

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  Fig. 7 RT-PCR analysis. AAV-hPPI-transduced (lane 1) and control (lane 2) hMSCs.

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  Fig. 8 Immunocytochemistry. Arrows indicate insulin-positive staining (brown-red).

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  Fig. 9 Insulin and C-peptide production. AAV-transduced hMSCs, untransduced hMSCs, and HIT-T15 cells are shown. Insulin production of cells at 1 week (a), 2 weeks (c), and 3 weeks (e) post-transduction and C-peptide production at 1 week (b), 2 weeks (d), and 3 weeks (f) post-transduction were tested. Insulin secretion from 1-3 week(s) post-transduction maintained a similar level or decreased slightly (c). C-peptide secretion from 1 week to 3 weeks post-transduction (d). Data were tested using ANOVA (a~g, p < 0.005).

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  Fig. 10 Relative viability of AAV-infected cells. MTT assay was used (p < 0.005).

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