Generation of insulin-producing human mesenchymal stem cells using recombinant adeno-associated virus.
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.
Kim
Jeong Hwan
JH
Department of Medical Engineering, Yonsei University College of Medicine, 134, Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea.
Park
Si-Nae
SN
Suh
Hwal
H
D000328:Adult; D000818:Animals; D002096:C-Peptide / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D004274:DNA, Recombinant; D000229:Dependovirus / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism*; D007763:Lac Operon / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D011384:Proinsulin / Q000235:genetics* / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D002096:C-Peptide; D004274:DNA, Recombinant; D007328:Insulin; D011498:Protein Precursors; C013737:preproinsulin; D011384:Proinsulin; D001616:beta-Galactosidase