Electroporation-mediated gene transfer of SOX trio to enhance chondrogenesis in adipose stem cells.
OBJECTIVE
The aim of the present study was to determine if the electroporation-mediated gene transfer of SOX trio enhances the chondrogenic potential of adipose stem cells (ASCs).


METHODS
ASCs were transfected with SOX trio genes using an electroporation technique and cultured for 3 weeks. The pellets were analyzed for DNA and glycosaminoglycan (GAG) analysis, and the gene and protein expression of SOX-5, SOX-6, SOX-9, type 1 collagen (COL1Al), type 2 collagen (COL2Al) and type 10 collagen (COL10A1) using real-time PCR and Western blot analysis. Further in vivo studies were carried out by subcutaneous transplantation of pellets in severe combined immunodeficiency (SCID) mice for 3 weeks.


RESULTS
The gene transfer efficiency was high (approximately 70%). Transfected ASCs showed high expression of corresponding genes after 21 days, and each SOX protein was detected in ASCs transfected with the corresponding gene. The chondrogenic differentiation of ASCs, as demonstrated by GAG levels and Safranin-O staining, showed significant enhancement when SOX trio were co-transfected, while subsets with single gene transfer of SOX-5, -6, or -9 did not show significant elevation. SOX trio co-transfection enhanced COL2A1 mRNA, but did not increase COL1A1 and COL10A1 mRNA. Type II collagen protein dramatically increased, and type X collagen decreased with co-transfection of the SOX trio. When pellets were implanted in the subcutaneous pouch of SCID mice for 3 weeks, ASCs co-transfected with SOX trio demonstrated abundant proteoglycan, significantly reduced mineralization.


CONCLUSIONS
The electroporation-mediated transfection of SOX trio greatly enhances chondrogenesis from ASCs, while decreasing hypertrophy.
Department of Orthopaedics, Dongguk University Ilsan Hospital, Republic of Korea. gunil@duih.org
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D000273:Adipose Tissue / Q000166:cytology*; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000235:genetics / Q000502:physiology*; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D018274:Electroporation; D006025:Glycosaminoglycans / Q000032:analysis; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055747:SOX Transcription Factors / Q000235:genetics / Q000378:metabolism*; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D055755:SOXD Transcription Factors / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods
D015415:Biomarkers; D006025:Glycosaminoglycans; D055747:SOX Transcription Factors; C490301:SOX5 protein, human; C486270:SOX6 protein, human; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D055755:SOXD Transcription Factors; D003094:Collagen