225 AUTOLOGOUS TRANSPLANTATION OF MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE IN ANIMAL

Abstract

The present study was carried out for isolation and culture of adipose tissue-derived mesenchymal stem cells of goat (gADSC) and dogs (1 dog was suffering from hip dysplasia and another dog from paraplegia) and their characterisation with different markers. Adipose tissue of goat and dog were aseptically isolated and treated with collagenase for 2 h in a CO₂ incubator. The enzymatic digested cells were filtered through a 41-µm filter and cells were resuspended in cell culture flask containing medium DMEM/F12, 10% fetal bovine serum, and 50 μg mL^–1 gentamycin. In vitro-cultured ADSC were characterised by amplification of mesenchymal stem cell (MSC)-specific surface marker genes of CD44, CD29, and CD166 in PCR and by immunocytochemistry of MSC-specific marker of CD44. For in vitro chondrogenesis, ADSC at passage 3 were incubated in DMEM/F12 containing 100 nM dexamethasone, 1.25 μg mL^–1 BSA, and 10 ng mL^–1 BMP-4 ITS (insulin-transferrin-selenium) for 3 wk. Chondrogenic differentiation cells were confirmed by Safranin O staining and positive expression of chondrocyte-specific marker genes Aggrecan: primers F-TTGGACTTTGGCAGAATACC and R-CTTCCACCAATGTCGTATCC, and Collagen II: primers F-AACCCTGGAACTGACGGAAT and R-CTCACCCGTTTGACCTTTCG in PCR. Dog ADSC-derived chondrocytes were aseptically injected at 1 × 10⁶ cells kg^–1 of BW into dogs with hip dysplasia and paraplegia. Both dogs recovered well after 1 month of autologous transplantation and were able to move freely. Then, 10 dogs having massive wounds were injected with heterologous undifferentiated mesenchymal stem cells at 1 × 10⁶ cells kg^–1 of BW and all dogs were cured in an average of 20 days. Then, the paralyzed and fractured dogs were further treated with undifferentiated MSC at 1 × 10⁶ cells kg^–1 of BW and most of the dogs were cured properly. These findings may have implications for defining the physiological roles of ADSC in arthritis, some orthopaedic problems, joint regeneration, and neurological disorders and several new applications leading to novel therapeutic opportunities.