208 Effect of growth factors and reprogramming molecules on induction to multipotency of dermal fibroblasts from colocolo (Leopardus colocolo)

Abstract

Reprogramming of terminally differentiated cells to higher plasticity levels can be achieved with small molecules. This can be of value for somatic cell nucleus transfer, deriving multi and pluripotent cells and conservation purposes. Recently, induced mesenchymal stem cells were derived from differentiated human and mouse cells by using small molecules and growth factors. The pampas cat or colocolo (Leopardus colocolo) is a South American felid categorrized as near threatened by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Major historical threats to the pampas cat include illegal hunting, habitat loss or transformation, and conflict retaliation for poultry predation. Here, we tested 5-azacytidine (an epigenetic modifier) and A8301 (a potent inhibitor of transforming growth factor-β type I receptor superfamily), linked to platelet-rich plasma (PRP) and platelet-derived growth factor (PDGF-B) to induce changes in the expression of pluripotency genes and differentiation capacity of colocolo fibroblasts towards other mesodermal lineages. For this, dermal fibroblasts were treated with (I) 5-azacytidine + PRP + A8301 + VitC, or (II) 5- azacytidine + VitC + A8301 + PDFG for 12 days. On Days 0, 5, and 12 of reprogramming, expression of OCT4, NANOG, E-cadherin and SNAIL was evaluated by reverse transcription-PCR, and tri-lineage differentiation was induced. For treatment I, no statistical difference was found in the expression of OCT4 and NANOG. Chondrogenic and osteogenic differentiation was observed. In treatment II, significant expression of OCT4 and NANOG (P < 0.05) was induced, and reprogrammed fibroblasts were differentiated into chondrogenic and osteogenic lineages. Immunohistochemistry positivity for OCT4 was detected in treatment II. In summary, we showed that dermal fibroblasts of pampas cat can be reprogrammed into cells with multipotent characteristics, particularly when a cocktail of 5-azacytidine + VitC + A8301 + PDFG was used. Treatment I probably failed because of other growth factors and proteins present in PRP, which might inhibit successful reprogramming or activate other pathways leading to a nonmultipotent phenotype. Further refinements of these protocols are required to improve the reprogramming protocol. This in turn should help us obtain cells that can be used in nucleus transfer or cellular therapies in endangered felid species.