197 INDUCED PLURIPOTENT STEM CELLS (iPS) DERIVED FROM EQUINE UMBILICAL CORD CELLS USING LENTIVIRUS VECTOR Stemcca

Abstract

Research on induced pluripotent stem cells (iPS) emerged to overcome the limitations of embryonic stem cells, such as ethical issues, security, compatibility, and availability. The nuclear reprogramming induced by viral vectors aims to induce differentiated cells to an embryonic pluripotent state. The iPS cells can be generated using retroviral vector expressing Oct4, Sox2, Klf4 and c-Myc, but produces much genomic integration (GI) which limit its use for therapeutic purpose. Alternatively, lentiviral vectors have been used to be safe and equally effective in producing iPS. Despite several cell types can be reprogramed, there is no information of which is the best cell type to be used in the generation of iPS. The umbilical cord is a reserve of multipotent mesenchymal stem cells and may present a greater reprogramming efficiency compared with fibroblasts in the generation of iPS. Here we describe the use of a single lentiviral vector composed by the combination of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) for the generation of iPS cells using equine umbilical cord (UC) cells. Therefore, samples were collected from 5 equine UC at birth. The umbilical matrices were subjected to enzymatic digestion in a solution of 0.004% collagenase diluted in PBS, and the cells obtained by filtration were plated in plastic culture bottles with 5 mL of DMEM supplemented with 20% fetal calf serum, antibiotics, and antimycotics, followed by incubation at 37°C in a 100% humid atmosphere at 5% CO₂ in air. When the cells reached 40% of confluence and a concentration of 10⁵ cells, these cells were transduced with 50 μL Human Stemcca cre-excisable constitutive polycistronic (oskm) lentivirus (EMD Millipore Corp., Billerica, MA, USA) produced according manufacturer's protocol plus 8 ng mL^–1 polybrene (hexadimethrine bromide, Sigma, St. Louis, MO, USA). The culture medium was renewed 12 h after incubation. Five days after transduction, cells were transferred to murine embryonic fibroblasts (MEF) feeder layer and cultured for 14 days in a specific medium for iPS. The morphologically similar colonies to the embryonic stem cells were visualised after two weeks of infection. When the clones were well established two mechanical and two enzymatic passages were performed. Cells were re-expanded under new MEFs and submitted to alkaline phosphatase activity detection (Leukocyte Alkaline Phosphatase Kit, Sigma) according to manufacturer's recommendations. Briefly, cell cultures were fixed, incubated with a mixture of alkaline naphthol AS-BI with fast red violet LB. Red labelling insoluble deposits indicated the sites of alkaline phosphatase activity. In all cultures tested (n = 10) the expression of alkaline phosphatase was detected. The cell culture samples will still be tested for gene expression of pluripotency factors. The combination of all factors in a single transcript was efficient for reprogramming cells from the umbilical cord and allowed the derivation of mesenchymal cells in equine iPS. The use of a single lentiviral reprogramming vector represents a powerful tool for the study of iPS technology and its possible therapeutic application.