Immunofluorescence and flow cytometry on characterization of equine induced pluripotent stem cells

The induced pluripotent stem cells (iPS) are adult cells genetically reprogrammed to a state similar to embryonic stem cells. Lentiviral vectors are used as a safe and effective way of producing iPS. The umbilical cord (UC) is a reserve of multipotent mesenchymal stem cell and therefore may represent an efficient source of cells to be reprogrammed. To characterize reprogrammed cells specific positive markers for pluripotent clones are used such as Oct-4, Nanog, Sox-2, TRA-1-60, TRA-1-81, SSEA-1, and SSEA3 SSEA-4. In this context, the aim of this study was to characterize, by immunofluorescence and flow cytometry methods, cells from equine cord matrix after reprogramming with lentiviral vector. For this, five samples of horses UC were collected at birth. Umbilical matrices were subjected to enzymatic digestion in a solution of 0.004% collagenase diluted in PBS, and cells obtained by filtration were plated into plastic culture flasks with 5 mL DMEM supplemented with 20% fetal bovine serum, antibiotics and antimycotics, followed by incubation at 37°C in a 100% wet, 5% CO2. When cells reached 40% confluence, a concentration of 105 cells were transfected with lentiviral human-vector EF1α 50mL STEMCCA (OKSM) (Millipore, SCR544), which contains the transcription factor OCT-4, SOX2, C-MYC and KLF-4 to generate iPS cells, according to the manufactureru0027s protocol, plus 8 ng/mL polybrene (hexadimethrine bromide, Sigma). The culture medium was renewed 12 hours after incubation. Five days after transduction, the cells were transferred into a subculture of mouse fibroblasts and cultured for 14 days in a specific medium for iPS. The first colonies were visualized after two weeks of the infection. When the clones were well established two mechanical passages and two enzymatic were made. After the re-establishment and proliferation of colonies, they were subjected to immunostaining protocols and analyzed by flow cytometry. Immunocytochemistry was performed in 6- well plates containing arround 20 to 30 colonies per well. For flow cytometry 200.000 cells were analyzed for each of the antibodies. In both techniques the following antibodies were used: Oct-4, Nanog, Sox -2, TRA- 1-60 and TRA- 1-81. We noticed that, in the immunofluorescence, horse umbilical cord cell’s colonies scored positive for Oct-4, Sox-2 and TRA-1-81, as in flow cytometry, colonies were positive for Oct-4, Nanog, TRA-1-60 and TRA-1 - 81. Although the markers used were specific to pluripotent cells, we did not observe a correspondence between the two techniques. Despite this, we can conclude that the equine umbilical cord cells were successfully reprogrammed and presented characteristics of pluripotent cells.