CHARACTERIZATION OF PARTIALLY REPROGRAMMED EQUINE AND BOVINE iPS CELLS IN DIFFERENT CULTURE CONDITIONS.

MICAELA NAVARRO; CARLA CAMPETELLA; ADRIAN MUTTO; CAROLINA BLUGUERMANN

CIUD AUTON D BUENOS AIRES

Jornada; JORNADAS DE LA SOCIEDAD ARGENTINA DE BIOLOGÍA; 2018

SOCIEDAD ARGENTINA DE BIOLOGIA

Induced pluripotent stem (iPS) cells can be produced by introducing four transcriptional factors ? Oct4, Klf4, Sox2 and c-Myc ? into adult somatic cells. These cells are similar to embryonic stem cells (ESC) considering their gene and protein expression profile. Although reprogramming protocols are well established for murine and human cells, this is not the case for domestic species such as equine and bovine. Taking this into consideration, the aim of this work was to reprogram equine and bovine fibroblast and analyze their pluripotent state in different culture media. We used lentiviral particles carrying STEMCCA plasmid to induce reprogramming. Two days after infection, equine and bovine infected fibroblasts were cultured in different conditions using irradiated murine embryonic fibroblast (iMEF) as a feeder layer. The different culture conditions tested were: DMEM + 10% fetal bovine serum (FBS) (M1), DMEM + 10% FBS + leukemia inhibition factor (LIF) (M2), knock out DMEM + knock out serum replacement + LIF (M3). To evaluate cells pluripotency, we checked colony morphology, performed a colorimetric alkaline phosphatase assay, and analyzed the expression of endogenous and exogenous pluripotency genes (PG) by PCR. Equine and bovine cells from all conditions started growing as compact colonies with define borders 10 to 15 days after viral infection. In both cases, cells showed a high nuclear/cytoplasmic ratio characteristic of pluripotent cells. However, cells cultured in M1 and M2 lost their morphology after the first passage. On the contrary, clones cultured in M3 were passaged more than 10 times although we could not expand them, with the exception of one equine clone (A5) from which we were able to obtain a large number of cells. Alkaline phosphatase expression was stronger for A5 clone in comparison with other M3 clones tested. When the expression of PG was analyzed, we observed that colonies cultured in M3 expressed all the endogenous PG tested and had silenced the exogenous expression of Oct4 and Sox2 (evaluated at passage 2 and 9) excluding equine clone A5 which retained expression of exogenous Sox2. This clone was also able to differentiate in vitro and to express genes from mesoderm and endoderm germ layers. These results indicate that we successfully obtained ?partially? reprogrammed equine and bovine iPS cells in M3 culture conditions. Cells were able to sustain their undifferentiated state only if exogenous PG expression persisted, which is in accordance with previous reports. Once expression from exogenous PG is silenced, endogenous expression of PG is not enough to maintain the pluripotent state of iPS cells cultured in M3 conditions. Based on this, we consider that the main roadblock to obtain ?fully? reprogrammed equine or bovine iPS cells could be surpassed if the appropriate culture conditions are found.