389 Culture of Equine Inner Cell Mass Using Equine Mesenchymal Cells as a Feeder Layer

Abstract

Stem cells can originate from embryo, fetus, and adults tissues. Adult stem cells are present in almost all organs and are responsible for tissue regeneration and repair. The mesenchymal stem cells (MSC) are multipotent cells present in the bone marrow. The plasticity of these cells allows them to be used in cell therapy because they have the potential to differentiate into several tissues with mesenchymal origin. On the other hand, embryonic stem cells (ESC) have the ability to differentiate into the 3 embryonic tissues (endoderm, mesoderm, and ectoderm) generating all kinds of tissue in the living organism. Embryonic stem cells can be maintained in their undifferentiated state when cultured in vitro in the presence of the leukemia inhibitory factor (LIF) and over a feeder layer composed normally by mouse fibroblasts. Because of the small number of articles studying equine ESC, the current experiment aimed to compare the utilization of equine fibroblasts and equine MSC as feeders for inner cell mass (ICM) culture. Eighteen blastocysts were collected on Day 7 after ovulation from 5 fertile mares that had been artificially inseminated. The embryos were placed in a petri dish with Dulbecco’s phosphate buffered saline + 10% FCS, and the ICM was mechanically removed using 2 insulin needles. The ICM was then transferred to one well of 24 well plates containing either an equine fibroblast monolayer (9 embryos) or a MSC monolayer (9 embryos). Both feeders layers were inactivated with 10 μg mL-1 mitomycine C, and the cell concentration was 140 000 cells/well. The culture media utilized was the DMEM/F12 with 20% FCS, 1% essential amino acids, 1% nonessential amino acids, 0.1 mM β-mercaptoetanol, 10 ng mL-1 LIF, penicillin, streptomycin, and amphotericin B. The ICM was cultured during 5 days for the adherence to the plate, and the media was change every 2 days. Data was analyzed by ANOVA. Results indicated no significant differences between the use of equine fibroblasts or MSC as feeder layer to support equine ICM in culture. The expansion of the ICM cells was observed after 10 to 12 days in culture in 44.4% (4/9) of ICM cultured in MSC monolayer and 55.5% (5/9) of the ICM cultured over a fibroblast layer. The formation of embryoid body-like structures were observed after the second passage (5-7 days in culture) in 50% (2/4) of embryos using MSC feeders and in 40% of embryos (2/5) for which fibroblasts were used. Cultures were then positively marked with the Oct 4 antibody for characterization of the undifferentiated lineage. The results of the current experiment showed that although further patronization of the culture system is still needed, both fibroblasts and MSC are suitable as feeders for the culture of equine ICM when aiming to establish ESC lineage in vitro. Financial support was provided by FAPESP and CNPq.