111 EQUINE AMNIOTIC EPITHELIAL OR BONE MARROW MESENCHYMAL STEM CELLS DIFFERENTLY SUPPORT IN VITRO EMBRYO DEVELOPMENT IN A BOVINE IN VITRO CULTURE MODEL

Abstract

There are indications that the culture system and the medium composition can affect embryo quality. In fact, various studies have been shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In light of this, recently, some studies used co-culture with mouse embryonic fibroblasts in the effort to improve the development of bovine and ovine in vitro-derived embryos. Despite the progress in equine IVM and ICSI technologies and the different culture conditions reported for preimplantation development of ICSI fertilized horse oocytes, the yield of blastocysts remained low. In the present study we investigated the benefits of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSC) or equine amniotic epithelial stem cells (AE-SC) on blastocyst development. This study employed the bovine embryo as a model and represents the initial step towards standardization of a protocol for the culture of equine embryos in our laboratory. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and layered over Hystopaque^™ 1.080, then centrifuged for 20 min at 400g and 4°C. Cell pellets were resuspended in 10 mL Dulbecco Modified Earle’s Medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, penicillin (100 U mL^–1) and streptomycin (100 μg mL^–1) and seeded in 24-well plates. Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fractions were incubated at 37°C with 0.05% trypsin for 45 min. Separated AE cells were plated on 25 cm² flask in standard culture media containing 10 ng mL^–1 epidermal growth factor. Seven hundred fifty cumulus–oocyte complexes with a homogeneous cytoplasm and two or more layers of cumulus cells were used. After IVM and IVF cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured for up to Day 7: 1) co-culture with granulosa cells (control); 2) co-culture with BM-MSC; 3) co-culture with AE-SC. The culture medium was TCM 199 + 10% fetal bovine serum, pyruvate and gentamicin at 38.5°C in 5% CO₂. Statistical analyses was performed by chi square test. Blastocysts developmental rates were similar among control, AE-SC and BM-MSC (35%, 41% and 30%, respectively), but the co-culture with AE-SC gave a significantly greater percentage of blastocysts compared to BM-MSC (P < 0.05). In conclusion, despite the absence of a significant increment in blastocysts attainment using stem cells as feeders for embryo culture, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSC. It can be suggested that these cells secrete biologically active substances including signaling molecules and growth factors of epithelial nature different from those of the BM cells of mesenchymal origin.

Regione Lombardia is acknowledged for the “Dote Ricercatori” fellowship to V.M.