Isolation and characterization of mesenchymal stem cells from bovine amniotic fluid at different gestational ages.

Introduction - Amniotic fluid (AF) is a reservoir of multipotent mesenchymal stem cells (MSCs), very promising cells for tissue engineering in clinical application. The aim of this work was to isolate and characterize bovine MSCs from AF (bAF-MSCs) as alternative sources of primitive multipotent stem cells in a species that could be an attractive large-animal model for biomedical and biotechnology researches. Materials and Methods - Samples were recovered, at slaughterhouse, from 18 pregnant cows at different gestational ages established on crown-rump length (1: 0-105 d; 2: 106-160 d; 3: 161-200 d; 4: 201-240 d; 5: 241 d-term of pregnancy). Cells were isolated by centrifugation and cultured in DMEM-TCM 199 (1:1) plus 10% FBS. At passage (P) 4, chondrogenic, osteogenic and adipogenic differentiation were induced (n=16) and evaluated morphologically and cytologically (Alcian Blue, Von Kossa and Oil Red O stainings respectively). Phenotypic characterization for stem cell markers was performed at P3 or P4 by flow cytometry for CD105, CD90, CD45 and CD14 (n=16), by immunocytochemistry (ICC) for Oct4, SSEA1, SSEA4 and α-SMA (n=4) and by RT-PCR analysis for Oct4, Nanog and Sox2 (n=7). In both ICC and RT-PCR, bovine fibroblasts were used as negative control. Results – Mean collected AF volume was 35 ml and 200-3636 cells/ml were recovered. Cells were isolated from 16/18 samples. At P0, cell populations appeared heterogeneous and we could identify 4 different cell types: round, spindle-shaped, fibroblastoid and large-flat cells. These different populations were observed at every gestational age analyzed. Through passages, only round cells (n=15/16) predominated. In only one sample, spindle-shaped cells prevailed. After 30 d of culture mean cell doublings were 14,1±3,2 and mean doubling time was 57,7±27,9 hrs without differences related to gestational age or sample (p>0.05). Cells were able to differentiate into osteocytes and adipocytes; chondrogenesis was clearly obtained only from samples recovered within 140 d of pregnancy (n=9). Percentage of cells positive for CD90 was 73,0±17,2, for CD105 45,8±35,7, for CD14 5,6±11,2 and for CD45 13,4±11,3; no differences were observed among different gestational ages (p>0,05). ICC demonstrated low presence of SSEA4 positive cells (0,05%) in 1 sample, the presence of α-SMA in all 4 samples (mean 11,8±14.1%, range 2.5-32.4%) and the lack of Oct4 and SSEA1. RT-PCR analysis, performed at P4, showed expression of Nanog in 1/7 sample and weak expression in 4/7 samples. Low expression of SOX2 was detected in 2/7 samples. Conclusions - Based on these preliminary results, in the bovine AF there is an heterogeneous cell population containing also multipotent MSCs, identified by their differentiation ability and phenotypic characteristics typical of MSCs. Unlike human AF-MSCs, only a fraction of bAF-MSCs are positive for some pluripotency markers. Current work is ongoing to further characterize other cell phenotypes selected after long term culture.