Isolation and in Vitro Characterization of Bovine Amniotic Fluid Derived Stem Cells at Different Trimesters of Pregnancy
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Amniotic fluid (AF) is a source 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 cells isolated from bovine AF as alternative sources of primitive multipotent stem cells in a species that could be a large-animal model for biomedical and biotechnology researches. Samples were recovered, at slaughterhouse, from 39 pregnant cows at different trimesters of pregnancy and cells were cultured in vitro. At passages (P) 3 and 7 differentiation towards chondrogenic, osteogenic and adipogenic lineages was induced. Flow cytometry analysis for CD90, CD105, CD73, CD44, CD34, CD45 and CD14 was performed, immunocytochemistry (ICC) for Oct4, SSEA4, α-SMA, Vimentin, N- and E- Cadherin and CK and qPCR analysis for OCT4, NANOG and SOX2 were carried out. The cell yield was significantly higher in the first trimester compared to the second and the third one (P < 0.05). Cells were isolated from 25/39 samples and cell population appeared heterogeneous. Two main cell types were identified in samples from all trimesters: round- (RS) and spindle-shaped (SS) cells. 17/25 samples showed both populations (mixed, MX). Both cell types showed MSC-markers and differentiation capability with some variability related to the passages. The SS-population also expressed low levels of stemness markers such as NANOG and SSEA4 but not OCT4. Bovine AF shows a heterogeneous cell population containing also MSCs, multipotent cells that represent an intermediate stage between embryonic stem cells and adult ones.
KeywordsAmniotic fluid Bovine Mesenchymal stem cells Differentiation Pluripotency
This research was funded by grant PRIN 2009 from MIUR and EPIHEALTH, EU FP7 n°278418.
The authors wish to thanks Professor Giuseppe Sarli (Pathological Anatomy Service, DIMEVET) for Vimentin clone 9 (DAKO M0725) antibody, and Professor Alessandra Scagliarini (Public Health and Animal pathology, DIMEVET) for positive controls for immunostaining analysis on CK protein. Thanks are also due to Dott. Chiara Sartori for the help given with first molecular analyses and to the students Valentina Montanari and Maria Chiara Corlianò for the help given during the culture and the differentiation of the samples.
The authors indicate no potential conflicts of interest.
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