Mesenchymal stem cells from amnion and amniotic fluid in the bovine

Amnion and amniotic fluid (AF) are noncontroversial and inexhaustible sources of mesenchymal stem cells (MSCs) that can be harvested noninvasively at low cost. As in humans, also in veterinary field, presumptive stem cells derived from these tissues reveal as promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. The aim of this work is to obtain and characterize, for the first time in bovine species, presumptive MSCs from the epithelial portion of the amnion (AECs) and from the AF (AF-MSCs) to be used for clinical applications. AECs display a polygonal morphology, whereas AF-MSCs exhibit a fibroblastic-like morphology only starting from the second passage, being heterogeneous during the primary culture. For both lines, the proliferative ability has been found constant over the ten passages studied and AECs show a statistically lower (P<0.05) doubling time with respect to AF-MSCs. AECs express MSC-specific markers (ITGB1 (CD29), CD44, ALCAM (CD166), ENG (CD105), and NT5E (CD73)) from P1 to P3; in AF-MSCs, only ITGB1, CD44, and ALCAM mRNAs are detected; NT5E is expressed from P2 and ENG has not been found at any passage. AF-MSCs and AECs are positive for the pluripotent markers (POU5F1 (OCT4) and MYC (c-Myc)) and lack of the hematopoietic markers. When appropriately induced, both cell lines are capable of differentiating into ectodermal and mesodermal lineages. This study contributes to reinforce the emerging importance of these cells as ideal tools in veterinary medicine. A deeper evaluation of the immunological properties needs to be performed in order to better understand their role in cellular therapy.