Spheroids from equine amnion mesenchymal stem cells: an in vitro study

Alessandra Coli, Maria Rita Stornelli, Francesca Nocchi, Roberta Lamanna, Mariacarla Iorio, Simone Lapi, Patrizia Urciuoli, Carla Lenzi, Elisabetta Giannessi


Background: Equine amnion mesenchymal stem cells (EAMSCs) from amnion isolated after the foal birth represented an alternative source of easy collection of mesenchymal cells used in equine regenerative medicine.

Methods: These cells grown as 2D culture in α-MEM medium supplemented with EGF were differentiated in adipogenic, chondrogenic and osteogenic cells. Half a million cells as pellet were left in 15ml tubes with the same differentiation media for 20 days. After the pellets were collected, embedded in paraffin for morphological study.

Results: 2D culture showed EAMSCs  with an embryonic phenotype (C-kit+, CD105+, Oct-4+) and a differentiation potential in adipogenic, chondrogenic and osteogenic multipotent cells. By a reproducible method of 3D culture, at day 20 the Authors evidenced a formation of small aggregated spheroids gradually gathering. In cross sections the surface of spheroid evidenced flattened cells embedded in a red matrix by Alizarin staining and occasionally a core of calcium precipitation. A network of apoptotic or necrotic cells in a not mineralized matrix was present into the center of nodules.  The 3D spheroids appeared larger (mean diameter of 605±53 µm for gathering spheroids and 1486±79 µm for spheroids already gathered) than those from standard monolayer cultures (mean diameter of 200 ± 73 µm).

Conclusions: EAMSCs cultured in 3D method preserve their in vitro multipotent differentiation than adherent 2D culture method. These EAMSCs included in extracellular matrix not mineralized at day 20 seem to be a good source of MSCs for tissue repair and regeneration in equine medicine.


Equine; Amnion; Mesenchymal stem cells; Cell culture; Spheroid; Immunohistochemistry


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DOI: http://dx.doi.org/10.18035/emj.v3i1.299


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