Objective: The purpose of this work is to analyze stem cells properties and the osteogenic potential of sheep amniotic epithelial cells (AEC), to evaluate the ability of osteogenic differentiation and their potential use in tissue regeneration. Methods : AEC were isolated from 3 months sheep. Cell morphology and expression of surface markers, in particular stem cell markers, were analyzed by flow citometry and fluorescence microscopy at 1, 6 and 12 passages of expansion in vitro. Cells were cultivated in an osteogenic medium for osteogenic differentiation. Differentiated cells were analyzed for calcein deposition and extracellular matrix deposition by fluorescence. Osteogenic differentiation was checked by Alizarin Red and RT-PCR evaluation of gene expression of molecules related to bone tissue. These cells after differentiation were implanted in experimental defects in vivo and analyzed by microscopic investigation after 45 days in order to evaluate their osteoregenerative potential. Results: Flow citometry analysis showed the presence of typical markers for AEC: Oct ¾, Nanog, Sox2 and TERT. Culture passage has moderate effects on cellular conditions, some adhesion molecules gradually disappear from cell surface, expression of Sox2 and TERT is reduced, NANOG increased. It has been observed a marked calcein deposit in the extracellular matrix, increased ALP activity, OCN and COL1 expression and mineralization of the matrix as revealed by Alizarin Red. Conclusions: Our in vitro studies confirm the hypotheses about the high potential of AEC to differentiate into multiple cell lines. The results obtained confirms that AEC can undergo rapid and extensive osteogenic differentiation and extracellular matrix mineralization in vitro. AEC introduced in pre-clinical experimental bone lesions still maintains stemness characteristics as they are still recognizable in the implant site for 45 days. Histological analysis demonstrated after 45 days, the possibility to identify abundant new bone formation in continuity with the existing bone, index of integration
Sheep amniotic epithelial cells: stemness characteristics and osteogenic potential
TETE', Stefano;
2012-01-01
Abstract
Objective: The purpose of this work is to analyze stem cells properties and the osteogenic potential of sheep amniotic epithelial cells (AEC), to evaluate the ability of osteogenic differentiation and their potential use in tissue regeneration. Methods : AEC were isolated from 3 months sheep. Cell morphology and expression of surface markers, in particular stem cell markers, were analyzed by flow citometry and fluorescence microscopy at 1, 6 and 12 passages of expansion in vitro. Cells were cultivated in an osteogenic medium for osteogenic differentiation. Differentiated cells were analyzed for calcein deposition and extracellular matrix deposition by fluorescence. Osteogenic differentiation was checked by Alizarin Red and RT-PCR evaluation of gene expression of molecules related to bone tissue. These cells after differentiation were implanted in experimental defects in vivo and analyzed by microscopic investigation after 45 days in order to evaluate their osteoregenerative potential. Results: Flow citometry analysis showed the presence of typical markers for AEC: Oct ¾, Nanog, Sox2 and TERT. Culture passage has moderate effects on cellular conditions, some adhesion molecules gradually disappear from cell surface, expression of Sox2 and TERT is reduced, NANOG increased. It has been observed a marked calcein deposit in the extracellular matrix, increased ALP activity, OCN and COL1 expression and mineralization of the matrix as revealed by Alizarin Red. Conclusions: Our in vitro studies confirm the hypotheses about the high potential of AEC to differentiate into multiple cell lines. The results obtained confirms that AEC can undergo rapid and extensive osteogenic differentiation and extracellular matrix mineralization in vitro. AEC introduced in pre-clinical experimental bone lesions still maintains stemness characteristics as they are still recognizable in the implant site for 45 days. Histological analysis demonstrated after 45 days, the possibility to identify abundant new bone formation in continuity with the existing bone, index of integrationI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
