Stemness characteristics and osteogenic potential of sheep amniotic epithelial cells

Abstract

We set out to characterize stemness properties and osteogenic potential of sheep AEC (amniotic epithelial cells). AEC were isolated from 3-month-old fetuses and expanded in vitro for 12 passages. The morphology, surface markers, stemness markers and osteogenic differentiation were inspected after 1, 6 and 12 passages of expansion, with an average doubling time of 24 h. AEC clearly expressed the stemness markers Oct-3/4 (octamer-binding protein-3/4), Nanog, Sox2 and TERT (telomerase reverse transcriptase) and displayed low levels of global DNA methylation. Culture had moderate effects on cell conditions; some adhesion molecules progressively disappeared from the cell surface, and the expression of Sox2 and TERT was slightly reduced while Nanog increased. No changes occurred in the levels of DNA methylation. Cells organized in 3D spheroids were used for IVD (in vitro differentiation). Within these structures the cells developed a complex intercellular organization that involved extensive intercellular coupling despite continuous cell migration. Marked deposition of calcein in the ECM (extracellular matrix), increased ALP (alkaline phosphatase) activity, expression of bone-related genes (osteocalcin) and the matrix mineralization shown by Alizarin Red staining demonstrate that AEC can undergo rapid and extensive osteogenic differentiation. AEC introduced in experimental bone lesions survived in the site of implantation for 45 days and supported consistent bone neoformation, thus showing promising potential applications in osteogenic regenerative medicine.

Rat full term amniotic fluid harbors highly potent stem cells

“…The constitutive existence of Tert gene in AFSCs has accredited the cells for having high proliferation capacity, hence, would be good for future applications. Nevertheless, some studies have also reported a short expression period of Tert gene, as the expression was found to be lost or lowly expressed after certain passages (Mattioli et al, 2012;Mauro et al, 2010). Interestingly, the in vitro system of our study shows that the rat full term AFSCs have been successfully cultured to more than 90 passages without the sign of senescence (data not shown).…”

Gestational stage affects amniotic epithelial cells phenotype, methylation status, immunomodulatory and stemness properties

“…In sheep, several of the previously described antigens and pluripotency associated transcription factors have been confirmed to be expressed [3, 20], thus showing a conserved behavior of this class of amniotic-derived cells amongst medium-sized mammals. This important feature allowed the use of ovine AECs (oAECs) in experimentally-induced defects to evaluate their regenerative properties and safety on an animal model of high translational value, in particular, for muscle skeleton diseases [3, 24, 25].…”

“…A conserved phenotype and expression profile of stemness markers were, firstly, demonstrated by Mattioli et al [3] regarding in particular the pluripotency associated transcripion factors OCT-4, SOX-2 and NANOG; on the contrary, TERT expression was detected in sheep AECs but not in the human counterparts [15]. The study of Mattioli et al [3] had also demonstrated the in vitro and in vivo function exerted by oAECs in sustaining osteogenic tissue lineage regeneration, suggesting an alternative cell source for repairing skeletal tissues disease.…”

“…Among them, OCT-4, NANOG and SOX2 represent the main transcription factors that guarantee the maintenance of self-renewal and pluripotency properties [15, 17]. In addition, several evidences have demonstrated that AECs express epithelial and mesenchymal specific markers [18, 19], providing a clinical use of these cells for both tissue lineages [3, 16, 20, 21]. More recently, AECs have been shown to be able to differentiate into cells expressing germ cell specific markers [22] and to differentiate into granulosa cells for restoring folliculogenesis in a mouse model of premature ovarian failure [23].…”

“…This important feature allowed the use of ovine AECs (oAECs) in experimentally-induced defects to evaluate their regenerative properties and safety on an animal model of high translational value, in particular, for muscle skeleton diseases [3, 24, 25]. …”