of any ethical concern, providing a rich population of multipotent stem cells. Amongst them, HAECs represent a potentially unlimited source of functional pancreatic endocrine cells. Objective and hypotheses: The aim of our study is to culture HAECs in serum-free condition preserving their phenotypic and genetic traits. Then we verify the capacity of HAECs to differentiate into at least one mesodermic lineage and we evaluate their pancreatic differentiative potential. We hypothesize that HAECs, cultured in serum-free media may show the same or better differentiation rate of HAECs cultured in standardized serum-rich conditions when induced into insulin producing cells Methods: Placenta samples were collected, HAECs were isolated and cultured in standard serum-rich medium and serum-free optimized media. We used RT-PCR to assess stem cell markers. Mesodermic osteogenic induction was performed for each media using the same induction cocktail. Differentiation was evaluated through Alizarin red staining and RT-PCR for osteogenic gene expression. To study HAECs’ pancreatic differentiation ability, nicotinamide was added to each medium. Pancreatic markers expression and immune-phenotype profile were assessed respectively with RT-PCR and fluorescence-activated cell sorting analysis. Results: Serum-free media sustained HAECs’ growth and stem cell potential (OCT4, NANOG, SOX2). Alizarin red assay showed mineralization in all the culture conditions, confirmed by RT-PCR for key osteogenic markers such as OCN RUNX2 and COL1A1. Preliminary pancreatic induction revealed expression of stemness marker NESTIN, common in pancreatic progenitor cells, and pancreatic markers INSULIN and NKX2.2. Conclusions: These data indicate that serum is not essential for HAECs culture and differentiation. Serum-free culture conditions could simplify the transition from laboratory to clinical practice. For this reason HAECs might be a possible and reliable source of insulin producing cells in clinical applications
Human amniotic epithelial cells (HAECs): a reliable source of insulin producing cells? / Okere, Bernard; Patianna, VIVIANA DORA; S., Saraceni; Predieri, Barbara; Paolucci, Paolo; Iughetti, Lorenzo. - In: HORMONE RESEARCH IN PAEDIATRICS. - ISSN 1663-2818. - STAMPA. - 78(suppl 1):(2012), pp. 21-21. (Intervento presentato al convegno 51th Annual Meeting of the European Society for Paediatric Endocrinology tenutosi a Leipzig nel September 20–23, 2012).
Human amniotic epithelial cells (HAECs): a reliable source of insulin producing cells?
OKERE, Bernard;PATIANNA, VIVIANA DORA;PREDIERI, Barbara;PAOLUCCI, Paolo;IUGHETTI, Lorenzo
2012
Abstract
of any ethical concern, providing a rich population of multipotent stem cells. Amongst them, HAECs represent a potentially unlimited source of functional pancreatic endocrine cells. Objective and hypotheses: The aim of our study is to culture HAECs in serum-free condition preserving their phenotypic and genetic traits. Then we verify the capacity of HAECs to differentiate into at least one mesodermic lineage and we evaluate their pancreatic differentiative potential. We hypothesize that HAECs, cultured in serum-free media may show the same or better differentiation rate of HAECs cultured in standardized serum-rich conditions when induced into insulin producing cells Methods: Placenta samples were collected, HAECs were isolated and cultured in standard serum-rich medium and serum-free optimized media. We used RT-PCR to assess stem cell markers. Mesodermic osteogenic induction was performed for each media using the same induction cocktail. Differentiation was evaluated through Alizarin red staining and RT-PCR for osteogenic gene expression. To study HAECs’ pancreatic differentiation ability, nicotinamide was added to each medium. Pancreatic markers expression and immune-phenotype profile were assessed respectively with RT-PCR and fluorescence-activated cell sorting analysis. Results: Serum-free media sustained HAECs’ growth and stem cell potential (OCT4, NANOG, SOX2). Alizarin red assay showed mineralization in all the culture conditions, confirmed by RT-PCR for key osteogenic markers such as OCN RUNX2 and COL1A1. Preliminary pancreatic induction revealed expression of stemness marker NESTIN, common in pancreatic progenitor cells, and pancreatic markers INSULIN and NKX2.2. Conclusions: These data indicate that serum is not essential for HAECs culture and differentiation. Serum-free culture conditions could simplify the transition from laboratory to clinical practice. For this reason HAECs might be a possible and reliable source of insulin producing cells in clinical applications
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