Objectives: Human placenta is available as a discarded tissue, which provides a rich population of multipotent stem cells. Amongst them, HAECs represent a promising resource for regenerative medicine. In the present study we cultured HAECs in serum-free and defined media preserving their phenotypic and genetic traits. Then we verified the mesodermic differentiation capacity of HAECs and finally evaluated their pancreatic differentiative potential. We hypothesize that HAECs, cultured in such conditions, may show the same or better differentiation rate of HAECs cultured in standardized serum-rich media 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 qPCR 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 qPCR, fluorescence-activated cell sorting analysis or immune-fluorescence. 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 qPCR for key osteogenic markers such as OCN, RUNX2 and COL1A1. Preliminary pancreatic induction revealed expression of stemness marker NESTIN, typical of pancreatic progenitor cells, and pancreatic markers INSULIN and PDX1. Conclusion: 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 reliable, ethical-free source of insulin producing cells in clinical applications.
Human amniotic epithelial cells as a reliable source for diabetes stem-cell therapy / Okere, Brenard; Patianna, Viviana; Saraceni, Serena; Predieri, Barbara; Paolucci, Paolo; Iughetti, Lorenzo. - In: PEDIATRIC DIABETES. - ISSN 1399-5448. - STAMPA. - 13 (Suppl. 17):(2012), pp. 17-18. (Intervento presentato al convegno The 38th Annual Meeting of the International Society for Pediatric and Adolescent Diabetes (ISPAD) tenutosi a Istanbul, Turkey nel 10-13 October 2012).
Human amniotic epithelial cells as a reliable source for diabetes stem-cell therapy
PREDIERI, Barbara;IUGHETTI, Lorenzo
2012
Abstract
Objectives: Human placenta is available as a discarded tissue, which provides a rich population of multipotent stem cells. Amongst them, HAECs represent a promising resource for regenerative medicine. In the present study we cultured HAECs in serum-free and defined media preserving their phenotypic and genetic traits. Then we verified the mesodermic differentiation capacity of HAECs and finally evaluated their pancreatic differentiative potential. We hypothesize that HAECs, cultured in such conditions, may show the same or better differentiation rate of HAECs cultured in standardized serum-rich media 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 qPCR 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 qPCR, fluorescence-activated cell sorting analysis or immune-fluorescence. 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 qPCR for key osteogenic markers such as OCN, RUNX2 and COL1A1. Preliminary pancreatic induction revealed expression of stemness marker NESTIN, typical of pancreatic progenitor cells, and pancreatic markers INSULIN and PDX1. Conclusion: 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 reliable, ethical-free source of insulin producing cells in clinical applications.
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