RESUMO
RESUMEN: Las células epiteliales del amnios (hAECs) son células madre pluripotenciales; tienen capacidad de diferenciarse en células de las tres capas embrionarias. Como tales, se utilizan en algunas terapias regenerativas en medicina. Este estudio tiene por objetivo describir un protocolo de aislación de las células epiteliales del amnios (hAECs) a partir de placentas humanas de partos por cesárea, así como su caracterización y comportamiento in vitro. Se aislaron hAECs de 20 placentas de partos por cesárea con un protocolo optimizado. Se caracterizaron las células mediante citometría de flujo, microscopia óptica y de fluorescencia, y se evaluó la proliferación de las células mediante MTT a los 1, 3, 5 y 7 días con y sin β-mercaptoetanol en el medio de cultivo. El análisis histológico del amnios mostró un desprendimiento prácticamente completo de las células después de la segunda digestión del amnios. El promedio de células obtenidas fue de 10.97 millones de células por gramo de amnios. Las hAECs mostraron una proliferación limitada, la cual no fue favorecida por la adición de β-mercaptoetanol en el cultivo. Se observó un cambio de morfología espontanea de epitelial a mesenquimal después del cuarto pasaje. Las células epiteliales del amnios pueden ser aisladas con un protocolo simple y efectivo, sin embargo, presentan escasa capacidad proliferativa. Bajo las condiciones de este estudio, la adición de β-mercaptoetanol no favorece la capacidad proliferativa de las células.
SUMMARY: human amnion epithelial cells (hAECs) are pluripotent stem cells; they have the ability to differentiate into cells of the three embryonic layers, and are used in various regenerative therapies in medicine. This study aims to describe a protocol for the isolation of amnion epithelial cells (hAECs) from human placentas from cesarean delivery, as well as their characterization and culture conditions in vitro. hAECs were isolated from 20 cesarean delivery placentas with an optimized protocol. The cells were characterized by flow cytometry, light and fluorescence microscopy, and the proliferation of the cells was evaluated by MTT at 1, 3, 5 and 7 days with and without β-mercaptoethanol in the culture medium. Histological analysis of the amnion showed a practically complete detachment of the cells of the underlying membrane after the second digestion. The average number of cells obtained was 10.97 million cells per amnion. The hAECs perform a limited proliferation rate, which was not favored by the addition of β-mercaptoethanol in the culture. A spontaneous morphology change from epithelial to mesenchymal morphology is exhibited after the fourth passage. The epithelial cells of the amnion can be isolated with a simple and effective protocol, however, they present little proliferative capacity. Under the conditions of this study, the addition of β-mercaptoethanol does not favor the proliferation of the cells.
Assuntos
Humanos , Separação Celular/métodos , Células Epiteliais/citologia , Âmnio/citologia , Citometria de Fluxo , MicroscopiaRESUMO
The aim of this study was to analyze the suitability of pluripotent stem cells derived from the amnion (hAECs) as a potential cell source for revitalization in vitro. hAECs were isolated from human placentas, and dental pulp stem cells (hDPSCs) and dentin matrix proteins (eDMPs) were obtained from human teeth. Both hAECs and hDPSCs were cultured with 10% FBS, eDMPs and an osteogenic differentiation medium (StemPro). Viability was assessed by MTT and cell adherence to dentin was evaluated by scanning electron microscopy. Furthermore, the expression of mineralization-, odontogenic differentiation- and epithelial-mesenchymal transition-associated genes was analyzed by quantitative real-time PCR, and mineralization was evaluated through Alizarin Red staining. The viability of hAECs was significantly lower compared with hDPSCs in all groups and at all time points. Both hAECs and hDPSCs adhered to dentin and were homogeneously distributed. The regulation of odontoblast differentiation- and mineralization-associated genes showed the lack of transition of hAECs into an odontoblastic phenotype; however, genes associated with epithelial-mesenchymal transition were significantly upregulated in hAECs. hAECs showed small amounts of calcium deposition after osteogenic differentiation with StemPro. Pluripotent hAECs adhere on dentin and possess the capacity to mineralize. However, they presented an unfavorable proliferation behavior and failed to undergo odontoblastic transition.