ABSTRACT
BACKGROUND:Placental mesenchymal stem cel s with rich sources are similar to bone marrow mesenchymal stem cel s in terms of morphology, surface markers and differentiation potential, which are one of ideal mesenchymal stem cel s in human body. However, there are few studies addressing the ultrastructure and phagocytotic function of human placental mesenchymal stem cel s and its physiological role in the the placenta has been little explored. OBJECTIVE:To investigate the ultrastrcture and phagocytotic function of placental mesenchymal stem cel s. METHODS:Placental mesenchymal stem cel s obtained from five placentae of normal pregnancy were cultured in vitro and observed for ultrastructure under transmission electron microscope. The fluorescent beads were added in the supernatant for 3 hours, and then the phagocytosis of placental mesenchymal stem cel s was evaluated by flow cytometry. RESULTS AND CONCLUSION:Under the transmission electron microscope, placental mesenchymal stem cel s had large nuclei with prominent nucleoli. In the cytoplasm, a plenty of rough endoplasmic reticula was seen, dilated or stacked. The cytoplasm was also rich in Golgi apparatus and lysosomes. The cel surfaces were covered by microvil i. The intercel ular junctions could be seen occasional y. A part of cel s from these five samples could phagocytose fluorescence beads, which ranged from 49.6%to 18.4%. The ultrastructural characteristics of placental mesenchymal stem cel s suggested these cel s were active to synthesize and secrete proteins and had phagocytotic function, indicating placental mesenchymal stem cel s may play a role in keeping the balance of micro-environments and clean the foreign substances in the placenta.
ABSTRACT
BACKGROUND:Cytotrophoblasts in placental cell components plays an important role in fetal immunological tolerance.Placental mesenchymal stem cells(pMSCs)have potential of multiple differentiation and inhibition of lymphocyte proliferation.However,conventional methods cannot acquire a large amount of purified human cytotrophoblasts or pMSCs.OBJECTIVE:To establish a method to obtain large placenta tissue,and harvest plenty of cytotrophoblasts and pMSCs with high purity and activity.METHODS:Human placenta tissues were dissected,minced,and dissociated in trypsin and DNAse I.The dissociation was performed in three stages of incubation at 180 r/min for 20 minutes at 37 ℃ The digesting suspension was filtered using a 200 mesh strainer before separated by Percoll gradients.The cytotrophoblast cells and pMSCs fractions were collected respectively.Fibroblasts of cytotrophoblast cells fraction were removed by differential adhesion.The pMSCs were seeds on 75-cm2 flask directly for culture.The dissociation of placenta tissue was observed.The number of harvesting cytotrophoblasts was quantified and Cytokeratin 7 expression was tested.The pMSCs primary culture time,cell passage,induced osteoblast differentiation were observed.The cell surface makers were also detected.RESULTS AND CONCLUSION:After digesting in trypsin and DNAse I,there was only little residue left.(5.48±1,98)×10~8 cytotrophoblasts were obtained after differential adhesion.(90±4.36)% of these cells were positive for Cytokeratin 7.At 19-21 days after pMSCs reached approximately 90% confluency,the cell number was(1.96±0.24)×10~6.The subcultre cells could be passaged again in 4 or 5 days.Flow cytometric analysis of pMSCs showed that the cells expressed CD29,CD44 and HLA-ABC intensively and were negative for CD34,CD45,CD14 and HLA-DR.pMSCs differentiated into osteoblast-like cells after induction,which stained bright salmon pink by Alizarin Red.Dissociating the placenta tissue in trypsin and DNAse I in combination with discontinuous Percoll gradient separation obtained a large number of cytotrophoblasts and pMSCs recovered from placenta tissue,with high purity and activity.