| Background: Neurodegenerative disease is a state which is the losses of structure and function in brain and spinal cord neurons.This disease affects people’s memory and motion.The etiology and pathogenesis of this disease are still unclear.Neurons are not regenerative.Once the neurons are damaged,they are unrecoverable.At present,the treatments of neurodegenerative diseases only depend on a few of medicines in the clinic.Medicines can only delay the course of these diseases and treat the relieve symptoms.Neural Stem Cells(NSCs)can self-renew and have the ability to differentiate into neurons,astrocytes and oligodendrocytes.In view of the irreversible damage of neurons caused by neurodegenerative diseases,Neural stem cells as the treatment of neurodegenerative diseases has become the most popular treatment in recent years.However,it is difficult to obtain a large number of neural stem cells in the clinic.Mesenchymal Stem Cells(MSCs)as a source of seed cells have become a hot topic for scholars at home and abroad.Human Placenta Derived Mesenchymal Stem Cells(hPDMSCs)have rich resources and these cells are easy to collect.HPDMSCs have low immunogenicity,and have a variety of differentiation potentials.It has been reported in the literature that hPDMSCs have the ability to differentiate into nerve cells.However,the differentiated nerve cells have limited renewable capacity.It is unclear whether the hPDMSCs can be induced to differentiation into neural stem cells.Objective: Through the isolation,culture and identification of human placental mesenchymal stem cells,to study the ability of human placental mesenchymal stem cells to differentiate into neural stem cells and provide a theoretical basis for stem cell treatment of neurodegenerative diseases.Methods: The placenta of pregnant women with full-term cesarean section was selected from the Maternal and Child Health Hospital of Haidian District,Beijing.Human Placenta Derived Mesenchymal Stem Cells were isolated by mechanical separation and enzymatic digestion of the placenta.The cells were cultured,passaged and cryopreserved in the medium with 10% FBS.The surface specific markers of placenta mesenchymal stem cells were detected by flow cytometry.The cells were cultured with osteogenic,chondrogenic and adipogenic induction medium for 21 days and then these cells were dyed with alizarin red,toluidine blue,and oil red O to observe the potential differentiation of placental mesenchymal stem cells.Adding neurotrophic factor bFGF and EGF to N2 and B27,the differentiation of placental mesenchymal stem cells into neural stem cells included three steps.After the density of differentiated neural stem cells were over 90%,they were digested with 0.0125% trypsin,and these cells were detected by immunofluorescence to observe the expression of surface specific markers which were included SOX1,PAX6 and Nestin.Results: Human placental mesenchymal stem cells which were isolated and cultured in vitro were similar to fibroblasts in a fusiform shape.These cells were adherent.Flow cytometry showed that hPDMSCs were highly expressed CD90,CD73 C,CD105 and CD146.However the cells did not express CD34,CD31,CD45 RA and HLA-DR.HPDMSCs were cultured in the osteogenic differentiation complete medium for 21 days.Then cells were stained with red calcined nodules after staining with alizarin red.Cells were stained blue by toluidine blue staining after hPDMSCs were cultured in the chondrogenic differentiation complete medium for 21 days.After hPDMSCs were cultured in the adipogenic differentiation complete medium for 21 days,cells showed red round lipid droplets which were stained with oil red O.After nerve induction,cell body shrank and the refraction of the cells became stronger.The cells grew from fibroblasts to spherical cells of varying sizes.After immunofluorescence detection,hPDMSCs did not express SOX1,PAX6 and Nestin.However neural stem cells differentiated from hPDMSCs were highly expressed SOX1,PAX6 and Nestin.Conclusions: In this experiment,human placental mesenchymal stem cells were successfully isolated by mechanical and enzymatic digestion.The biological characteristics of hPDMSCs were similar to those of other sources of mesenchymal stem cells.The isolated cells had the potential to differentiate into osteogenic,chondrogenic and adipogenic cells.Neural stem cells can be successfully obtained after induction by neurotrophic factors.The successful induction provided a theoretical basis in the clinic for obtaining a large number of neural stem cells to treat neurodegenerative diseases. |
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