ABSTRACT
Human dental pulp stem cells (hDPSCs) could be differentiated into neuron like-cells under particular microenvironments. It has been reported that a wide range of factors, presented in cerebrospinal fluid (CSF), playing part in neuronal differentiation during embryonic stages, we herein introduce a novel culture media complex to differentiate hDPSCs into neuron-like cells. The hDPSCs were initially isolated and characterized. The CSF was prepared from the Cisterna magna of 19-day-old Wistar rat embryos, embryonic cerebrospinal fluid (E-CSF). The hDPSCs were treated by 5% E-CSF for 2 days, then neurospheres were cultured in DMEM/F12 supplemented with 10-6 μm retinoic acid (RA), glialderived neurotrophic factor and brain-derived neurotrophic factor for 6 days. The cells which were cultured in basic culture medium were considered as control group. Morphology of differentiated cells as well as process elongation were examined by an inverted microscope. In addition, the neural differentiation markers (Nestin and MAP2) were studied employing immunocytochemistry. Neuronal-like processes appeared 8 days after treatment. Neural progenitor marker (Nestin) and a mature neural marker (MAP2) were expressed in treated group. Moreover Nissl bodies were found in the cytoplasm of treated group. Taking these together, we have designed a simple protocol for generating neuron-like cells using CSF from the hDPSCs, applicable for cell therapy in several neurodegenerative disorders including Alzheimer’s disease.
ABSTRACT
Human dental pulp stem cells (hDPSCs) could be differentiated into neuron like-cells under particular microenvironments. It has been reported that a wide range of factors, presented in cerebrospinal fluid (CSF), playing part in neuronal differentiation during embryonic stages, we herein introduce a novel culture media complex to differentiate hDPSCs into neuron-like cells. The hDPSCs were initially isolated and characterized. The CSF was prepared from the Cisterna magna of 19-day-old Wistar rat embryos, embryonic cerebrospinal fluid (E-CSF). The hDPSCs were treated by 5% E-CSF for 2 days, then neurospheres were cultured in DMEM/F12 supplemented with 10-6 μm retinoic acid (RA), glialderived neurotrophic factor and brain-derived neurotrophic factor for 6 days. The cells which were cultured in basic culture medium were considered as control group. Morphology of differentiated cells as well as process elongation were examined by an inverted microscope. In addition, the neural differentiation markers (Nestin and MAP2) were studied employing immunocytochemistry. Neuronal-like processes appeared 8 days after treatment. Neural progenitor marker (Nestin) and a mature neural marker (MAP2) were expressed in treated group. Moreover Nissl bodies were found in the cytoplasm of treated group. Taking these together, we have designed a simple protocol for generating neuron-like cells using CSF from the hDPSCs, applicable for cell therapy in several neurodegenerative disorders including Alzheimer’s disease.
ABSTRACT
Objective: Cerebrospinal fluid [CSF] has a broad range of molecules and neurotrophic factors essential for neurogenesis. Bone marrow mesenchymal stem cells [BMSCs] are multipotent stem cells that can differentiate into the cells with neural-like phenotype under the induction of appropriate growth factors. According to the significant role of retinoic acid [RA] in neurogenesis, this study aims to differentiate BMSCs into neuronlike cells using CSF, RA, and the combination of CSF and RA
Methods: Rat BMSCs were isolated and characterized. The CSF was prepared from the cisterna magna of 19-day-old Wistar rat embryos. The BMSCs were induced by either 5% CSF [CSF group], 10-6 microM RA [RA group], or CSF plus RA [CSR group] for 12 days. Morphology of differentiated cells was examined by inverted microscope and axonal outgrowth measured using Image J software. In addition, the expression of neural-specific markers [Nestin and MAP-2] was examined by immunocytochemistry
Results: We observed specific-neuronal morphology in the differentiated cells. The maximum axon length was seen in the CSR group on the 12th day of induction. Immunocytochemistry results showed that the neural progenitor marker [Nestin] was expressed in all treated groups. However, MAP-2, as a mature neural marker, was only expressed in the CSR group
Conclusion: The findings suggest that CSF accompanied RA lead to differentiation of cells with neuronal and glial phenotypes from BMSCs in vitro