Role of cerebrospinal fluid in differentiation of human dental pulp stem cells into neuron-like cells / 대한해부학회지

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.

Role of cerebrospinal fluid in differentiation of human dental pulp stem cells into neuron-like cells / 대한해부학회지

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.

Condition medium of cerebrospinal fluid and retinoic acid induces the transdifferentiation of human dental pulp stem cells into neuroglia and neural like cells / 대한해부학회지

Cerebrospinal fluid (CSF) contains several molecules which are essential for neurogenesis. Human dental pulp stem cells (hDPSCs) are putatively neural crest cell-derived that can differentiate into neurons and glial cells under appropriate neurotrophic factors. The aim of this study was to induce differentiation of hDPSCs into neuroglial phenotypes using retinoic acid (RA) and CSF. The hDPSCs from an impacted third molar were isolated by mechanical and digestion and cultured. The cells have treated by 10⁻⁷µM RA (RA group) for 8 days, 10% CSF (CSF group) for 8 days and RA with CSF for 8 days (RA/CSF group). Nestin, microtubule-associated protein 2 (MAP2), and glial fibrillary acidic protein immunostaining were used to examine the differentiated cells. Axonal outgrowth was detected using Bielschowsky's silver impregnation method and Nissl bodies were stained in differentiated cells by Cresyl violet. The morphology of differentiated cells in treated groups was significantly changed after 3–5 days. The results of immunocytochemistry showed the presence of neuroprogenitor marker nestin was seen in all groups. However, the high percentage of nestin positive cells and MAP2, as mature neural markers, were observed at the pre-induction and induction stage, respectively. Nissl bodies were detected as dark-blue particles in the cytoplasm of treated cells. Our findings showed the RA as pre-inducer and CSF as inducer for using in vitro differentiation of neuron-like cells and neuroglial cells from hDPSCs.

[ influence of cerebrospinal fluid accompanied by retinoic acid on differentiation of bone marrow mesenchymal stem cells into neuron-like cells in vitro]

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

Expression of spermatogonial and pluripotency markers in spermatogonial stem cells after treatment with different culture factors

As condition and component of culture determine fate map of spermatogonial stem cells [SSCs], the aim of this study was to evaluate of growth factors GDNF, LIF and RA on proliferation and differentiation of SSC. SSCs were cultured in two groups: The first group GDNF and LIF and the second group RA. The number of clumps and colony formation was monitored during 1 month in culture. To identification of the colony, stained with PLZF using immunostaining. Pluripotency gene Oct 4 and neural markers MAP2, NeuroD and Nestin were analyzed by RT-PCR. In the presence of GDNF and LIF, cells proliferated rapidly and many compact clumps were appeared whereas after exposure to RA cells formed small clumps. The results of immunocytochemistry shows PLZF was detected in the group GDNF and LIF. RT-PCR showed high level expression Oct 4 in the group GDNF and LIF whereas neural markers MAP2, NeuroD and Nestin were expressed in the group RA. GDNF and LIF are essential for self-renewal and colony formation of SSCs that confirm the stem cells activity of these cells but RA inhibits stem cell activity of SSCs and induces neural differentiation of these

Functional concentrations of BMP4 on differentiation of mouse embryonic stem cells to primordial germ cells

Bone morphogenetic protein 4 [BMP4] has a significant role in primordial germ cells [PGCs] differentiation from mouse embryonic stem cell [mESC]. The aim of this study is to determine the best concentration of BMP4 at a time of two days on differentiation PGCs from mESC. To differentiate PGCs, embryoid bodies [EBs] from mESCs were cultured in concentrations of 0, 5 and 10 ng/ml BMP4 for two days. Germ cell markers Oct4 [Pou5f1], Stella [Dppa3] and Mvh [Ddx4] were analyzed by flow cytometry, immunocytochemistry and reverse transcriptase polymerase chain reaction [RT-PCR]. Flow cytometry data demonstrated most Mvh-positive cells were observed only in the treated groups. Immunocytochemistry of EBs in the treated groups identified cells positive for Mvh. PCR results showed expression of Oct4 in the control group and treated groups. Stella and Mvh were expressed only in the treated groups. Low concentrations of BMP4 during two days had an optimal effect on differentiation of PGCs from mESC