Anat Cell Biol.  2020 Sep;53(3):292-300. 10.5115/acb.19.241.

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

• Goudarzi G
• Hamidabadi HG
• Bojnordi MN
• Hedayatpour A
• Niapour A
• Zahiri M
• Absalan F
• Darabi S

• KMID: 2507642
• DOI: http://doi.org/10.5115/acb.19.241

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.

Keyword

Human dental pulp stem cells; Cerebrospinal fluid; Cell transdifferentiation; Alzheimer

Figure

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  Fig. 1 Phase contrast microscopy photograph. (A) Fbroblast like cells of human dental pulp stem cells with spindle-shaped morphology were observed. (B) Morphological modifications of human dental pulp stem cells 8 days after induction by the inverted microscope. Scale bars=100 µm (A, B).

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  Fig. 2 The comparison between the mean viability rates at experimental and control groups in days (D) D2, D4, D6, and D8 (P<0.05).

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  Fig. 3 Cresyl violet staining. Human dental pulp stem cells at experimental groups on day 8. Scale bar=10 µm.

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  Fig. 4 Bielschowsky’s silver staining at experiment group in days 4 (A) and 8 (B) (Scale bars=10 µm). (C) Comparing the rate of axon growth between the experimental group on days 4 and 8 with control group (P<0.01).

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  Fig. 5 Immunocytochemistry of neuron-like differentiated cells from human dental pulp stem cells. Expression of nestin the (A) control and (B) experimental groups on day 3, (C) expression of MAP2 in the experimental group on day 8. Scale bars=10 µm (A–C).

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  Fig. 6 Real-time polymerase chain reaction (PCR) analysis for neural markers during in vitro differentiation of human dental pulp stem cells (n=3 biological samples, mean±standard deviation; *P<0.05, **P<0.01, ***P<0.001).

Reference

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