Anat Cell Biol 2020; 53(3): 292-300
Published online September 30, 2020
Copyright © Korean Association of ANATOMISTS.
1Department of Brain and Cognitive Sciences, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, 2Department of Anatomy & Cell Biology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, 3Immunogenetic Research Center, Department of Anatomy & Cell Biology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, 4Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, 5Research Laboratory for Embryology and Stem Cells, Department of Anatomical Sciences, School of Medicine, Ardabil University of Medical Sciences, Ardabil, 6Department of Anatomical Sciences, School of Medical Sciences, Bushehr University of Medical Sciences, Bushehr, 7Abadan School of Medical Sciences, Abadan, 8Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran
Correspondence to:Hatef Ghasemi HamidabadI
Department of Anatomy & Cell Biology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari 33044756, Iran
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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), glial-derived 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.
Keywords: Human dental pulp stem cells, Cerebrospinal fluid, Cell transdifferentiation, Alzheimer
Degeneration of neurons is one of the features of neurodegenerative disease that have a major impact at quality of patient’s life. Considering that this type of disorder are rapidly rising, it is important to access a strategy to disease prevention. A treatment that has recently attracted the attention of researchers is the differentiation of stem cells into neuron-like cells under specific microenvironment and transplanted them to the lesion. However, none of them cannot prevent the progression of disease. In this way, the material that seems useful is cerebrospinal fluid (CSF). CSF produced by Choroid plexus with epithelial and vascular structure. To date, the findings has revealed that CSF has some microenvironment which are necessary for neurogenesis such as transforming growth factor-β (TGF-β), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and basic fibroblast growth factor (b-FGF) . Several research have shown the role of CSF factors on proliferation and differentiation of mesenchymal stem cells (MSCs) into neuron-like cells through the interacting by specific receptor on the cell surface. This findings suggest that the tissue microenvironment can induce the differentiation [2,3]. According to our previous study, we investigated the influence of CSF accompanied by retinoic acid (RA) on differentiation of MMSCs into neuron-like Cells
In this experimental study, the third molar teeth without caries were collected under confirmed guidelines set by Dental Clinic of Mazandaran University of Medical Science with informed consent of the participants and in according to Helsinki Declaration. The surfaces of the teeth were cleaned by phosphate buffered saline (PBS), then the pulp tissue was gently separated and cut into small pieces under sterile conditions and enzymatically digested in digestion solution which contained the trypsin 0.25% (Gibco, Gran Island, NY, USA) enzyme, in the next step centrifuged at 800 rpm at 4°C for 5 minutes, then the supernatant was removed. The isolated cells were grown in DMEM/F12 (Gibco-Life Technologies, Invitrogen, Paisley, Scotland, UK) supplemented with 15% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA), L-glutamine (Gibco), Penicillin (100 units/ml) and Streptomycin (100 mg/ml) (both from Sigma), half of the culture medium was renewed every 2–3 days that coincides with monitoring the cells in terms of growth and morphological features via inverted microscope [11,15].
CSF was collected from 19-day-old Wistar rat embryos. In sterile conditions, CSF was collected from Cisterna magna region and transferred into sterile micro tubes. After centrifugation (14,000 rpm, 5 minutes, room temperature), the supernatant was kept in –80ºC temperature [1,16,17].
Neurospheres formation of hDPSCs was performed as reported by Darabi et al. . Briefly, 2×105 the hDPSCs cells were added on a 24-well culture plate in medium containing serum-free DMEM/F12 with 5% embryonic cerebrospinal fluid (E-CSF) for 2 days. Then, the neurospheres were cultured in the 6-well plate in DMEM/F12 supplemented with 10-6 RA, 5% FBS, 2% B27, 100 ng/ml glial-derived neurotrophic factor (GDNF, Invitrogen, Paisley, Scotland) and 200 ng/ml BDNF (Invitrogen, Paisley, Scotland) for 6 days. In addition, 2×105 the hDPSCs cells were cultured on a 24-well culture plate in basic culture medium for 2 days, and then the neurosphers were transferred into the 6-well plate in DMEM/F12 supplemented with 5% FBS, 2% B27, 100 ng/ml GDNF and 200 ng/ml BDNF for 6 days. The cells which were cultured in basic culture medium were considered as control group.
The cell metabolic activity of isolated hDPSCs quantitatively was assesed by the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) according to manufacturer instructions (Sigma-Aldrich). Briefly, hDPSCs were seeded (4×104 cells/well, in triplicate) in a 6-well plate. After incubation, under standard conditions of temperature and humidity, removed the supernatant and the cells were exposed to 50 μl of Dimethyl sulfoxide (Sigma-Aldrich), then placed onto a roller for 5–10 minutes to dissolve the formazan crystals. Finally, the absorbance measured at 570 nm by a Cytofluor 4000 plate reader (PerSeptive Biosystems, Framingham, MA, USA) .
Morphologically differentiated cells were daily analyzed by inverted microscope (Eclipse-TS100; Nikon, Tokyo, Japan). In addition, the longest protrusion in each cell was considered as neurite out growth and the length of these processes was measured on days 4 and 8 after differentiation by Image J 1.41 software (National Institute of Health, Bethesda, MD, USA).
Expression of neuronal markers, Nestin and MAP2, was evaluated in differentiated cells using Immunocytochemistry. After three times washing with PBS, differentiated cells were fixed using 4% PFA. Then, the fixed cells were washed with PBS three times after 15 minutes. In the next step, to penetrate cells and inhibition of non-specific antigens, probably attached to secondary antibodies, they were incubated with Triton X-100 (Sigma-Aldrich) for 30 minutes and bovine serum albumin (BSA, Sigma-Aldrich) for 45 minutes at room temperature, respectively. Then, the cells were incubated for one hour with primary monoclonal antibodies. After three times washing with PBS, the cells were kept with suitable secondary antibodies conjugated with Phycoerythrin (PE, Sigma-Aldrich) for one hour at 37°C. Then, DAPI (Sigma-Aldrich) was used for staining the nucleus. Finally, the neural markers were examined using fluorescent microscopes (Eclipse-TE600, Nikon) and imaging took place .
Total RNA was harvested from cell groups at different stages of cell differentiation using Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), after quantification, 5 μg RNA was reverse-transcribed into cDNA using oligo dT primer and reverse transcriptase (Fermentas), according to the manufacturers’ instructions. In this regard, random primers (Fermentas) were used, under standard conditions. Real-time polymerase chain reaction (PCR) amplifications were conducted for 3 minutes at 95°C and PCR cycling conditions were 95°C, 30 seconds; 60°C, 45 seconds; and 72°C, 45 seconds; for 40 cycles and 72°C for 7 minutes for the final extension. For the real-time PCR analysis, the final products were separated on a 1.5% agarose gele (Fermentas) and detected with ethidium bromide, visualized and photographed on a UV transluminator. The sequence of the primers which are considered in this experiment are described in Table 1.
Statistical analysis was performed by SPSS for Windows, Version 16.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA and Chi Square test were used to analyze the data based on the comparison of the mean quantitative variables. In addition, significant level was considered as
At the onset of cell culture, the hDPSCs were appeared elongated-shaped. The cells could be proliferated consistently and formed colony. Following, the cells were cultured and the sub-cultured cells exhibited fibroblast-like morphology and their phenotypes were kept during passages 3–5 (Fig. 1A).
Our morphological assay proved that specific neuronal morphological changes were observed with bipolar and multipolar soma, large euchromatin nuclear, clear nucleolus and a cytoplasm rich in granules in experimental group (Fig. 1B). It should be noticed that induced cells in some cases established their cytoplasmic processes and expanded synapses with adjacent cells. In addition, button like areas were found at the end point of cellular processes that may indicate the functional connection between neurons. These structures were only observed in treated group on day 8 (Fig. 1B). In contrast, cells which were cultured in basic culture medium, exhibited spindle-shaped fibroblast like-cell phenotype.
The percentages of viable cells in the experimental group in all days was higher than in the control group. However, there is not seen a significant differences between two groups (
According to the results of Cresyl violet staining, the most of the neuron-like cells showed positive reaction. Moreover, spherical objects similar to Nissl bodies were found within the cytoplasm of these cells (Fig. 3).
One of the most important indicators of neuronal phenotype are presence of long process as axon in differentiated cells observed and were measurable in the treatment groups from 4th day (Fig. 4A, B). The diameter of processes with the same size or larger than cell body was measured in micrometer (μm). The maximum length of axons was measured on day 8 in the experimental group compared to control group (Fig. 4C).
In order to identify neuroprogenitor cells and neuron-like cells, Nestin and MAP2 markers were applied, respectively. A high percentage of neural progenitors cells were detected in the experimental group. However, the expression of MAP2, known as a marker for mature neurons, was only observed in the experimental group (Fig. 5).
Real-time PCR analysis revealed that higher expression of Oct4 and Sox2 (pluripotency genes) were seen at first day of neurospheres formation and their expresstion were remarkably decreased from day 4 to day 8 in experimental group than control group. However, the level of nestin expresstion was gradually increased from day 2 to day 4 in experimental group. Whereas, the expresstion NF-M was increased from day 4 to day 8. In addition, the expresstion NF-H was not seen within first-two days of induction. Interestingly, their expression were considerably increased from day 4 to day 8 (Fig. 6).
Neurodegenerative disease is the progressive disorder that causes the patient’s disability and it imposes the high costs on patients and communities every year. Recently, the researchers sought to reveal the early pathogenic hallmark in AD and tried to block it . In this way, they tried to prevent the death of neurons or progenitor neuron stem cells. But, the key point to consider is that the pathology of neurogical disorders begins long before the onset of clinical symptoms. So, without pre-clinic treatment, apoptosis is observed in neural progenitor stem cells and mature neurons before the appearance of cognitive symptoms . Moreover, scientists have designed various therapeutic drugs, but unfortunately, the results have not been promising to replace the damaged neurons, they only relieve some of the physical or mental symptoms [10,22]. In contrast, stem cell therapy is one of the most important strategies in medicine [23,24]. One of the important factors to consider when choosing a cell source is the ability of cells to differentiate into the neuron-like cells
In conclusion, 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.
This project was funded by a grant from Mazandaran University of Medical Sciences, Sari, Iran (grant No. 1813).
Conceptualization: HGH. Data acquisition: GG, HGH. Data analysis or interpretation: MNB, AH, AN. Drafting of the manuscript: MZ, FA, SD. Critical revision of the manuscript: GG. Approval of the final version of the manuscript: all authors.
No potential conflict of interest relevant to this article was reported.