EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Stem cells from human exfoliated deciduous teeth are a population of multipotent mesenchymal stem cells that can self-renew and actively secrete a broad spectrum of trophic and immunomodulatory factors. Brain-derived neurotrophic factor is able to induce stem cells to neural differentiation to repair the nervous system. However, the mechanism of brain-derived neurotrophic factor-induced neural differentiation in stem cells from human exfoliated deciduous teeth remains unclear.
Materials and Methods: In this study, we focused on brain-derived neurotrophic factor and investigated its effects on neural differentiation in stem cells from human exfoliated deciduous teeth. We cultured stem cells from human exfoliated deciduous teeth under various different brain-derived neurotrophic factor concentrations. We then analyzed the effects of brain-derived neurotrophic factor to the neural differen-tiation and associated signaling pathways in stem cells from human exfoliated deciduous teeth.
Results: We demonstrated that a high concentration of brain-derived neurotrophic factor could induce neural differentiation in stem cells from human exfoliated deciduous teeth, and brain-derived neurotrophic factor also increased the expression levels of neural differentiation-related proteins in stem cells from human exfoliated deciduous teeth, which was associated with the suppression of growth factor receptor-bound protein 2/extracellular signal-regulated kinase 1 and 2 signaling pathways.
Conclusions: Knockdown of growth factor receptor-bound protein 2 inhibited the neural differentiation of stem cells from human exfoliated deciduous teeth via changes in growth factor receptor-bound protein 2/extracellular signal-regulated kinase signaling pathways, but this phenotype could be rescued by overexpression of extracellular signal-regulated kinase. Our findings suggested that brain-derived neurotrophic factor can regulate the differentiation of stem cells from human exfoliated deciduous teeth through the growth factor receptor-bound protein 2/extracellular signal-regulated kinase signaling pathway.

Key words : Brain-derived neurotrophic factor, Extracellular signal-regulated kinase, Growth factor receptor-bound protein 2, Neural differentiation, Tooth-derived stem cells

Tooth-derived cells are easily obtained and provide a convenient and safe way to generate stem cells for clinical application in the future, with little or no trauma. It is thus a reasonable and simpler alternative for storage of tooth-derived stem cells rather than other tissues. Stem cells from human exfoliated deciduous teeth (SHED) are easily obtained and cultured for experimental research and clinical application. It is highly recommended to purify stem cells from young and healthy children, since the cultured cells will be in good condition and will proliferate very well.
Brain-derived neurotrophic factor is a member of the neurotrophin family and plays a pivotal role in the survival, differentiation, and synaptic plasticity of neurons in the mammalian nervous system, serving as a neurotransmitter modulator.¹ Brain-derived neurotrophic factor is mainly located in the olfactory bulb, cortex, and hippocampal formation in the brain. It has also been reported that BDNF is expressed at the synaptic terminals.^2,3 Brain-derived neurotrophic factor can be released into the extracellular space, which is triggered by multiple neural electrical activities.⁴ These effects are mediated through activation of tyrosine protein kinase TrkB receptor-signaling pathways, such as phospholipase γ 1 pathways, phosphatidylinositol-3-kinase/Akt, and the Ras/mitogen-activated protein kinase.⁵
It has been reported that some methods can also enhance the effect of BDNF,^6,7 although there is not sufficient evidence on the clinical efficacy.^8,9 Although data from animal studies have demonstrated that direct BDNF administration could produce positive and satisfactory results, the invasive nature of these studies renders these approaches less clinically applicable.^10,11 The intraperitoneal administration of the BDNF-HIV fusion-peptide resulted in an remarkable spatial memory improvement.^12-16 Gene therapy could also facilitate BDNF expression.^17-19 However, until now, it has remained problematic to produce the safety, site-specific, and adjustable function of BDNF.²⁰
In this study, we isolated stem cells from exfoliated deciduous teeth of healthy children, focused on BDNF, and investigated the BDNF effects on neural differentiation in SHED. By cell transfection and Western blot analysis, we explored the mechanism through which BDNF regulates the differentiation of SHED.

Materials and Methods

Human tissue and cells Dental pulps were extracted from normal exfoliated human deciduous teeth of 6- to 10-year-old children (5 cases) under local anesthetics. All protocols on human tissue and cells were approved by the Ethics Committee of Affiliated Hospital of Jinggangshan University. Written informed consent was collected from all patients and guardians.The pulps separated from a remnant crown were digested with 3 mg/mL collagenase type I and 5 mg/mL dispase at 37 °C for 50 minutes. Cells were collected, seeded onto 3-cm plates, and then incubated at 37 °C in 5% CO2. The culture medium was alpha-minimal essential medium (GIBCO) supplemented with 15% fetal bovine serum, 100 μM L-ascorbic acid 2-phosphate, 2 mM L-glutamine, and 100 U of antibiotic-antimycotic agent.

Cells were cultured in 6-well plates and incubated with primary antibody (CD34 monoclonal antibody [581], CD3458104, 2022, Invitrogen; anti-CD45 [human] clone HI30 antibody, MABF1602, 2022, Sigma-Aldrich; CD90 rabbit monoclonal antibody, AF1636, 2022, Beyotime; endoglin/CD105 monoclonal antibody, 2D5E8, 2022, Proteintech) overnight. The cells were then incubated with a second antibody (fluorescein isothiocyanate goat anti-human immunoglobulin G [H+L], A0556, 2022, Beyotime). Finally, the cells were analyzed by flow cytometry.

Immunocytochemistry analysis

The SHED were incubated for 3 days and fixed with 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100 in phosphate-buffered saline for 20 minutes. Then, β-tubulin III primary antibodies were diluted in blocking buffer (10 mg/mL bovine serum albumin) and incubated overnight at 4° C. Cells were counterstained with 4',6-diamidino-2-phenylindole for 3 minutes.²¹ The stained cells on the coverslips were analyzed under a fluorescent microscope.

Small interfering RNA transfection

We followed the normal approach for siRNA transfection of SHED.²² Briefly, cells were seeded into a 6-well plate and transfected with 80 nM of a siRNA using jetPRIME reagent. Scrambled siRNA was used as the negative control.

Western blot analysis

Total proteins were extracted with radioimmuno-precipitation assay buffer and loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrop-horesis gel. Proteins were then transferred onto polyvinylidene difluoride membranes. We then incubated the primary and secondary antibodies with polyvinylidene difluoride membranes, followed by signal detection with a chemilu-minescence system.²²

Immune characterization of stem cells from human exfoliated deciduous teeth cultures

Tooth extraction was performed, and SHED cultures were performed, which were highly positive (>95% of the population) for mesenchymal stem cells (CD90/Thy-1) and endothelial cells (CD105). According to the flow cytometry results, mesenchymal stem cell markers could be detected on the surfaces of more than 90% of the purified and cultured SHED, including CD105, CD90, CD34, and CD45 (Figure 1).

Brain-derived neurotrophic factor promotes the neural differentiation of stem cells from human exfoliated deciduous teeth

To evaluate the effect that BDNF promotes the neural differentiation of SHED, we treated the SHED with various concentrations of BDNF. Immunofluo-rescence staining of β-tubulin III was performed to measure the neural differentiation of SHED on various days after neurotrophin administration (Figure 2). The result demonstrated that BDNF promotes the neural differentiation of SHED in a dose-dependent manner.

Brain-derived neurotrophic factor increased the expression levels of neural differentiation-related proteins in stem cells from human exfoliated deciduous teeth

To better show the molecular changes and that BDNF promotes the neural differentiation of SHED, we treated the SHED with various concentrations of BDNF, after which several markers for neural differentiation were detected (Figure 3). The results demonstrated that the protein levels of neuroepithelial stem cell protein, β-tubulin III, neural cell adhesion molecule, and neuronal differentiation 1 transcription factor were all significantly upregulated after treatment with various concentrations of BDNF. The data indicated that BDNF increased the expression levels of neural differentiation-related proteins in SHED and promoted the neural differentiation of SHED in a dose-dependent manner (Figure 3).

Brain-derived neurotrophic factor activates the growth factor receptor-bound protein 2/extracellular signal-regulated kinase signaling pathway

Next, we examined the effects of BDNF on growth factor receptor-bound protein 2 (GRB2) and the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway in SHED. As shown in (Figure 4), BDNF upregulated the phosphorylation levels of ERK1/2 in SHED and the protein level of GRB2 as well, which indicated that GRB2 activated the ERK1/2 pathway. The expression levels of the total proteins in the ERK pathway are shown in (Figure 4). These results suggested that BDNF activates the GRB2/ERK signaling pathway.

Brain-derived neurotrophic factor promotes the neural differentiation of stem cells from human exfoliated deciduous teeth through regulation of the growth factor receptor-bound protein 2/extracellular signal-regulated kinase signaling pathway

To further evaluate whether BDNF affected the neural differentiation of SHED through the GRB2/ERK pathway, we performed the knockdown of GRB2 by using siRNA to inhibit the activity of GRB2. Blockage of GRB2 was also significantly inhibited neural differentiation (Figure 5), and this effect could be rescued by overexpression ERK in SHED. Western blot data showed that the protein level of GRB2 also recovered by overexpression ERK in SHED. Together, these data indicated that BDNF promoted the neural differentiation of SHED through regulation of the GRB2/ERK signaling pathway.

The use of SHED is a simple and convenient resource for multipotent, self-renewing mesenchymal stem cells and provides a good opportunity to obtain stem cells, considering every child loses teeth. If needed, these cells can be used to treat injuries or diseases in the future, and this is a much better option than discarding teeth. Moreover, in this manner, risks for conditions that may lead to immune reactions are avoided.
Brain-derived neurotrophic factor is vital in the neural differentiation of neural stem cells and may have therapeutic potential for neural regeneration. We examined the effects of BDNF on the GRB2 and ERK1/2 pathways in SHED. In this study, we focused on BDNF and investigated its effects on neural differentiation in SHED. We found that BDNF upregulated the phosphorylation levels of ERK1/2 in SHED and the protein level of GRB2. All the results demonstrated that a proper concentration of BDNF induced neural differentiation in SHED and that BDNF regulates the expression of the neural differentiation-related proteins in SHED, which was associated with the suppression of the GRB2 and ERK1/2 signaling pathways.
The neuroprotective and pro-neurogenesic effects of BDNF have been well studied. Here, we also demonstrated that BDNF can induce neural differentiation in SHED. Taken together, our findings suggest that BDNF can regulate differentiation of SHED through the GRB2/ERK signaling pathway, thus offering a promising strategy for neural injury and recovery in the future.

High concentrations of BDNF could induce neural differentiation in SHED, and BDNF also increased the expression levels of neural differentiation-related proteins in SHED, which was associated with the suppression of GRB2 and ERK1/2 signaling pathways.

Volume: 20 Issue: 11 November 2022

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