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Conditioned-medium of stem cells from human exfoliated deciduous teeth prevent apoptosis of neural progenitors

Purpose This study aimed to evaluate the neuroprotective ability of the conditioned medium of stem cells from human exfoliated deciduous teeth (CM-SHED) to prevent glutamate-induced apoptosis of neural progenitors. Materials and methods Neural progenitors were isolated from two-day-old rat brains, and the conditioned medium was obtained from a mesenchymal stem cell SHED. Four groups were examined: neural progenitor cells cultured in neurobasal medium with (N + ) and without (N-) glutamate and glycine, and neural progenitor cells cultured in CM-SHED with (K + ) and without (K-) glutamate and glycine. Results The expression of GABA A1 receptor (GABAAR1) messenger RNA (mRNA) in neural progenitor measured by real-time quantitative PCR. GABA contents were measured by enzyme-linked immunosorbent assay, whereas the apoptosis markers caspase-3 and 7-aminoactinomycin D were analysed with a Muse® cell analyzer. The viability of neural progenitor cells in the K + group (78.05 %) was higher than the control group N- (73.22 %) and lower in the N + group (68.90 %) than in the control group. The K + group showed the highest GABA content, which significantly differed from that in the other groups, whereas the lowest content was observed in the N + group. The expression level of GABAAR1 mRNA in the K + group was the highest compared to that in the other groups. CM-SHED potently protected the neural progenitors from apoptosis. Conclusions CM-SHED may effectively prevent glutamate-induced apoptosis of neural progenitors.


Introduction
Neural death is a major symptom in neurodegenerative diseases such as Alzheimer's, Parkinson's, multiple sclerosis, and Huntington's disease. Apoptosis results from a trigger that activates a signal transduction pathway leading to cell death. Some triggers for apoptosis in chronic neurodegenerative diseases include excitotoxicity mediated by increased extracellular glutamate levels, increased formation of free radicals, induced DNA damage, increased expression of genes encoding the p53 protein, and damage to the plasma membrane (Kermer et al., 2004, Taylor et al., 2008. Excitotoxicity is an essential component of the pathogenesis of brain damage during the perinatal period. This process is initiated by elevated extracellular glutamate levels, which lead to hyperactivity of the glutamate receptors, increasing calcium (Ca 2+ ) influx and ultimately resulting in apoptosis (Allen et al., 2004, Almeida et al., 2005. Glutamate can be converted into gamma-aminobutyric acid (GABA) by the enzyme glutamic acid decarboxylase (GAD). Thus, GAD reduces extracellular glutamate levels. In mature neurons, GABA is a major inhibitory neurotransmitter of the central nervous system, whereas in neural progenitors, GABA can cause chlorine (Cl -) efflux, resulting in depolarization which leads to neural excitation (Brooks-Kayal and Russek 2012, Egusa et al., 2012). To perform its function, GABA must bind its receptor. The GABA A1 receptor (GABAAR1) is the most common GABA receptor in the brain, which may inhibit apoptosis when activated. Caspase-3 is expressed as its inactive form procaspase-3, which has a nitrosylated cysteine residue in its catalytic site. During apoptosis, denitrosylation of this cysteine residue converts procaspase-3 into its active form. Activation of GABAAR1 may inhibit this denitrosylation, thereby maintaining caspase-3 in its inactive form and inhibiting apoptosis (Esmaeili et al., 2014). Glutamate receptor hyperactivity causes an increase in Ca 2+ influx into cells, which triggers apoptosis (Finucane et al., 1999, Nechushtan et al., 1999, Pawlowski and Kraft 2000, Banke and Traynelis 2003, S Smaili et al., 2011. One of the treatment options under development for neurodegenerative diseases is using stem cells. Previous studies have reported that human adipose derived stem cells (hADSCs) are capable of differentiating directly into dopaminergic neurons (Spitzer 2006). Stem cells from human exfoliated deciduous teeth (SHED) are adult stem cells derived from the deciduous dental pulp tissue. Stem cell lines were first isolated from human deciduous teeth by tooth exfoliation. (Wei et al., 2012). SHED have the following advantages: the retrieval process is not invasive, and cell availability is unlimited (Miura et al., 2003, Wei et al., 2012, Khademizadeh et al., 2019. SHED also possess neuroregenerative and neuroprotective capabilities owing to the secretion of their metabolites into their conditioned medium (CM) (Kerkis andCaplan 2012, Inoue et al., 2013). One such metabolite is GAD. In this study, we assessed the neuroprotective potency of the conditioned medium of stem cells from human exfoliated deciduous. This study provides a scientific basis for the use of CM-SHED to treat neurodegenerative diseases, which has not been previously reported.
Neural progenitors were isolated as described in previous studies (Lee et al., 2009, Zainuri et al., 2018. The materials used for neural progenitor cell isolation were as follows:

Ethics statement
This study has been approved by the National Board of Health Research and Development Jakarta, Indonesia (approval number LB.02.01 / 5.2 / KE.055).

Statistical analysis
If the data distribution was normal, analysis of variance (ANOVA) was carried out to determine the differences between more than two groups. In case of abnormal data distribution, the Kruskal-Wallis test followed by the Mann-Whitney test was performed.

Results
The neural progenitors' biomarkers PSA-NCAM + and A2B5-were characterized using flow cytometry. Among the five fractions, only fractions two, four, and five had a population of PSA-NCAM + and A2B5-cells above 80 %. (Fig. 1).
This study showed that the groups of neural progenitors cultured with CM-SHED (K + and K-) had higher viability than those cultured without CM-SHED (N + and N-). After treatment, the percentage of live neural progenitors in the N + group was lower than that in the other groups. The effect of CM-SHED on the viability of neural progenitors is demonstrated in Table 2. While the viability of neuron progenitors in the K + group (78.05 %) was higher than that in the N-control group (73.22 %), the N + group had a lower viability (68.90 %).
Among all the of experiments, the highest and lowest level of GABA was observed in the K + and N + group, respectively (Fig. 2). There was a significant difference (p < 0.05) in the GABA content between the K + group and the other groups.
GABAAR1 content was observed to be significantly higher in the N + group than the other groups (p < 0.05) (Fig. 3). The expression of GABAAR1 mRNA in the K + group was higher than that in the N-group (p < 0.05), whereas in the N + and K-groups, the levels were lower than the N-group (p < 0.05) (Fig. 4).

Discussion
In the present study, we found that neural progenitors cultured with CM-SHED were more viable than those cultured without CM-SHED were. This is presumably due to the role of CM-  (Inoue et al., 2013). Other studies have reported that the addition of CM-SHED might inhibit apoptosis, reduce tissue loss in the brain, and improve neurological function in mice with ischemic hypoxia (Mita et al., 2015, Wang et al., 2018. Another study reported that the upregulation of the phosphoinositide-3kinase (PI3K)/Akt pathway prevents neuronal apoptosis via regulation of BCL2 and BAX expression (Yamagata et al., 2013). Yamaza et al. reported the ability of SHED to express  the transforming growth factor-b1 (TGF-b1) and TGF-b2 receptor, platelet-derived growth factor (PDGF) receptor, extracellular signal-regulated kinases (ERK), phosphorylated ERK, Akt, and phosphorylated Akt (Yamaza et al., 2010). This study showed no significant difference regarding GABA content between the K-and N-groups, which were not subjected to glutamate induction. This result might be caused by normal glutamate content in the CM. The addition of glutamate and CM-SHED containing GAD catalyzes the glutamate-to-GABA conversion, resulting in high GABA content. Miura et al. found that SHED could express the GAD enzyme (Miura et al., 2003). Another study reported the ability of SHED to express neurotrophic factors, such as brainderived neurotrophic factor (BDNF) (Esmaeili et al., 2014). One previous study reported that BDNF might enhance the release of GABA via the mitogen-activated protein kinase (MAPK) signaling pathway and cyclic adenosine monophosphate-response element binding protein (CREB) transcription factors (Yamagata et al., 2013).
The expression of GABAAR1 mRNA was highest in the K + group compared to that in the other groups. This is probably due to the GABA-mediated activation of the MAPK pathway and phosphorylation of CREB at the serine 133 residue. CREB transcription factors are active when bound to other CREB family members, forming a heterodimer structure. Thereafter, they bind to the cAMP response element (CRE) in the promoter region of GABAAR1, which has a TGACGTCA motif, resulting in GABAAR1 mRNA transcription. It has been proven that the administration of muscimol, a GABAAR1 agonist, causes a subunit change from c2 to non-c2 in the GABAAR1 synaptic region, resulting in decreased GABAergic synapse strength (Yamaza et al., 2010). In the present study, the lowest GABA content was found in the N + group, presumably due to the binding of GABA to Fig. 2 Effect of conditioned medium of stem cells from human exfoliated deciduous teeth (CM-SHED) with or without the addition of glutamate and glycine on gamma-aminobutyric acid (GABA) levels in neural cell culture. *Significant difference, p < 0.05. Fig. 3 Effect of conditioned medium of stem cells from human exfoliated deciduous teeth (CM-SHED) with or without the addition of glutamate and glycine on the level GABA A1 receptor (GABAAR1) protein in neural cell culture. *Significant difference, p < 0.05. GABAAR1. GABAAR1 content was highest in the N + group. Another possibility for the reduced GABA content in the N + group is the negative feedback from GABAAR1. It has been reported that GABAAR1 activation lowers the GAD65 content via the BDNF/ tropomyosin receptor kinase B (TrkB) signaling pathway. Decreased levels of GAD65 cause a reduction in GABA (Obrietan et al., 2002). The high concentration of GABAAR1 in the N + group was also thought to be due to BDNF, which increases GABAAR1 protein expression on the cell surface via the TrkB, protein kinase C (PKC), and PI3K pathways. In each pathway, BDNF exerts positive feedback on GABAAR1 (Matsuzaki et al., 1999). The present study reports a decreased GABAAR1 mRNA expression in the N + group compared to that in the control group. This decrease could be either because the protein was not required, or may be induced by BDNF that decreases the transcription of GABAAR1 mRNA via the Janus kinase/ signal transducer and activator of transcription (JAK/STAT) pathway. BDNF activates the phosphorylation process in STAT3 signaling, thereby initiating the formation of inducible cAMP early repressor (ICER), which binds to inactive, phosphorylated CREB (pCREB), thus inhibiting the binding of pCREB to CRE in the promoter of GABAAR1 (Matsuzaki et al., 1999, Lund et al., 2008, Porcher et al., 2011. There was no significant difference in the content of GABAAR1 protein between the N-and K-groups, because GABA content was similar in both the groups. Khalilov et al. reported a dynamic change in progenitor neuron GABAAR1 regulation before and after the peak of giant depolarizing potentials (GDPs) (Khalilov et al., 2015). The increase in GABA reported in the K + group may be due to postoperative GDP inhibition of GABAAR1 activity, compensating for excitatory glutamate activity and resulting in neuroprotection.
In the present study, the suspected postoperative GDP inhibition of GABAAR1 activity in the N + group could not overcome the excitation from glutamate induction, leading to apoptosis. In the K + group, where there was an increase in GABA due to CM-SHED, the suspected postoperative GDP inhibition of GABAAR1 activity in the N + group could not overcome the excitation from glutamate induction, leading to apoptosis.can compensate for the excitatory activity by glutamate resulting in neuroprotection. The primary limitation of this study is that we did not identify the composition of CM-SHED. Hence, further studies are warranted to identify the proteins in CM-SHED that play a role in neuroprotection. Further, a similar molecular docking study to that reported by Asadollahi et al. (Asadollahi et al., 2019) might be needed to analyse the interaction between GABAAR1 and GDPs.

Conclusion
CM-SHED may effectively prevent glutamate-induced apoptosis of neural progenitors, as indicated by the expression level of GABAAR1 mRNA being highest in the K + group. This study reveals that stem cells from human exfoliated deciduous teeth are a resource, which may be used for regenerative therapy in the medical-dental field.

Ethical statement
This study 'Conditioned-Medium of Stem Cells from Human Exfoliated Deciduous Teeth Prevent Apoptosis of Neural Progenitors" obtained ethical approval from National Board of Health Research and Development with the ethical approval number LB.02.01 / 5.2 / KE.055.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Fig. 4 Effect of conditioned medium of stem cells from human exfoliated deciduous teeth (CM-SHED) with or without the addition of glutamate and glycine on expression of mRNA subunit of GABA A1 receptor (GABAAR1) in neural cell culture. *Significant difference, p < 0.05.