Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells

Abstract Objectives Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons. Methodology After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied. Results Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, β-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs. Conclusion These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.


Introduction
Neurodegenerative disorders are characterized by irreversible progressive loss of neurons in the brain, which brings suffering to millions of people worldwide.
Regeneration of the brain via endogenous neurogenesis is very limited and restricted to its region. 1,2 Neural stem/progenitor cells (NSPCs), which are selfrenewing, multipotent cells residing in the regenerative area of the brain, have been intensively studied for their potential use to regenerate damaged nervous tissue. During brain tissue repair, the endogenous NPSCs infiltrate into the lesion site to process the neurogenesis and replace the lost tissue. However, the brain fails to regenerate functional tissue due to the low incidence of NSPCs in adult humans and the lack of structural support for the migration of the NPSCs. 3 Thus, transplantation of exogenous sources of stem cells with neuronal potential-combined with the presence of secretome, growth factors, and supportive extracellular matrix-has been suggested as a plausible approach for neurodegenerative disease treatment. 4,5,6 Apart from the brain, NSPCs can be derived from various types of stem cells via culturing in a serum-free medium consisting of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) that enable neural specification and maintain proliferative activity, followed by subsequent terminal differentiation into neurons or glial cells. 7,8 NSPCs are usually cultured on a non-adhesive surface to form a free-floating clustering of cells known as a neurosphere. 7 The neurosphere cultures also present an extracellular matrix typically enriched in the brain tissue and expresses synaptic and ion transport machinery. 9 Formation of the neurosphere depends on many factors already shown in several studies, such as passage number of initial cells, 10 type of culture vessel surface, 11 time of culture, 12 and method of neurosphere dissociation. 13 However, these factors have been investigated independently and each study used cells from different sources. Thus, the standard protocol that researchers should adopt for maximizing the neurosphere formation that contributes to the highest potential of neuronal differentiation has not yet been determined.
Human dental pulp stem cells (DPSCs) are mesenchymal stem cells located in a perivascular niche that play an essential role in dentin and pulp regeneration. 14,15 During mastication or injury of the teeth, DPSCs respond to biochemical cues from extracellular matrix change, neighboring cells, and physical signals from mechanical stresses, resulting in proliferation, and both odontogenic and osteogenic differentiation. 16 According to embryonic development, DPSCs originate from the ectomesenchyme, since they express neural crest cell markers, 17 which indicate the same origin as the nervous system. This brought a lot of attention from researchers to the study of neuronal differentiation potential of cells as a regenerative source for neurodegenerative disease.
Despite DPSCs having been differentiated into multiple types of neurons, the methods of NSPC induction are highly varied according to the desired terminal differentiation. 18 The most noticeable variable is the duration of neurosphere formation, which could be from 4 to 15 days. 19,20 The passage number of cells used for NSPC induction is also slightly different amongst studies; however, they are mostly considered to be of early passage. 21,22 Nevertheless, there are no specific guidelines regarding optimal conditions for a high yielding NSPCs induction of DPSCs.
Our study aims to demonstrate a standard method to induce DPSCs into NSPCs and differentiate them into neuronal cells. We explored the factors involved in the neurosphere formation, including culture vessel surface, duration of the culture, methods to dissociate the neurospheres, and passage number of DPSCs.
We also continued the study on the effect of DPSCs Isolation, maintenance, and characterization of human dental pulp stem cells The human dental pulp stem cells (DPSCs) were isolated via outgrowth method, cultured, and characterized as described in our previous study. 23 Briefly, pulp tissues were submerged in a cell culture medium containing Dulbecco's modified Eagle's medium (DMEM, HyClone, Fisher Scientific, Loughborough, UK), 10% fetal bovine serum (FBS, Biochrome, Berlin, GY), 100 U/mL Penicillin (Gibco, Thermo Fisher Scientific), and 100 μm/mL Streptomycin (Gibco), then maintained in a humidified atmosphere with 37°C and 5% CO 2 .
The medium was changed every other day until the confluence of the outgrowth cells reached 80%, then they were sub-cultured or harvested using 0.

Statistical analysis
All experiments were done in triplicate. Statistical tests were performed with GraphPad Prism 5 software.
The data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used when comparing differences between groups and Tukey's test was applied for multiple comparisons.
An independent t-test was used when comparing the differences between the two treatments. P-values <0.05 were considered statistically significant.

Results
Cells isolated from pulp tissue of human permanent teeth show mesenchymal stem cell characteristics.
The isolated cells exhibited a spindle-like shape similar to fibroblasts ( Figure 1A). The cells highly expressed mesenchymal stem cells markers CD44 (0.63±0.12%) ( Figure 1B). The colonies were observed when they were cultured in low density, demonstrating that the cells were capable of self-renewal. ( Figure   1C). The cells were differentiated into osteoblast and adipocyte, which is indicated by alizarin red staining of mineralized nodules ( Figure 1D) and oil red O staining of lipid droplets ( Figure 1E), respectively. Thus, the cells can be defined as DPSCs.
Neurosphere culture requires culture vessel inhibiting cell attachment  an amount of NSPC or seeding a 2D culture by dissociation. 24 In our study, mechanical dissociation by repeated pipetting, with various times and enzymatic digestion by Trypsin and Accutase, were compared for the efficiency of neurosphere re-forming. A 100time pipetting generated the highest neurosphere number and the efficiency decreased when repeated pipetting times were increased ( Figure 4). However, the number of the re-forming neurosphere from the 100-time pipetting was significantly lower compared to enzymatic dissociation. Comparing two enzymes, the neurospheres generated from Accutase disaggregation were significantly higher than Trypsin disaggregation ( Figure 4). Therefore, dissociation of neurospheres by Accutase resulted in significantly higher viable neurospheres than trypsin and mechanical dissociation.    The spatiotemporal expression of Nestin makes it an excellent marker to identify NSPCs, and has been Dissociation of the neurosphere is needed for the amplification of the NSPC population and neuronal differentiation. It is a critical process to preserve the viability of the NSPCs. Mechanical dissociation by repeated pipetting is convenient and cheap, since it requires equipment that is already available for routine lab work. Mechanical dissociation generates traumatic force that damage cells, resulting in an over 40% reduction of viable cells. 35 Trypsin is a protease commonly used to digest cell adhesion molecules to detach cells from plastic ware. Accutase, a gentler enzyme, has been developed to replace trypsin and is useful for flow cytometry analysis, since it preserves most cell epitopes (http://www.accutase.com/ accutase.html). Accutase is understood to preserve more surface molecules, such as receptors of growth factors. As a result, cells are more responsive to EGF and bFGF, and are more viable than cells dissociated with trypsin. 33 The study showed that Accutase is an effective enzyme for dissociation of DPSCs-derived neurospheres.

Passage number does not affect NSPC induction but reflects neuronal differentiation outcomes
To obtain sufficient stem cell numbers for study or therapeutic use, the cells must be subcultured several

Conclusions
In our study, we propose a guideline for neurosphere generation from DPSCs. The culture requires modified vessel surfaces to inhibit cellular attachment and promote 3D spheroid formation. A 5-day culture duration is an optimal time allowing high proliferative NSPCs to form neurospheres. Dissociation of the neurospheres should be done using Accutase digestion to obtain a high number of viable cells.