Characterization of neural stem cells derived from human stem cells from the apical papilla undergoing three-dimensional neurosphere induction

Abstract Objectives The endogenous repairing based on the activation of neural stem cells (NSCs) is impaired by neurodegenerative diseases. The present study aims to characterize human stem cells from the apical papilla (hSCAPs) with features of mesenchymal stem cells (MSCs) and to demonstrate the neuronal differentiation of hSCAPs into NSCs through the formation of three-dimensional (3D) neurospheres, verifying the structural, immunophenotyping, self-renewal, gene expression and neuronal activities of these cells to help further improve NSCs transplantation. Methodology The hSCAPs were isolated from healthy impacted human third molar teeth and characterized as MSCs. They were then induced into 3D-neurospheres using a specific neural induction medium. Subsequently, the intra-neurospheral cells were confirmed to be NSCs by the identification of Nissl substance and the analysis of immunofluorescence staining, self-renewal ability, and gene expression of the cells. Moreover, the neuronal activity was investigated using intracellular calcium oscillation. Results The isolated cells from the human apical papilla expressed many markers of MSCs, such as self-renewal ability and multilineage differentiation. These cells were thus characterized as MSCs, specifically as hSCAPs. The neurospheres induced from hSCAPs exhibited a 3D-floating spheroidal shape and larger neurospheres, and consisted of a heterogeneous population of intra-neurospheral cells. Further investigation showed that these intra-neurospheral cells had Nissl body staining and also expressed both Nestin and SOX2. They presented a self-renewal ability as well, which was observed after their disaggregation. Their gene expression profiling also exhibited a significant amount of NSC markers (NES, SOX1, and PAX6). Lastly, a large and dynamic change of the fluorescent signal that indicated calcium ions (Ca2+) was detected in the intracellular calcium oscillation, which indicated the neuronal activity of NSCs-derived hSCAPs. Conclusions The hSCAPs exhibited properties of MSCs and could differentiate into NSCs under 3D-neurosphere generation. The present findings suggest that NSCs-derived hSCAPs may be used as an alternative candidates for cell-based therapy, which uses stem cell transplantation to further treat neurodegenerative diseases.


Introduction
Neurological disorders affecting the central nervous system (CNS) were cited by the Global Burden of Disease Study and lead to permanent disability and mortality.These disorders result from abnormalities in bodily structures and biochemical or physiological properties, which cause the loss of functional neurons. 1 They highly affect neural stem cells (NSCs), neural progenitor cells (NPCs), and the adult neurogenesis ability. 2 Unfortunately, the endogenous repair of tissue through progenitor cells is limited to 2 active regions: the subventricular zone (SVZ) and the subgranular zone (SGZ). 3There is, however, an alternative treatment to neuronal regeneration: modified-exogenous NSCs transplantation. 4Although this procedure is promising, the use of NSCs from the fetal brain, adult brain, or high potency-embryonic stem cells (ESCs) results in donor site morbidity and generates ethical concerns. 5searchers have been working on techniques to make this treatment ethical, such as the characterization of human stem cells from the apical papilla (hSCAPs) of developing teeth as MSCs. 6These hSCAPs can differentiate into specialized cells under optimal induction environments, such as those of adipogenic, osteogenic, and neurogenic lineages. 7The hSCAPs have demonstrated their potential for neuronal differentiation through the expression of neuronalassociated markers. 8These cells are ectomesenchymal stem cells.They originate from migratory neural crest stem cells and present a superior and committed neuronal differentiation ability. 9Moreover, the hSCAPs can be obtained from dental waste through noninvasive procedures, which means they are easily accessible. 10e formation of neurospheres is a functional approach that provides a three-dimensional (3D) microenvironment for the differentiation of NSCs. 11tablishing the optimal conditions for the induction of neurospheres requires specific growth factors, such as the basis fibroblast growth factor (bFGF) and the epidermal growth factor (EGF), 12 an appropriate induction period and unique cell culture vessels that trigger the formation of NSCs. 13Interestingly, 3D-neural induction shows superior neuronal differentiation compared with the monolayer method. 14ese factors indicate that hSCAPs could be excellent candidates to generate NSCs under the formation of 3D-neurospheres, allowing medical professionals to overcome the limitations of modified-exogenous NSC transplantation.
The present study aims to characterize hSCAPs with properties of MSCs, then to differentiate them into NSCs through the generation of 3D-neurospheres and to further verify their structural, immunophenotyping, self-renewal, gene expression, and neuronal activities to identify the potential of this approach for the treatment of neurodegenerative diseases.

Cell detection and culture
A dual enzymatic digestion method was used to detect hSCAPs, as previously described. 7,9The teeth were first placed in a proliferation medium consisting (T-75 cm 2 flask, Nunc TM , Thermo Scientific, Waltham, MA, USA) and cultured in a proliferation medium in an incubator at 37°C with 5% CO 2 and 95% humidity.
The proliferation medium was changed every 2 days until an 80% confluence rate was reached.Sub-culturing by trypsinization with 0.05% trypsinethylenediaminetetraacetic acid (EDTA) (Gibco, Life Technologies) was then performed. 15The Compact Cell Culture Microscope CKX3 (Olympus, Hamburg, Germany) was used to observe cell morphology and assess plastic-adherence ability.

Cells derived from migratory neural crest stem cells
The uncharacterized cells at passage 3 were seeded in 24-well plates (Nunc™, Thermo Fisher Scientific) at a density of 2x10 4 cells/well and cultured in the proliferation medium until they reached an 80% confluence rate.They were confirmed to be derivative of migratory neural crest stem cells through the immunocytochemistry staining of β-III tubulin and Nestin.
The adipogenic induction medium was changed every 2 days.Lipid droplets were verified with Oil Red O staining, as previously described. 7The culture medium was then discarded and the cells were washed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich)

Osteogenic differentiation
The uncharacterized cells at passage 3 were seeded in 24-well plates at a density of 2x10 4 cells/ well and cultured in the proliferation medium until they reached an 80% confluence rate.They were induced into osteogenic differentiation after being cultured for 4 weeks in an osteogenic induction medium consisting of MEM, 10% FBS, 1% antibiotic-antimycotic, 50 µg/ mL ascorbate-2-phosphate (Sigma-Aldrich), 0.1 µM dexamethasone, and 10 mM β-glycerophosphate (Sigma-Aldrich).The osteogenic induction medium was changed every 2 days.The calcification of an extracellular matrix was observed with an Alizarin red staining solution, used as previously described. 7e cultured medium was then quickly removed and the cells were washed with PBS and fixed with 4%

Neurogenic differentiation
The uncharacterized cells at passage 3 were seeded in 24-well plates at a density of 2x10 4 cells/ well and cultured in the proliferation medium until they reached an 80% confluence rate.The neurogenic differentiation was induced using two phases of a neuronal induction medium, described in a previous study. 7For 24 hours, the cells were induced into neurogenic differentiation with the phase I neuronal induction medium, which consisted of Dulbecco's

Modified Eagle Medium supplemented with Nutrient
Mixture F-12 (Ham) (DMEM/F-12, Gibco, Life Technologies), 10% FBS, 1% antibiotic-antimycotic, 10 ng/mL bFGF (Gibco, Life Technologies), and 500 µM β-mercaptoethanol (Sigma-Aldrich).After this treatment, the cells were cultured for 6 hours in the phase II neuronal induction medium, which consisted of DMEM/F-12, 2% dimethyl sulfoxide (Sigma-Aldrich), 1 % a n t i b i o t i c -a n t i m y c o t i c a n d 1 0 0 µ M b u t y l a t e d hy d r ox ya n i s o l e ( S i g m a -A l d r i c h ) .
The Nissl substance of a typical neuronal cell marker was stained with Cresyl violet and observed under the Compact Cell Culture Microscope CKX3.

Analysis of cell-surface antigen molecules
The uncharacterized cells at passage 3 were seeded in a T-75 cm 2 flask at a density of 1x10 6 cells and cultured in the proliferation medium until they reached an 80% confluence rate.Subsequently, cells at the density of 5x10 5 cells were harvested and had their

Colony-forming unit fibroblast
T h e u n c h a ra c t e r i z e d c e l l s a t p a s s a g e 3 were seeded in 6-well plates (Nunc™, Thermo Fisher Scientific) at a density of 500 cells/well and cultured in the proliferation medium for 7 days.The medium was changed every 2 days.The colonies of these cells were observed using Giemsa staining, as previously described. 7The culture medium was quickly discarded and the cells underwent PBS washing and fixation with 4% paraformaldehyde in PBS for 30 minutes.They were fixed with methanol (EMSURE ® , MERCK) for 10 minutes and washed with distilled water.Then, 1% of Giemsa solution (Sigma-Aldrich) was incubated at room temperature for 30 minutes and washed several times with PBS, until the excess staining was fully removed.The purple colonies of these cells were observed under the Compact Cell Culture Microscope CKX3.

Neurosphere induction
To generate neurospheres, the characterized hSCAPs at passage 5 were induced at a density of 6.

Cresyl violet staining
The neuronal cells derived from hSCAPs, the characterized hSCAPs, and the neurospheres were fixed in 4% paraformaldehyde at room temperature
All of the antibodies were diluted with 5% BSA (in PBS with 0.05% Tween-20 [Sigma-Aldrich]).After this primary incubation, the cells were incubated at room temperature for 4 hours with the following secondary antibodies: Goat anti-mouse Alexa Flour plus 488 (1:1,000; Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) and

Self-renewal ability
To demonstrate the self-renewal ability of these NSCs, the neurospheres at passage 1 were collected and enzymatically dissociated with Accutase (Gibco, Life Technologies) for 3 minutes, in an incubator at 37°C with 5% CO 2 and 95% humidity.Subsequently, the dissociated intra-neurospheral cells were resuspended with the neural induction medium, seeded in the Costar ® ultra-low attachment multiple well-plate, size 24 well, and kept inside it.Half of the medium was changed every two days.After 5 days, the morphology of neurospheres at passage 2 was observed under the Compact Cell Culture Microscope CKX3.

RT-qPCR
The characterized hSCAPs at passage 4 and the neurospheres at passage 1 were collected, lysed and had their total RNA extracted with a High Pure RNA Isolation Kit (Roche, Basel, Switzerland).The RNA was quantified using the Nanodrop™ 2000/2000c spectrophotometers (Thermo Scientific).The extracted RNA was reverse-transcribed into cDNA with the Transcriptor First Stand cDNA Synthesis Kit (Roche) and qPCR was performed with the CFX96 real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the KAPA SYBR FAST qPCR kits (Sigma-Aldrich).
The thermocycling conditions for the qPCR were: 95°C for 180 seconds, followed by 40 cycles of 95°C for 3 seconds and of 60°C for 30 seconds.The primer pairs used for qPCR (Integrated DNA Technologies, the Gemini Singapore Science Park II, Singapore) are listed in Figure 1.The gene expression level was calculated using the 2 -ΔΔCt method. 16

Intracellular calcium oscillation
The intracellular calcium influx of the neurospheres was evaluated to identify their potential neuronal activity.The intracellular calcium influx was assessed in accordance with descriptions by previous studies. 7,17e neurospheres at passage 1 were incubated with DMEM/F-12, 1% antibiotic-antimycotic, 0.08% pluronic acid (Invitrogen), and 3 µM Fluo-3 AM (Invitrogen), which is a fluorescence chelator of intracellular calcium (Ca 2+ ), at 37°C with 5% CO 2 and 95% humidity for 60 minutes.The neurospheres

Statistical analysis
Data are expressed as the mean ± standard deviation (SD) of the three experimental replicates.

Different groups were compared by the unpaired
Student's t-test, conducted by the GraphPad Prism (San Diego, CA, USA).The differences in which *p<0.05 were considered statistically significant.

Characterization of hSCAPs
An analysis was conducted to determine whether the cells isolated from human apical papilla tissues exhibited characteristics commonly associated with MSCs.The cells had a fibroblast-like shape and could grow on plastic-adherent culture vessels (Figure 2A).
The immunofluorescence staining confirmed that the cells were derivative of migratory neural crest stem cells, as they positively stained for β-III tubulin (Figure 2B) and Nestin (Figure 2C).Moreover, under appropriate differentiation-inducing conditions, the cells differentiated into adipocytes, osteocytes, and neuronal cells: they presented accumulated lipid droplets (Figure 2D), secreted calcified nodules (Figure 2E), and exhibited Nissl substance (Figure 2F), as revealed through Oil Red O, Alizarin red, and Cresyl violet staining, respectively.Moreover, the flow cytometry profiling of the cell-surface antigen molecules in these cells demonstrated positive markers for MSCs, including CD73, CD90, CD105, and CD146, as indicated by the high intensity of histograms.A great portion of the cells did not express CD34 and co-expressed CD34-, CD73+, CD90+, CD105+, and CD146+ (Figure 1G).Colonies of isolated cells with positively stained Giemsa dye, used to evaluate selfrenewal properties, were formed (Figure 1H).Taken together, the isolated cells derived from human apical papilla tissue exhibited properties of MSCs and were verified to be hSCAPs.

Generation of neurospheres
Placed inside an ultra-low attachment multiple-well plate, regularly filled with a neutral induction medium, the characterized hSCAPs changed morphology, transforming themselves, on day 1, from typical fibroblast-like shapes (Figure 3A) into free-floating cells (Figure 3B).These cells then aggregated, forming 3D-spheroid clusters known as 'neurospheres'.The size of the neurospheres increased in a time-dependent manner on days 3 (Figure 3C) and 5 (Figure 3D).These neurospheres consisted of intra-neurospheral cells with NSC properties, which were subsequently characterized.

Identification of Nissl substance
The presence of the Nissl body, a typical neuronal substance, was confirmed by the Cresyl violet staining.The hSCAPs presented a typical fibroblast-like shape morphology, a pale purple background, characteristic of a nucleus (black arrows), and a dark spot, typical of a nucleolus (black asterisks) (Figure 4A).Interestingly, the cell body in the cluster of intra-neurospheral cells presented an intense purple substance (white arrows), which indicated typical neuronal substance (Figure 4B) and was consistent with the neuronal cells derived from hSCAPs under the two-dimensional neuronal differentiation (Figure 4F).These results suggest that the neurospheres consisted of neuronal cells.

Immunofluorescence phenotyping of NSCs
To have their NSC properties assessed, neurospheres underwent an immunofluorescence staining of their NSC markers and were compared with the characterized hSCAPs.First, the characterized hSCAPs were stained with DAPI to have their nuclei located (Figure 5A).
In the hSCAPs, the expression of Nestin (Figure 5B (Figure 7A).These neurospheres at passage 1 then underwent a treatment with the Accutase enzyme and disaggregated into individual cells (Figure 7B).
Interestingly, the intra-neurospheral cells could be re-aggregated in the ultra-low attachment multiplewell plate and become neurospheres at passage 2, which indicates their self-renewal ability, typical of NSCs (Figure 6C).

Gene expression profiling
At the molecular level, it was discovered that the neurospheres presented higher expressions of NES, SOX1, and PAX6 than the hSCAPs.These three markers are known to indicate the presence of NSCs.
This suggests that the hSCAPs differentiated into NSCs through the 3D-neurosphere induction.

Neuronal activity
Intracellular calcium oscillation was detected in the typical neuronal cells, indicating that neuronal

Discussion
Adult neurogenesis in the mammalian brain was shown to be regulated by the physiological and biological processes of NSCs, such as cell proliferation, cell differentiation, cell fate determination, cell survival, maturation, integration of the generated neuronal cells into the existing circuitry, and functional input reception. 18The potential compartments where NSCs could be located are the SVZ of the lateral ventricle and the SGZ of the hippocampal dentate gyrus, which are defined as the actively restricted regions for adult neurogenesis. 19However, their endogenous repairing by their NSCs is limited by damages in the CNS. 3 In this study, NSCs exhibited a self-renewal ability, which resulted in their aggregation into clusters of cells 20 that differentiated into neurons, oligodendrocytes, and astrocytes under optimal in vitro inducing conditions. 21Therefore, the transplantation of exogenous NSCs could allow for the replacement of degenerated neurons and the regeneration of injured CNSs. 4 However, the collection of NSCs from the SVZ and SGZ of adult brains was shown to cause donor site morbidity, harming donors and creating ethical concerns. 5It is also complicated to isolate and cultivate the NSCs from these regions: cells can be isolated from adult brains, but at a deficient number. 22The present study aimed to overcome these limitations by choosing an alternative tissue to collect cells from, and resulted in the discovery of cells that could neuronally differentiate into NSCs without causing donor site morbidity and ethical concerns.
Dental-derived mesenchymal stem cells are a promising resource for neuronal regeneration processes, due to their neuronal differentiation ability. 23evious researches have described hSCAPs as a novel population of post-natal multipotent stem cells residing in the apical papilla tissue of immature permanent teeth. 6The present study analyzed stem cells located Characterization of neural stem cells derived from human stem cells from the apical papilla undergoing three-dimensional neurosphere induction the expression of cell-surface antigen molecules. 24e hSCAPs derived from the developing root of immature permanent teeth and consisted of a large population of early stem cells that exhibited superior properties compared with those of other adult stem cells derived from mature tissue, including the potential for differentiation and the ability to selfrenew. 25The collection of these hSCAPs took place through a non-invasive process: the cells were easily accessible and provided by dental waste. 10Additionally, an in vitro study demonstrated that the secretome of hSCAPs has neurotrophic factors that can trigger the neurite outgrowth of human neuroblastoma cells and an in vivo study showed that these neurotrophic factors can enhance the regeneration of sciatic nerve injuries. 26The results of the present study indicate hSCAPs from dental waste as an alternative resource to generate NSCs with less ethical concerns and donor site morbidity risks.

Various strategies to differentiate cells into neurons
have been recently developed, including epigenetic modification, small molecules, psychotropic drugs, and enriched medium cocktails with chemical inducers, 27 but the placement of MSCs in a neuronal induction medium containing chemical inducers provided a faster neuronal differentiation rate than other methods. 28The generation of neurospheres was defined as a potential in vitro model for studying CNS disorders. 29The present study effected the neuronal differentiation of characterized hSCAPs into NSCs through a 3D neurosphere induction process, which took place within a neural induction medium containing bFGF and EGF in low-adherent culture vessels for 5 days.To form neurospheres, specimens require supplementation with specific growth factors, including bFGF and EGF, and a m i c r o e n v i r o n m e n t s u i t a b l e f o r n e u r a l induction and further neuronal maturation. 11,12,30study found that, after undergoing a spheroidbased 3D neural induction method, NPCs derived from human-induced pluripotent stem cells (hiPSCs) expressed high concentrations of Nestin/PAX6 and differentiated into neuronal cells, exhibiting a longer neurite outgrowth than that of cells undergoing a 2D monolayer method.31 Moreover, the formation of 3D spheres was shown to induce a higher neurogenic potential in the hSCAPs than the 2D method, resulting in overall higher neurite numbers, mean neurite lengths, total neurite lengths, and expressions of neurogenic-associated genes.14 During the neurosphere induction process, the size of the neurospheres greatly increased, and fiveday intervals generated a greater number of viable neurospheres.13 Furthermore, it was found that at 5 days of culture, a primary neurosphere culture originating from newborn Sprague-Dawley rats contained NSCs with a healthy morphology, but at 8 to 9 days of culture these NCSs revealed a dark area formed by dead intra-neurospheral cells.32 The present study investigated specific parameters and cellular structures to elucidate the characterization of in vitro-induced neurons.33 First, an analysis of cell morphology revealed that characterized hSCAPs presented typical fibroblast-like shapes, while the neurospheres they originated had a 3D-spheroidal form.This finding is similar to those of recent studies that investigated neurospheres derived from human dental pulp stem cells (hDPSCs) 12,13 and hiPSCs.31 Second, it was found that the Nissl body, an intensely basophilic granular consisting of a rough endoplasmic reticulum, was only present in neurons.34 In vitrodifferentiated neuronal cells derived from hSCAPs 7 and hDPSCs 35 were recently shown to present Nissl bodies, which were found through a staining process with Cresyl violet dye.Moreover, the hippocampal area of a rat model exhibited the organization of cells with Nissl bodies, which were characterized as neurons.36 Therefore, the identification of the Nissl body can be used to verify the characteristics of typical neuronal cells.In the present study, the Cresyl violet staining process revealed that the Nissl body was present in intra-neurospheral cells, which evidenced their neuronal phenotype.
Certain studies found that biomarkers for embryonic and adult neurogenesis were necessary to characterize the gene and protein expressions of NSCs. 37NES, a protein-encoding gene, was shown to encode the Nestin protein, which is expressed in diving cells during the early stages of development of the nervous system. 38The SOX1 gene encodes a transcription factor that exerts an essential role in neurogenesis. 39X2 encodes the transcription factor essential for self-renewal and is critical in maintaining NSCs. 40PAX6 encodes one of the critical embryonic transcription factors, which regulates CNS morphogenesis and is widely expressed in the neuroectoderm. 41From the sixth to the eighth day of a neurosphere induction process, hDPSCs and human gingival mesenchymal stem cells (hGMSCs) generated NCSs that, compared with those in undifferentiated hDPSCs and hGMSCs, exhibited greater expressions of NES and SOX1, and a lower expression of PAX6, respectively. 42These results imply that these induced cells presented the same NSC Characterization of neural stem cells derived from human stem cells from the apical papilla undergoing three-dimensional neurosphere induction profile, and that a long-time neurosphere induction might affect the expression of PAX6.On the fifth day of the neurosphere induction process conducted in this study, neurospheres presented higher expressions of NES, SOX1, and PAX6, than the hSCAPs that were used as the negative control-and the intraneurospheral cells were thus proven to be NSCs.Other studied found that in vitro-induced neurospheres derived from hDPSCs expressed Nestin, 13,17 and that neurospheres derived from stem cells of bovine adipose tissue expressed Nestin, SOX2, and β-III tubulin (neurogenic-associated protein). 43Moreover, it was found that in vivo NSCs derived from neurospheres of embryonic brain cells E14.5 to E16.5 were positive for Nestin staining, thus confirming their NSC properties. 44e neurospheres analyzed in the present study consisted of a heterogeneous population with different immunofluorescence staining patterns, and the co-expression of Nestin and SOX2 in the intraneurospheral cells verified their profiling as NSC.
Subsequently in this study, the ability of the NCSs to self-renew, represented by the re-formation of the neurospheres, was demonstrated through their disaggregation with Accutase.Studies with neurospheres derived from hDPSCs found that the dissociation these clusters with Accutase allowed for the re-forming of more viable cells than the mechanical and enzymatic (trypsin) dissociations. 13Moreover, neurospheres generated from the primary culture of adult rat SVZ and hippocampus cells formed more new neurospheres after undergoing disaggregation with Accutase than after being dissociated with trypsin. 45e present study showed that a new passage of neurospheres was formed after neurospheres underwent Accutase dissociation within a neural induction medium in an ultra-low attachment culture system.These results indicate that the neurospheres presented the ability of self-renewal.
Previous studies have investigated functional neuronal networks, intercellular communication 12,46 and intracellular signalling 17,47 to further verify the functional profile of neuronal cells.Intracellular calcium oscillation is a technique used to investigate the status of Ca 2+ influx. 48Ca 2+ are essential ions that get internalized into neurons during vesicular neurotransmitter-releasing activity. 49The activity of intracellular calcium transients can be alternately represented as neuronal activity, which is closely correlated to electrical activity recorded with a whole-cell patch clamp. 50In the present study, the neuronal activity of intra-neurospheral cells was analyzed through the visualization of intracellular calcium oscillation.The Fluo-3 AM, a calcium indicator, was used to detect the activity of intracellular calcium signalling during neurotransmission. 51h e d y n a m i c c h a n g e s i n t h e f l u o r e s c e n c e intensity of intra-neurospheral cells consisted of continuous high-intensity peaks and wide intervals.
In contrast, steady baseline patterns of low intensity were observed in the hSCAPs used as the negative control.Since these findings on intracellular calcium oscillation were consistent with previous studies that presented the functional profilings of neuronal cells derived from hDPSCs 17,35,52 and of hiPSCs derived from dentate gyrus neuronal progenitors, 53 the intraneurospheral cells analyzed in the present study were proven to be functional neuronal cells.However, further investigation with electrophysiological tests and neurogenic maturation analyses is still necessary to thoroughly characterize the neuronal profile of these cells.
The findings of this study reinforce the potential of in vitro-induced NSCs derivated from hSCAPs, which can be used as an alternative resource for neuronal regeneration processes, replacing animal models and reducing limitations such as donorsite morbidity and ethical concerns.The present study aimed to develop an efficient method for the production of neural commitment of hSCAPs in largescale expansion, specifically in a serum-free medium.
However, a preclinical study on the safety and efficacy of in vitro-induced NSCs derived from hSCAPs must be done before applying these findings to in vivo transplantation.Transplantation of neurospheres into damaged CNS areas should also be performed in future studies to explore factors such as host integration, cell survival, and neuronal differentiation in vivo models.
The results of this study demonstrated the MSC properties of hSCAPs and the neuronal profiling of NSCs derived from characterized hSCAPs that underwent a 3D neurosphere induction process.These properties were revealed through the analysis of cell morphology, protein expression (by immunofluorescence staining), self-renewal ability, gene expression, and intracellular calcium oscillation.These results suggest that NSCs derived from hSCAPs can be used for exogenous transplantation in stem cell-based therapies for neurodegenerative diseases.Characterization of hSCAPs … and CD146+ (Figure 1G).… and CD146+ (Figure 2G).

Gene expression profiling
These three markers are known to indicate the presence of NSCs.
These three markers are known to indicate the presence of NSCs (Figure 7).
Where it reads, page 7 Where it reads, page 10 The intra-neurospheral cells obviously revealed calcium ions signal.(A''-D'') The hSCAPs presented a low and narrow dynamic change of calcium ions intensity (red, orange, and yellow lines).Importantly, higher and wider dynamic changes of calcium ions intensity were observed at intra-neurospheral cells (pink, dark blue, and light blue lines).Data were expressed as the mean intensity of calcium ions; n=3, Scale bar: A-D and A'-D' = 100 μm We obtained human impacted third molars from three Thai patients aged 15-20 years who visited the Oral and Maxillofacial Surgery Clinic at the Dental Hospital of Mahidol University in Thailand.A careful selection was performed, and only the teeth with healthy apical papilla tissue and no signs of caries, pulp necrosis, trauma, or periodontal disease were used in this study.The present experimental procedures were approved by the Ethics Committee on Human Rights Related to Human Experimentation of the Faculties of Dentistry and Pharmacy at Mahidol University, Thailand (approval no.MU-DT/PY-IRB 2021/016.0706),and written informed consent was obtained from all participants before their inclusion in the study.The present authors ensured that all the procedures were performed in accordance with the Declaration of Helsinki.

of
Minimum Essential Medium (MEM, Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies) and 1% antibiotic-antimycotic (Gibco, Life Technologies).They were then washed with 0.1 M phosphate buffer saline (PBS, Sigma-Aldrich, St. Louis, MO, USA).Afterward, the apical papilla tissue was dissected and digested twice in 3 mg/mL collagenase I (Worthington, Lakewood, NJ, USA) and 4 mg/mL dispase II (Sigma-Aldrich) at 37°C for 60 minutes.The sample was filtered through a 70 µm cell strainer (Falcon TM , Thermo Fisher Scientific, Waltham, MA, USA), seeded into a cell culture vessel Characterization of neural stem cells derived from human stem cells from the apical papilla undergoing three-dimensional neurosphere induction for 30 minutes.They were stained with 0.5 % of Oil Red O (Sigma-Aldrich) in an isopropanol (EMSURE ® , MERCK, Darmstadt, Germany) solution for 60 minutes at room temperature, then rinsed three times in deionized water (DI H 2 O).The lipid droplet ensembles were stained and observed under the Compact Cell Culture Microscope CKX3.
paraformaldehyde for 30 minutes.They were stained with 40 mM of an Alizarin red (Sigma-Aldrich) solution at room temperature for 20 minutes, then rinsed three times with DI H 2 O.The extracellular matrix calcification was observed under the Compact Cell Culture Microscope CKX3.

25x10 4 cells
/well and cultured in a neural induction medium composed of DMEM/F-12 supplemented with 2% B-27 (Gibco, Life Technologies), 20 ng/mL EGF (Gibco, Life Technologies), 20 ng/mL bFGF and 1% antibioticantimycotic, placed in a Costar ® ultra-low attachment multiple-well plate, size 24 well (Corning, NY, USA).The neurospheres were kept in the neural induction medium for 5 days.Half of the medium was changed every two days.The morphology of the neurospheres was observed and imaged with the Compact Cell Culture Microscope CKX3.Furthermore, the properties of NSCs in the neurospheres were detected through the identification of Nissl substance, made with Cresyl violet staining, the protein expression evaluation, made with immunocytochemistry, the analyses of selfrenewal abilities and gene expressions through the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the analysis of functional activity through intracellular calcium oscillation.
for 1 hour, then washed with PBS for 5 minutes and with double distilled water (ddH 2 O) for 1 minute.Subsequently, they were incubated with a Cresyl Violet Acetate working solution (Electron Microscopy Sciences, Hatfield, PA, USA) in a dark environment for 1 hour.Thereafter, the three specimens were washed with ddH 2 O, then with 90, 95, and 100% ethanol (EMSURE ® , MERCK), respectively (according to the order first cited in this section).The stained Nissl substance was observed under the Compact Cell Culture Microscope CKX3.
Characterization of neural stem cells derived from human stem cells from the apical papilla undergoing three-dimensional neurosphere induction donkey anti-rabbit Alexa Flour plus 594 (1:1,000; Invitrogen).Nuclei were counterstained and mounted with the Prolong™ Diamond antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen).The samples were observed under the Confocal Microscope Platforms STELLARIS 5 (Leica Microsystems, Wetzlar, Germany) and imaged with the Leica Application Suite X software (Leica microsystems).The fluorescent intensity in the cells was measured using the ImageJ software (NIH, Bethesda, MD, USA).
were subsequently washed with DMEM/F-12, 1% antibiotic-antimycotic, and PBS, then immediately put into Tyrode's solution (1 mM MgCl 2 , 2 mM CaCl 2 , 5 mM KCl, 25 mM HEPES, 30 mM glucose and 129 mM NaCl, pH 7.4; all from Sigma-Aldrich) and kept inside it.The characterized hSCAPs at passage 4 were used as a negative control.The neuronal activity was triggered with 50 mM KCI.Subsequently, the fluorescent intensity was recorded at excitation 506 nm for 120 seconds, with the Confocal Microscope Platforms STELLARIS 5, and the mean intensity of the fluorescent signals was assessed using the Leica Application Suite X software, which allowed for the interpretation of the neuronal activity.
), which indicated ectomesenchyme origins, along with the presence of SOX2 (Figure 5C) indicated that these cells had pluripotency.Interestingly, the individual intra-neurospheral cells presented their nuclei after undergoing DAPI staining (Figure 5A') and also expressed both Nestin (Figure 5B') and SOX2 (Figure 5C'), which represented the specific NSC markers.Double immunofluorescence staining (Nestin/SOX2) was performed on hSCAPs and neurospheres to verify the characteristics they shared with NSCs (Figure 5D-G and 5D'-G').The results demonstrated that while hSCAPs rarely co-expressed Nestin and SOX2 (Figure 5F), neurospheres did co-express the two (Figure 5F').Furthermore, the neurospheres exhibited different staining patterns, which indicates the heterogeneity of the intra-neurospheral cell population.The individual intra-neurospheral cells, which co-expressed Nestin, SOX2, and DAPI, were confirmed to be NSCs (Figure5G').Lastly, the analysis of fluorescence intensity demonstrated that hSCAPs presented a higher intensity of Nestin than of SOX2.The neurospheres exhibited the greatest SOX2 expression (Figure5H).Taken together, the present results indicate that hSCAPs can differentiate into NSCs after forming 3D neurospheres.Self-renewal abilityAfter undergoing neural induction for 5 days, the hSCAPs differentiated into neurospheres, which aggregated, forming a 3D-cluster of NSCs Characterization of neural stem cells derived from human stem cells from the apical papilla undergoing three-dimensional neurosphere induction

Figure 2 -
Figure 2-Characterization of hSCAPs.(a) The isolated cells can grow on plastic adherent culture vessels and reveal the typical fibroblastlike shape morphology.(b-c) The neural crest stem cells' derivative origin was demonstrated with β-III tubulin and nestin staining, respectively.(d) The number of isolated cells that expressed these markers (CD34−, CD73+, CD90+, CD105+, and CD146) are highly expressed.(e) The isolated cells can form colonies. (f-h) Multipotential differentiation abilities were demonstrated by adipogenesis, osteogenesis, and neurogenesis, respectively.Scale bars: a, f, g, and h = 100 µm, b and c = 50 µm, and e = 5 mm

Figure 3 -
Figure 3-Generation of neurospheres.(a) The hSCAPs presented the typical fibroblast-like shape.(b) Neurospheres exhibited freefloating aggregated cells and consisted of a cluster of intra-neurospheral cells.Scale bars: a and b = 100 µm

Figure 4 -Figure 5 -
Figure 4-Identification of Nissl substance.(a) The hSCAPs revealed the pale purple background of the nucleus (black arrows) and the violet spot of the nucleolus (black asterisks).(b) The cluster of intra-neurospheral cells exhibited an intense purple substance (white arrows) that indicates the Nissl body of neuronal cells marker.Scale bars: a and b = 100 µm

Figure 2 -Figure 2 - 9 Figure 5 -Figure 5 -
Figure 2-Characterization of hSCAPs.(a) The isolated cells can grow on plastic adherent culture vessels and reveal the typical fibroblastlike shape morphology.(b-c) The neural crest stem cells' derivative origin was demonstrated with β-III tubulin and nestin staining, respectively.(d) The number of isolated cells that expressed these markers (CD34−, CD73+, CD90+, CD105+, and CD146) are highly expressed.(e) The isolated cells can form colonies. (f-h) Multipotential differentiation abilities were demonstrated by adipogenesis, osteogenesis, and neurogenesis, respectively.Scale bars: a, f, g, and h = 100 μm, b and c = 50 μm, and e = 5 mm