An Overview of Protocols for the Neural Induction of Dental and Oral Stem Cells In Vitro

Abstract

To date, various adult stem cells have been identified within the oral cavity, including dental pulp stem cells, dental follicle stem cells, stem cells from apical papilla, stem cells from human exfoliated deciduous teeth, periodontal ligament stem cells, and mesenchymal stem cells from the gingiva. All of these possess neurogenic potential due to their common developmental origin from the embryonic neural crest. Besides the relative ease of isolation of these adult stem cells from readily available biological waste routinely produced during dental treatment, these cells also possess the advantage of immune compatibility in autologous transplantation. In recent years, much interest has been focused on the derivation of neural lineages from these adult stem cells for therapeutic applications in the brain, spinal cord, and peripheral nerve regeneration. In addition, there are also promising nontherapeutic applications of stem cell-derived neurons in pharmacological and toxicological screening of neuroactive drugs, and for in vitro modeling of neurodevelopmental and neurodegenerative diseases. Hence, this review will critically examine the diverse array of in vitro neural induction protocols that have been devised for dental and oral-derived stem cells. These protocols are defined not only by the culture milieu comprising the basal medium plus growth factors, small molecules, and other culture supplements but also by the substrata/surface coatings utilized, the presence of multiple culture stages, the total culture duration, the initial seeding density, and whether the spheroid/neurosphere formation is being utilized to recapitulate the three-dimensional neural differentiation microenvironment that is naturally present physiologically in vivo.

Introduction: A Diverse Array of Adult Stem Cells Identified Within the Oral Cavity

Over the last decade, the nascent field of regenerative medicine has progressed rapidly with the discovery of an increasing number of different types of multipotent adult stem cells from various somatic tissues throughout the human body.¹ Of these, the oral cavity has attracted much attention, as it has yielded a diverse array of adult stem cells that can be classified into two major categories: (1) dental stem cells that generate the dentin–pulp complex in vivo and (2) nondental oral stem cells that do not generate the dentin–pulp complex in vivo.^2–6 Dental stem cells encompass dental pulp stem cells (DPSCs),⁷ stem cells from human exfoliated deciduous teeth (SHED),⁸ and stem cells from apical papilla (SCAP).⁹ Nondental oral stem cells encompass dental follicle stem cells (DFSCs),¹⁰ periodontal ligament stem cells (PDLSCs),¹¹ and gingival mesenchymal stem cells (GMSCs).¹²

Even though all of these aforementioned dental and oral stem cells are mesenchymal in character with fibroblastic morphology, there is also much divergence in the properties of these cells, that is, differences in proliferative capacity, multilineage differentiation potential, and expression of cell surface receptors and other marker genes.^2–6 In common with other adult stem cells, dental and oral stem cells exhibit a high degree of population heterogeneity, and there is no consensus on the specific gene markers that define them as adult stem cells. In any case, the commonly adhered standards for identification and isolation of multipotent mesenchymal stem cells from bone marrow and adipose tissues are usually applied to dental and oral stem cells.^2–6 This is best exemplified by the minimal criteria for defining human multipotent mesenchymal stem cells established by the International Society for Cellular Therapy in 2006,¹³ which include the following: (1) the ability to rapidly adhere to plastic surface under standard culture conditions, (2) the ability to differentiate into the adipogenic, osteogenic, and chondrogenic lineages under appropriate inducing culture conditions, and (3) the expression of common mesenchymal stem cell-associated markers such as CD105, CD73, and CD90, together with the lack of expression of CD45, CD34, CD14, CD11b, CD79a, CD19, and HLA-DR surface molecules. In addition, clonogenicity—that is the ability to form adherent colonies from single cells plated at low densities, is also widely utilized by many laboratories as a criteria for identification of dental and oral stem cells.^2–6

A major advantage of dental and oral stem cells is their ease of isolation from readily available biological waste routinely produced during dental treatment that would otherwise be discarded, that is, extraction of impacted third molars (i.e., wisdom tooth) and erupted deciduous tooth (i.e., primary tooth) and their surrounding tissues.^2–6 This is much less invasive than the aspiration of bone marrow for the extraction of mesenchymal stem cells (BM-MSCs). Compared to hematopoietic stem cells from umbilical cord blood, dental and oral stem cells are generally more plastic with a more extensive multilineage differentiation potential.^2–6 Indeed, it was reported that some dental and oral stem cells expressed pluripotency markers not normally expressed in other adult stem cells, such as OCT4, SOX2, and MYC.¹⁴ One of the most intractable technical challenge faced in utilizing adult stem cells for therapy is their limited proliferative capacity and tendency to undergo senescence with prolonged ex vivo culture.^15,16 In this regard, the higher proliferative capacity of DPSC and SHED, compared to bone marrow and adipose-derived mesenchymal stem cells,^17,18 has attracted special attention for therapeutic applications. Yet another advantage is that dental and oral stem cells do not face ethical and immune-compatibility issues associated with embryonic and fetal stem cells, due to their ease of isolation from a readily available autologous source. To date, there have been no reports of teratoma or other cancer formation by transplanted dental and oral stem cells, unlike the case of human embryonic stem cells and induced pluripotent stem cells (iPSCs). Also absent are safety issues associated with genetic modification and use of viral vectors for the reprogramming of somatic cells into iPSCs.

While the overwhelming majority of previous studies have shown that dental and oral stem cells certainly have useful applications in various dental treatments, that is, periodontal and maxillofacial regeneration,^19,20 osseous integration of titanium implants,²¹ and dental pulp regeneration after endodontic treatment²²; a number of studies have emerged which suggest that such adult stem cells also have much potential for diverse nondental biomedical applications.^2–6 Much research has been focused on neural lineages derived from dental and oral stem cells, because these cells possess a much higher innate neurogenic potential than most other adult stem cells, due to their origin from the embryonic neural crest.^23,24 The focus of this review is on the in vitro differentiation of dental and oral stem cells into neural lineages for therapeutic applications in neurodegenerative diseases and repair of brain and spinal cord injuries, as well as for nontherapeutic applications in pharmacology, toxicology, and in vitro disease modeling.

Dental and Oral Stem Cells Originate from the Embryonic Neural Crest

The neural crest is a distinctive transient structure formed during vertebrate embryogenesis, which gives rise to the central nervous system,²⁵ as well as a diverse multitude of other lineages, including the dental mesenchyme.^26,27 Due to its crucial functional role in early development, it has often been considered the “fourth germ layer” in vertebrate embryogenesis,²⁸ in addition to the ectoderm, mesoderm, and endoderm. The formation of the neural crest is initiated at the junction between the epidermal ectoderm and the closing neural tube during the process of neurulation.²⁸ The distinct and unique subpopulation of cells that forms within the neural crest subsequently migrate along specific pathways to defined sites within the developing vertebrate embryo, undergoing an epithelial–mesenchymal transition during the process,²⁹ and differentiating into diverse functional lineages, including cells of the central nervous system and the dental mesenchyme.^25–27 Interaction between the dental mesenchyme and the stomodeal exoderm lining the interior of the developing oral cavity generates tooth and its surrounding tissue, including the periodontium, as well as gives rise to the various niches of dental and oral-derived adult stem cells.^30,31 The pioneering study of Chai et al. utilized transgenic reporter genes (Wnt1 and R26R) to track the progeny of the cranial neural crest during mandible and tooth development,³² and conclusively demonstrated that migrating neural crest cells gave rise to the condensed dental mesenchyme during mammalian embryogenesis. This was further confirmed by the study of Yamazaki et al.,³³ which also utilized a transgenic reporter gene (LacZ) to trace and validate the presence of neural crest-derived cells within the developing tooth.

Baseline Neural Marker Expression by Undifferentiated Dental and Oral-Derived Stem Cells in the Absence of Neural Induction

Due to their origin from the neural crest, it is not surprising that undifferentiated dental and oral-derived stem cells exhibit baseline expression of neural markers, even without having been subjected to neural induction. This was observed and validated by several studies. Foudah et al. demonstrated that a high percentage of undifferentiated human DPSCs and PDLSCs displayed spontaneous expression of mature neuronal markers βIII-tubulin and NeuN, as well as the neural stem cell marker Nestin.³⁴ Feng et al. reported that both SHED and DPSCs spontaneously expressed neural markers, such as βIII-tubulin, microtubule-associated protein 2 (MAP2), tyrosine hydroxylase (TH), and Nestin, with expression levels being lower in DPSCs compared to SHED.³⁵ However, exposure of DPSCs to recombinant Wnt1 increased expression levels of these aforementioned neural markers. Both DPSCs and SHED are from the same histological source (dental pulp), the only difference between them being donor age (i.e., DPSCs are derived from impacted third molars of mature adults, whereas SHED are derived from erupted deciduous tooth of younger children or teenagers); it was thus hypothesized that Wnt/β-catenin signaling played an important role in age-dependant neural differentiation of dental stem cells. Nevertheless, contrary results were reported by the study of Govindasamy et al.,³⁶ which showed that DPSCs displayed higher spontaneous expression of the neuroectodermal markers PAX6, GBX2, and Nestin, compared to SHED. Immunocytochemistry analysis by Martens et al. revealed that undifferentiated human DPSCs uniformly expressed neural markers βIII-tubulin, S100 protein, and synaptophysin, with a subset of the population displaying positive immune reactivity for galactocerebroside, neurofilament, and nerve growth factor (NGF) receptor p75.³⁷ In another study by Tamaki et al. on human DPSCs, DFSCs, and SCAP, it was demonstrated that these adult stem cells in the undifferentiated state were positively immunoreactive to antibodies against neural markers such as Nestin, βIII-tubulin, and neurofilament (NF)-200.³⁸ The study of Karaöz et al. verified the intrinsic neuroglia characteristics of human DPSCs by demonstrating that these cells in the undifferentiated state spontaneously expressed several specific neural markers associated with both mature neural lineages and neural stem/progenitor cells such as SOX2, tenascin C (TNC), ENO2, MAP2ab, c-FOS, NES, NEF-H, NEF-L, glial fibrillary acidic protein (GFAP), TUBB3, and connexin-43.³⁹ Expression of neural markers at the molecular level by undifferentiated dental and oral-derived stem cells may possibly correlate with the display of some differentiated neuronal functions. For example, in the study of Davidson,⁴⁰ the patch-clamp techniques were used to demonstrate the presence of a tetrodoxin-sensitive voltage-gated inward current in human dental pulp cells.

The Neuroregenerative Potential of Dental and Oral-Derived Stem Cells Has Been Validated by In Vivo Transplantation Studies

The neuroregenerative potential of dental and oral-derived stem cells has been validated by in vivo studies in which undifferentiated stem cells have been transplanted into live animal models. In particular, the transplantation of undifferentiated DPSCs has demonstrated much promise in the regeneration of various different types of neural lesions. In the study of Yang et al.,⁴¹ the transplantation of Nurr-1-positive cells isolated from human dental pulp into a rat stroke model facilitated the regeneration of damaged brain tissues. Sasaki et al. fabricated artificial nerve conduits by placing DPSCs within biodegradable poly-dl-lactide-co-glycolide tubes, and these demonstrated positive efficacy in repairing gaps in the facial nerves of rats upon implantation.⁴² Spyridopoulos et al. transplanted DPSCs into the transected intercostal nerves of pigs and observed the morphological and functional recovery of neural lesions through immunohistochemical and electrophysiological evaluation.⁴³ Mead et al. demonstrated that intravitreally transplanted DPSCs exerted a neuroprotective effect and stimulated axon regeneration of retinal ganglion cells in a rat optical injury model.⁴⁴

Currently, there is accumulating evidence to suggest that the positive neuroregenerative effects of transplanted DPSCs is more likely due to the paracrine effect of growth factors and chemokines secreted by these cells, rather than the DPSCs giving rise to neural tissues themselves. Huang et al. reported that the transplantation of rhesus monkey DPSCs into the hippocampus of healthy mice brain not only stimulated proliferation of endogenous neural cells but also induced the migration and homing of neural progenitors and mature neurons to the transplantation site, even though all of the transplanted cells had undergone apoptosis within 5 days of transplantation.⁴⁵ Similar results were reported by Leong et al., who demonstrated that transplantation of human DPSCs enhanced poststroke recovery in rats, even though only 2.3% of the transplanted cells survived and were engrafted in the host rats.⁴⁶ Arthur et al. demonstrated that transplanted adult human DPSCs in an avian embryonic model system could enhance endogenous axon guidance through the SDF1-CXCR4 chemokine axis.⁴⁷

Besides DPSCs, a number of studies have also investigated the neuroregenerative potential of other types of dental and oral-derived stem cells. Beigi et al. seeded SHED on a nanofibrous nerve guide and showed that this cell–biomaterial construct could stimulate axonal regeneration of rat sciatic nerves upon implantation in situ.⁴⁸ Li et al. demonstrated that human PDLSCs can be utilized as an alternative cell source to autologous Schwann cells for promoting regeneration of injured peripheral nerves.⁴⁹ Transplanted human DFSCs were shown to express the oligodendrocyte lineage marker in situ within a rat spinal cord injury model.⁵⁰

In Vitro Neural Differentiation of Dental and Oral-Derived Stem Cells: Therapeutic and Nontherapeutic Applications

Although transplantation studies with live animal models have conclusively demonstrated the neuroregenerative potential of dental and oral-derived stem cells, it must be noted that in most of these previous studies, undifferentiated stem cells were utilized. A major limitation to utilizing undifferentiated stem cells for neural regeneration is the tendency of these cells to undergo spontaneous differentiation into multiple lineages upon transplantation or grafting in vivo. Hence, the clinical efficacy of transplantation could be reduced due to only a small subfraction of the transplanted stem cells differentiating into neural lineages in vivo. Moreover, there is also a risk of the undifferentiated stem cells giving rise to undesired lineages at the transplantation site that may hamper neural regeneration, that is, fibrotic scar tissue. Predifferentiating stem cells into neural lineages in vitro could enable the expression of neuron-associated surface receptors and adhesion molecules before grafting in vivo, which may hasten integration with the host's nervous system, thereby enhancing the clinical efficacy of transplantation therapy. Indeed, in vitro differentiated neural lineages derived from stem cells have displayed much promise for the treatment of various neurodegenerative and neurodevelopmental diseases,^51,52 traumatic spinal cord and brain injuries,^53,54 stroke,⁵⁵ and peripheral nerve regeneration.⁵⁶ Besides clinical therapy, efficient in vitro protocols for neural differentiation of dental and oral-derived adult stem cells can also have diverse nontherapeutic applications, such as for in vitro neurotoxicity and neuropharmacological screening of new drugs and industrial chemicals, as well as for the in vitro modeling of neurodegenerative and neurodevelopmental diseases.^57,58

DPSCs: Neurogenesis In Vitro

Because the putative DPSC population is in fact heterogeneous, and not all cells have equal propensity to differentiate into the neural lineage, it may be advantageous to carry out fluorescence-activated or magnetic-affinity cell sorting to isolate out the cellular subpopulation that possesses greater neurogenic potential, before subjecting the cells to neurogenic differentiation in vitro. In the study by Dai et al.,⁵⁹ it was proposed that the isolated DPSC subpopulation expressing the p75 neurotrophin receptor could have greater therapeutic potential than unsorted DPSCs, due to their increased propensity for neuronal differentiation.

To date, there have been numerous studies that have carried out in vitro differentiation of DPSCs into neural lineages, utilizing a diverse array of highly varied protocols. As summarized in Table 1, the various in vitro neural differentiation protocols developed for DPSCs can be defined not only by the culture milieu comprising the basal medium plus growth factors, small molecules, and other culture supplements but also by the substrata/surface coatings utilized, the presence of multiple culture stages, the total culture duration, the initial seeding density, and whether spheroid/neurosphere formation is being utilized to recapitulate the three-dimensional neural differentiation microenvironment that is naturally present physiologically in vivo.^60–62 Xiao and Tsutsui reported that even without neural induction, DPSCs cultured as spheroids in suspension culture under serum-free conditions were capable of spontaneously differentiating into neural lineages in vitro, as confirmed by the expression of various neural markers.⁶³ Much variability exists in the use of spheroid suspension culture for the neurogenic differentiation of DPSCs under neural-inducing conditions. Gervois et al.⁶⁰ and Osathanon et al.⁶¹ utilized neurosphere formation with nonadherent culture dishes during the initial phase of their in vitro neural differentiation protocol, whereas the study of Karbanová et al.⁶² utilized neurosphere formation later at the end phase of the culture protocol. While Gervois et al.⁶⁰ later derived a monolayer of neural cells through the seeding of intact neurospheres, Osathanon et al.⁶¹ carried out manual dissociation of the neurospheres through manual pipetting to obtain a similar monolayer.

Table 1.

In Vitro Neural Induction Protocols Devised for DPSCs

					Culture milieu
Study	Spheroid/neurosphere formation	Single/multiple stages (duration)	Initial seeding density	Substrata/surface coating	Basal medium	Culture supplements	Growth factors	Small molecules	Neural markers analyzed	Application/functional assessment
Gervois et al.⁶⁰	Yes	Neurosphere formation (6–8 days)	7.5 × 10³ cells/cm²	Nonadherent tissue culture dishes	DMEM/F12 (1:1)	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	Penicillin (100 U/mL) + streptomycin (100 mg/mL)	—	—
		Neurogenic maturation (28 days)	Unspecified—seeding of intact neurospheres	Poly-l-ornithine (15 mg/mL) + laminin (2 mg/mL)	Neurobasal medium	B27 (2% v/v) + N2 (1% v/v)	NT-3 (30 ng/mL)	l-glutamine (2 mM) + dbcAMP (1 mM) + penicillin (100 U/mL) + streptomycin (100 mg/mL)	Immuno-cytochemistry for detection of NeuN, MAP2, NCAM, GAP.43, synaptophysin, synapsin I, GFAP, A2B5, and Nestin.Semiquantitative RT-PCR for detection of NCAM, MAP2, SGG10, and MASH1.Enzyme-linked immunosorbent assay for detection of VEGF, NGF, BDNF, and GDNF secretion.	Patch-clamp analysis of tetrodoxin and tetraethyl ammonium-sensitive voltage-gated Na⁺ and K⁺ channels
Osathanon et al.⁶¹	Yes	Neurosphere formation (7 days)	5.0 × 10⁵ cells per 60 mm petri dish	Nonadherent tissue culture dishes	Neurobasal medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	l-glutamine (2 mM) + penicillin (100 U/mL) + streptomycin (100 mg/mL) + amphotericin-B (5 mg/mL)	Immunocytochemistry for detection of βIII-tubulin in neurospheres.	—
		Neurogenic maturation (7 days)	Neurospheres were dissociated and seeded at 2.0 × 10⁴ cells per well of 24-well plate	Collagen type IV	Neurobasal medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	Retinoic acid (10 nM) + l-glutamine (2 mM) + penicillin (100 U/mL) + streptomycin (100 mg/mL) + amphotericin-B (5 mg/mL)	Semiquantitative RT-PCR for detection of SOX2, SOX9, βIII-tubulin, GABA receptors α2, β2, and β3, and HEY1 (notch signaling target).	Fluorescent detection of intracellular Ca²⁺ flux upon stimulation with NMDD
Karbanová et al.⁶²	Yes	Neurogenic expansion (21 days)	Unspecified	TCPS	Neurobasal medium	B27 (2% v/v) + FBS (1% v/v)	EGF (20 ng/mL) + bFGF (10 ng/mL)	l-glutamine (2 mM) + penicillin (100 U/mL) + streptomycin (100 mg/mL)	Western blot for detection of nucleostemin, Musashi1, and NG2.Immunocytochemistry for detection of Nestin, FORSE1, Bcrp1, A2B5, NG2, RC2, GFAP, O4, Pan-NF, and βIII-tubulin.	—
		Neurosphere formation (7 days)	Unspecified	Nonadherent tissue culture dishes	Neurobasal medium	B27 (2% v/v)	BMP-2 (50 ng/mL) + EGF (20 ng/mL) + bFGF (10 ng/mL)	l-glutamine (2 mM) + penicillin (100 U/mL) + streptomycin (100 mg/mL)	Immunocytochemistry for detection of Pan-NF, βIII-tubulin, Nestin, A2B5, and O4.	—
Osathanon et al.⁶¹	No	Stage 1 (24 h)	Unspecified (80% confluence)	TCPS	DMEM	FBS (20% v/v)	—	β-mercaptoethanol (1 mM)	—	—
		Stage 2 (24 h)	—	TCPS	DMEM	—	—	β-mercaptoethanol (3 mM) + BHA (200 μM)	Immunocytochemistry for detection of βIII-tubulin.Semiquantitative RT-PCR for detection of SOX2, SOX9, and βIII-tubulin.	—
Pisciotta et al.⁶⁴	No	Preinduction (24 h)	Unspecified—confluent monolayer	TCPS	α-MEM	FCS (10% v/v)	—	β-mercaptoethanol (1 mM) + l-glutamine (2 mM) + penicillin (100 U/mL) + streptomycin (100 mg/mL)	—	—
		Neural induction (unspecified duration—culture until neuronal-like morphology is appreciable)	—	TCPS	α-MEM	—	—	β-mercaptoethanol (10 mM) + DMSO (2% v/v) + BHA (200 μM)	Immunocytochemistry for detection of βIII-tubulin, MAP2, Synapsin, and GFAP (after 1 week).Western blot for detection of βIII-tubulin, MAP2, and GFAP (after 1 week).	—
Yalvac et al.¹⁴	No	Preinduction (24 h)	3000–5000 cells/cm²	Poly-d-lysine (100 mg/mL)	DMEM	FBS (20% v/v)	bFGF (5 ng/mL)	β-mercaptoethanol (1 mM)	—	—
		Neural induction (12 days)	—	Poly-d-lysine (100 mg/mL)	Neurobasal medium	B27 (2% v/v)	bFGF (50 ng/mL) + NGF (50 ng/mL)	—	Immunocytochemistry for detection of βIII-tubulin.	—
Ryu et al.⁶⁵	No	Preincubation (24 h)	Unspecified	TCPS	α-MEM	FBS (20% v/v)	—	β-mercaptoethanol (1 mM)	—	—
		Preinduction (5 h)	—	TCPS	α-MEM	—	—	β-mercaptoethanol (2 mM)	—	—
		Neural Induction (14 days)	—	TCPS	α-MEM	FBS (10% v/v)	bFGF (10 ng/mL) + EGF (10 ng/mL) + NGF (20 ng/mL)	DMSO (2% v/v) + BHA (200 μM)	Immunocytochemistry for detection of Nestin, MAP2, and NeuN.	—
Zhang et al.⁶⁶	No	Preinduction (24 h)	20,000 cells/cm²	TCPS	α-MEM	FBS (10% v/v)	—	β-mercaptoethanol (1 mM)	—	—
		Neural induction (12 days)	—	TCPS	α-MEM	—	—	β-mercaptoethanol (10 mM) + DMSO (2% v/v) + BHA (200 μM)	Immunocytochemistry for detection of NSE and NeuN.Semiquantitative RT-PCR for detection of NSE.	—
Király et al.^67,68	No	Epigenetic reprogramming (2 days)	20,000 cells/cm²	Poly-l-lysine	DMEM/F12 (1:1)	FCS (2.5% v/v)	bFGF (5 g/mL)	5-azacytidine (10 mM)	Quantitative RT-PCR for detection of Vimentin, Nestin, NGN2, N-tubulin, NSE, NF-M, and GFAP was assessed at six time points: (1) noninduced, (2) after 2 days epigenetic reprogramming, (3) first day of induction, (4) third day of induction, (5) after 1 day of maturation, and (6) after 3 days of maturation.Semiquantitative RT-PCR for detection of PAX6, En1, Dlx1, and MASH1 after neural maturation.	Transplant into rats with lesions to the forelimb motor cortex after neural maturation.Injection into the cerebrospinal fluid of new-born rats after neural maturation.Immunohistochemistry to detect Vimentin, N-tubulin, NF-M, NeuN, GFAP, GAD65/07, synaptophysin, and EAAC1 expression by engrafted cells.Patch-clamp analysis of functional activity of voltage-dependent Na⁺ and K⁺ channels.
		Neural induction (3 days)	—	Poly-l-lysine	DMEM/F12 (1:1)	ITS (1% v/v)	NGF (10 ng/mL) + bFGF (10 ng/mL) + NT-3 (30 ng/mL)	IBMX (250 μM) + Forskolin (50 μM) + TPA (200 nM) + dbcAMP (1 mM)
		Neural maturation (3–7 days)	—	Poly-l-lysine	Neurobasal-A	B27 (1% v/v) + N2 (1% v/v)	NT-3 (30 ng/mL)	dbcAMP (1 mM)
Chang et al.⁷¹Dopaminergic neurons	No	Stage 1 (4 days)	Unspecified	TCPS	DMEM/F12 (1:1)	N2 (1% v/v)	Noggin (300 ng/mL)	—	—	—
		Stage 2 (5 days)	—	TCPS	DMEM/F12 (1:1)	N2 (1% v/v)	BDNF (50 μg/mL) + SHH (50 μg/mL) + FGF-8b (50 μg/mL)	Ascorbic acid (200 mM)	—	—
		Stage 3 (3 days)	—	TCPS	DMEM/F12 (1:1)	N2 (1% v/v)	BDNF (50 μg/mL) + SHH (50 μg/mL)	Ascorbic acid (200 mM)	—	—
		Stage 4 (3 days)	—	TCPS	DMEM/F12 (1:1)	N2 (1% v/v)	BDNF (50 μg/mL) + GDNF (50 ng/mL) + TGF-β3 (2 μg/mL)	Ascorbic acid (200 mM) + dbcAMP (200 mM)	Immunocytochemistry for detection of NeuN, GFAP, Nestin, βIII-tubulin, O1, and TH.Semiquantitative RT-PCR for detection of Nestin and βIII-tubulin.	—
Chang et al.⁷¹ Motor neurons	No	Stage 1 (4 days)	Unspecified	TCPS	DMEM/F12 (1:1)	N2 (1% v/v) + NEAA (1% v/v)	—	All-trans retinoic acid (0.1 μM) + Heparin (1 mg/mL)	—	—
		Stage 2 (2 days)	—	TCPS	DMEM/F12 (1:1)	N2 (1% v/v) + NEAA (1% v/v)	SHH (100 ng/mL)	All-trans retinoic acid (0.1 μM)	—	—
		Stage 3 (3 days)	—	TCPS	DMEM/F12 (1:1)	N2 (1% v/v) + NEAA (1% v/v)	SHH (100 ng/mL)	All-trans retinoic acid (0.1 μM) + ascorbic acid (200 ng/mL) + dbcAMP (1 μM)	—	—
		Stage 4 (6 days)	—	TCPS	DMEM/F12 (1:1)	N2 (1% v/v) + NEAA (1% v/v)	BDNF (10 ng/mL) + GDNF (10 ng/mL) + IGF-1 (10 ng/mL)	Ascorbic acid (200 mM) + dbcAMP (1 μM)	Immunocytochemistry for detection of NeuN, GFAP, Nestin, βIII-tubulin, O1, and choline acetyl-transferase.Semiquantitative RT-PCR for detection of Nestin and βIII-tubulin.	—
Kanafi et al.⁷²	No	Stage 1 (3 days)	20,000 cells/cm²	TCPS	Neurobasal medium	B27 (0.5% v/v)	SHH (200 ng/mL) + FGF-8 (100 ng/mL) + bFGF (50 ng/mL)	—	Quantitative RT-PCR for detection of Engrailed1, Nurr1, Pitx3, Lmx1a, Nestin, and βIII-tubulin on day 0, 3, 6, and 9.Immunocytochemistry and flow cytometry for detection of Nestin, Musashi2, HNK1, Engrailed1, Nurr1, Pitx3, TH, βIII-tubulin, and Map2ab on day 9.	—
		Stage 2 (6 days)	—	TCPS	Neurobasal medium	B27 (0.5% v/v)	BDNF (50 ng/mL)	—		Measurement of intracellular Ca²⁺ flux and secretion of dopamine upon stimulation with KCl and ATP
Arthur et al.⁷³	No	Stage 1 (7 days)	Unspecified	TCPS	Neurobasal-A	B27 (2.0% v/v)	EGF (20 ng/mL) + bFGF (40 ng/mL)	Penicillin (100 U/mL) + streptomycin (100 mg/mL)	—	—
		Stage 2 (7 days)	—	TCPS	DMEM/F12 (1:1)	ITS (1% v/v)	bFGF (40 ng/mL)	Penicillin (100 U/mL) + streptomycin (100 mg/mL)	—	—
		Stage 3 (7 days)	—	TCPS	DMEM/F12 (1:1)	ITS (1% v/v)	bFGF (40 ng/mL)	Retinoic acid (0.5 M) + penicillin (100 U/mL) + strepto mycin (100 mg/mL)	Immunocytochemistry for detection of Nestin, βIII-tubulin, PSA-NCAM, NF-M, and NF-H.Quantitative RT-PCR for detection of Nestin, βIII-tubulin, NF-M, and NF-H.	Patch-clamp analysis of Na⁺ currents
Arthur et al.⁷³	No	Single stage (21 days)	Unspecified	TCPS	Neurobasal-A	B27 (2.0% v/v)	EGF (20 ng/mL) + bFGF (40 ng/mL)	Penicillin (100 U/mL) + streptomycin (100 mg/mL)	Immunocytochemistry for detection of Nestin, βIII-tubulin, PSA-NCAM, NF-M, and NF-H.Quantitative RT-PCR for detection of Nestin, βIII-tubulin, NF-M, and NF-H.	Patch-clamp analysis of Na⁺ currents
Feng et al.⁷⁴	No	Single stage (14 days)	Unspecified	Chitosan	DMEM/F12 (1:1)	B27 (2% v/v) + N2 (2% v/v)	BDNF (25 ng/mL) + NGF (40 ng/mL) + bFGF (25 ng/mL)	—	Semiquantitative RT-PCR and western blot for detection of Nestin, CNP, MAP2, and GFAP.Immunocytochemistry for detection of CNP, MAP2, and GFAP.	Tissue engineering on three-dimensional porous chitosan scaffolds
Feng et al.³⁵	No	Single stage (7 days)	1000 cells per well of 24-well plate	TCPS	DMEM/F12 (1:1)	B27 (2% v/v) + N2 (2% v/v)	Wnt1 (100 ng/mL) + BDNF (25 ng/mL) + NGF (40 ng/mL) + bFGF (25 ng/mL)	—	Western blot and immunocytochemistry for detection of βIII-tubulin, MAP2, Nestin, and TH.	—
Osathanon et al.⁷⁵	No	Single stage (7 days)	5.0 × 10⁵ cells per 60 mm petri dish	TCPS	Neurobasal medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	l-glutamine (2 mM) + penicillin (100 U/mL) + streptomycin (100 mg/mL) + amphotericin-B (5 mg/mL)	Immunocytochemistry for detection of βIII-tubulin.Semiquantitative RT-PCR for detection of SOX2, SOX9, βIII-tubulin, NF, and NMD.	—
Bressan et al.⁷⁶	No	Single stage (21 days)	Unspecified	Hydroxyapatite	DMEM/F12 (3:1)	FBS (1% v/v) + B27 (2% v/v)	NGF (10 μg/mL)	Penicillin (100 U/mL) + streptomycin (100 mg/mL)	Quantitative RT-PCR and immunocytochemistry for detection of S100, Nestin, βIII-tubulin, GFAP, and CNP.	—
Karaöz et al.⁷⁷	No	Single stage (3–5 days)	Unspecified (70% confluence)	Collagen type I	MEM with Earle's salt	Neural stem cell proliferation supplement (Stem Cell Technologies, Inc., Vancouver, Canada)	BDNF (10 ng/mL) + EGF (10 ng/mL) + bFGF (10 ng/mL)	IBMX (0.5 mM) + penicillin–streptomycin solution (1% v/v)	Immunocytochemistry for detection of GFAP, C-FOS, NF, HNK1ST, βIII-tubulin, Nestin, and γ-enolase.	—
He et al.⁷⁸	No	Single stage (4 days)	Unspecified (70–80% confluence)	TCPS	DMEM	FBS (20% v/v)	—	Hydrocortisone (1 μM) + DMSO (2% v/v) + BHA (200 μM) + Forskolin (10 μM) + β-mercaptoethanol (0.1 mM)	Immunocytochemistry for detection of NSE and GFAP.Semiquantitative RT-PCR for detection of GFAP.	—

α-MEM, minimum essential medium alpha modification; Bcrp1, breast cancer resistance protein 1; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; BHA, butylated hydroxyanisole; BMP-2, bone morphogenetic protein 2; CNP, 2′,3′-cyclic nucleotide 3′-phosphodiesterase; dbcAMP, dibutyrl cyclic adenosine monophosphate; Dlx1, distal-less homeobox 1; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; DPSCs, dental pulp stem cells; EGF, epidermal growth factor; En1, engrailed homeobox 1; FBS, fetal bovine serum; FCS, fetal calf serum; FGF-8, fibroblast growth factor 8; GABA, gamma-aminobutyric acid; GAP.43, growth-associated protein 43; GDNF, glial cell line-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; HNK1, human natural killer-1; HNK1ST, human natural killer-1 sulfotransferase; IBMX, 3-isobutyl-1-methylxanthine; IGF-1, insulin-like growth factor 1; ITS, insulin–transferrin–selenium; Lmx1a, LIM homeobox transcription factor 1 alpha; MAP2, microtubule-associated protein 2; MASH1, mammalian achaete-scute homolog 1; NCAM, neural cell adhesion molecule; NEAA, nonessential amino acids; NeuN, neuronal nuclear antigen; NF, neurofilament; NF-H, neurofilament heavy polypeptide; NF-M, neurofilament medium polypeptide; NG2, neuron-glial antigen 2; NGF, nerve growth factor; NGN2, neurogenin-2; NMD, neuromodulin; NSE, neuron-specific enolase; NT-3, neurotrophin 3; Nurr1, nuclear receptor-related 1 protein; PAX6, paired box 6; Pitx3, pituitary homeobox 3; PSA-NCAM, polysialylated-neural cell adhesion molecule; RT-PCR, reverse transcriptase-polymerase chain reaction; SHH, sonic hedgehog; TCPS, tissue culture polystyrene; TGF-β3, transforming growth factor β3; TH, tyrosine hydroxylase; TPA, 12-O-tetradecanoylphorbol 13-acetate; VEGF, vascular endothelial growth factor.

Another major variable in the protocols developed for the in vitro neurogenic differentiation of DPSCs is the presence of multiple stages in a different culture milieu.^{14,60–62,64–71} This may include the aforementioned neurosphere formation stage in suspension culture for the first 6–8 days,^60,61 as well as preinduction treatment with either β-mercaptoethanol^14,64–66 or 5-azacytidine.^67,68 In the study by Ni et al.,⁶⁹ it was shown that pretreatment with β-mercaptoethanol, an antioxidizing agent, could promote the survival of neural precursors, in addition to enhancing Nestin expression. In the study of Lu et al.,⁷⁰ it was demonstrated that a short 5-h duration of exposure to 5 mM β-mercaptoethanol could induce the expression of the neural markers—neuron-specific enolase (NSE) and NF-200 by adult adipose-derived stromal cells. The duration of preinduction treatment is usually 24 h and the concentration of β-mercaptoethanol utilized is usually 1 mM.^14,64–66 In addition, the study of Ryu et al.⁶⁵ incorporated a further 5-h duration of exposure to a higher concentration of β-mercaptoethanol (2 mM) before 14 days of neural induction culture. Some of these studies also incorporated the use of higher concentrations of β-mercaptoethanol (10 mM) in the neural induction culture, following the preinduction step.^64,66 Besides β-mercaptoethanol, the demethylating agent 5-azacytidine has also been utilized for preinduction treatment of DPSCs before neural induction culture.^67,68 The rationale is that the demethylation of genomic DNA through 5-azacytidine treatment could erase epigenetic memory, thus making DPSCs more pliable to differentiate into neural lineages. In two separate studies by Király et al.,^67,68 DPSCs were exposed to 10 mM of 5-azacytidine for 2 days in an epigenetic reprogramming step before neural induction culture.

Multistage neural induction protocols, without neurosphere formation or preinduction treatment with either β-mercaptoethanol or 5-azacytidine, have also been reported.^71–73 Chang et al.⁷¹ developed two separate four-stage protocols (15 days duration) for directing the differentiation of DPSCs into the motor and dopaminergic neural sublineages in vitro. A key difference between the two protocols is the utilization of all-trans retinoic acid for induction of DPSCs into motor neurons, which is absent in the protocol for dopaminergic neural differentiation.⁷¹ Several single-stage protocols for the in vitro neurogenic differentiation of DPSCs have also been reported,^{35,73,74–78} with total durations ranging from 3–5 days to 21 days.^73,77 Nevertheless, most of these were older studies and the current trend for in vitro neurogenic differentiation of adult stem cells tends to gravitate toward more complex multistage culture protocols, as these would more closely recapitulate the dynamic changing microenvironment of the stem cell niche during neural differentiation.

The majority of studies utilizing multistaged protocols for the neural induction/differentiation of DPSCs carried out analyses of neural marker expression only at the protocol endpoint. Nevertheless, there were a few exceptions. For example, both Osathanon et al.⁶¹ and Karbanová et al.⁶² also carried out analyses of neural marker expression after the first stage of their reported protocol. By contrast, the studies of Király et al.^14,68 and Kanafi et al.⁷² carried out neural marker analyses at various disparate time points throughout the entire duration of their reported protocols. These are summarized in Table 1.

Another major variable in the protocols developed for in vitro neurogenic differentiation of DPSCs is the substrata or surface coating utilized for cell culture. As summarized in Table 1, neurosphere suspension culture is usually carried out in nonadherent culture dishes,^60–62 while most of the adherent monolayer culture is usually carried out on bare tissue culture-treated polystyrene (TCPS), in which the naturally hydrophobic polystyrene surface is chemically modified to become hydrophilic, through increasing its negative charge.⁷⁹ Nevertheless, there were a number of studies through which the DPSCs were cultured on different substrata such as poly-l-ornithine with laminin,⁶⁰ collagen type IV,⁶¹ collagen type I,⁷⁷ poly-d-lysine,¹⁴ poly-l-lysine,^67,68 chitosan,⁷⁴ and hydroxyapatite.⁷⁶ It must be noted that the choice of these substrata for neural induction of DPSCs was based on earlier studies on the neural induction of other stem cell types, which are in turn arbitrarily based on experimental trial and error of commonly utilized substrates for cell culture.^80,81 Currently, within the scientific literature, there is a lack of systematic comparison of the various commonly utilized substrata for the neural induction of stem cells. The study of Ge et al.⁸¹ attempted to fill in this gap, by carrying out a systematic comparison of three substrata—poly-l-ornithine, poly-l-lysine, and fibronectin. The results showed that poly-l-ornithine significantly increased neural stem/progenitor cell proliferation and differentiation, compared to poly-l-lysine and fibronectin, and the underlying mechanism of the neural inductive effect of poly-l-ornithine involved the extracellular signal-regulated kinase (ERK) signaling pathway.⁸¹

The various single- and multistaged protocols developed for in vitro neurogenic differentiation of DPSCs also exhibited much variation in the culture milieu, which is composed of the basal medium plus various culture supplements, growth factors, and small molecules. As seen in Table 1, the overwhelming majority of studies utilized either the Neurobasal or Dulbecco's modified Eagle's medium (DMEM)/F12 medium, a few studies utilized Minimum Essential Medium Eagle, alpha modification (α-MEM),^64–66 while there was only one much earlier study that utilized MEM with Earle's salt,⁷⁷ and also two other studies that utilized DMEM alone.^61,78 The formulation of Neurobasal medium, developed by Brewer et al.⁸² is specially optimized for the survival of neural cells cultured in vitro and is designed to be used together with the commercially available B27 serum-free supplement that was also developed by Brewer et al.⁸² Enhanced survival of neural cells in the Neurobasal medium is achieved by having a lower osmolarity compared to DMEM, and by decreasing cysteine and glutamine concentrations together with the removal of toxic ferrous sulfate present in DMEM/F12.⁸² In addition, the Neurobasal medium has also been reported to inhibit the growth of glial lineage cells.⁸² It must be noted that the Neurobasal medium was originally developed for embryonic and fetal neural cells, and that there exists a slightly different variant developed specially for adult and postnatal neural cells termed the Neurobasal-A medium.⁸³ This was utilized by the studies of Király et al.^67,68 as well as by the study of Arthur et al.⁷³

Besides B27, other culture supplements that have been utilized for the neurogenic differentiation of DPSCs include N2, insulin–transferrin–selenium (ITS), nonessential amino acids (NEAA), fetal calf serum (FCS), and fetal bovine serum (FBS). The N2 supplement was developed by Bottenstein and Sato⁸⁴ for the serum-free culture of postmitotic neurons and consists of a mixture of insulin, transferrin, progesterone, selenium, and putrescine.⁸⁴ ITS is often supplemented in serum-free or low-serum culture. Its constituents—ITS—serve different functions in cell culture.⁸⁵ Insulin is known to enhance the uptake of glucose and amino acids, as well as promote intracellular transport, lipogenesis, and protein and nucleic acid synthesis. Transferrin is a chelator of free iron in solution and could suppress reactive oxygen species levels in cell culture. Selenite is also utilized as an antioxidant in culture media and is known to be a cofactor for glutathione peroxidase and other proteins. The only study that supplemented NEAA within the culture milieu for neurogenic differentiation of DPSCs was that of Chang et al.⁷¹ In a previous study by Dhara et al.,⁸⁶ it was reported that NEAA did not promote early neural differentiation but instead enhanced proliferation of neural precursors. As seen in Table 1, FCS and FBS have also often been supplemented in the culture milieu for neurogenic differentiation of DPSCs. Nevertheless, the current trend toward focusing on clinical applications would obviously gravitate away from serum and move toward serum-free culture milieu formulations.

There is also much variation in the array of protein-based growth factors supplemented within the in vitro culture milieu for neurogenic differentiation of DPSCs. As seen in Table 1, the two most commonly utilized growth factors are the epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Previous studies have reported that both EGF and bFGF are potent mitogens that stimulate proliferation of neural precursors.⁸⁷ The typical concentrations of EGF and bFGF utilized for neurogenic differentiation of DPSCs typically range from 10 to 50 ng/mL (Table 1). Less commonly utilized growth factors for the neural induction of DPSCs include bone morphogenetic protein 2 (BMP-2), transforming growth factor β3 (TGF-β3), glial cell line-derived neurotrophic factor (GDNF), neurotrophin 3 (NT-3), NGF, brain-derived neurotrophic factor (BDNF), sonic hedgehog (SHH), Wnt-1, fibroblast growth factor 8 (FGF-8), insulin-like growth factor 1 (IGF-1), and noggin. BMP-2, TGF-β3, and GDNF are members of the TGF-β superfamily of multifunctional proteins that regulates proliferation, differentiation, and other functions in various different cell types, in addition to playing keys roles in neurogenesis.⁸⁸ BMP-2 has previously been reported to induce neuronal differentiation in postmigratory neural crest cells,⁸⁹ through the transcription factor MASH1, and is utilized for neurosphere formation by DPSCs in the study of Karbanová et al.⁶² Both TGF-β3 and GDNF have been shown to promote neuronal survival^90,91 and were utilized by Chang et al.⁷¹ for the neural induction of DPSCs. NT-3, NGF, and BDNF are all members of the neurotrophin family of proteins and are known to play key roles in the process of neurogenesis.⁹² These three growth factors have been reported to induce neural differentiation of mesenchymal stem cells^93–95 in addition to DPSCs (Table 1). Developmental studies have implicated SHH in the induction of both motor and dopaminergic neurons within the embryonic neural tube,^96,97 and SHH was utilized in the multistaged neural induction of DPSCs by Chang et al.⁷¹ and Kanafi et al.⁷² Both Wnt-1 and FGF-8 have previously been utilized for neural induction of bone marrow-derived human mesenchymal stem cells,^98,99 in addition to DPSCs, as reported by the studies of Chang et al.,⁷¹ Kanafi et al.,⁷² and Feng et al.³⁵ Other miscellaneous growth factors that have been utilized for neural induction of DPSCs include IGF-1 and noggin,⁷¹ both of which have also been shown to induce neural differentiation of mesenchymal stem cells.^100,101

The last major component of the culture milieu for neural induction of DPSCs includes a diverse array of various small molecules (Table 1). These include β-mercaptoethanol and 5-azacytidine utilized for preinduction treatment, which has been discussed earlier, as well as other small molecules without specific neuroinductive effects such as antibiotics (penicillin and streptomycin) and antimycotics (amphotericin-B), energy supplements such as l-glutamine, and vitamins such as ascorbic acid (vitamin C). Other small molecules with reported neuroinductive effects include retinoic acid,¹⁰² dibutyrl cyclic adenosine monophosphate (dbcAMP),¹⁰³ 3-isobutyl-1-methylxanthine (IBMX),¹⁰³ dimethyl sulfoxide (DMSO),¹⁰⁴ butylated hydroxyanisole (BHA),¹⁰⁵ forskolin,¹⁰⁶ 12-O-tetradecanoylphorbol 13-acetate (TPA),¹⁰⁷ and hydrocortisone.¹⁰⁸ Varying combinations of these neuroinductive small molecules have been utilized in the various protocols that have been devised for neural differentiation of DPSCs (Table 1), and it is presently unclear which of these would represent the optimal combination for promoting neurogenesis of DPSCs.

Of the numerous studies that have carried out neural induction/differentiation of DPSCs, only a few of these performed functional assessment of the derived neural lineages through a variety of different techniques. These include electrophysiological analysis of ion (K⁺ or Na⁺) channels based on the patch-clamp technique,^60,67,73 fluorescent detection of intracellular Ca²⁺ flux upon stimulation with neurotransmitters,⁶¹ and transplantation into live animal models followed by immunohistochemical detection of neural marker expression by the engrafted cells.^67,68

PDLSC: Neurogenesis In Vitro

Similar to DPSCs, PDLSCs also constitute a highly heterogeneous population of cells with varying propensity to undergo neural differentiation. Hence, it may be advantageous to subject PDLSCs to fluorescence-activated or magnetic-affinity cell sorting based on specific surface markers that predispose these cells to undergo neurogenesis. One of these potential markers is the gap junction protein connexin-43. Pelaez et al. reported that the purified PDLSC subpopulation expressing connexin-43, also strongly expressed various pluripotency-associated transcription factors such as OCT4, Nanog, and Sox2, in addition to a number of neural crest-specific markers such as Sox10, p75, and Nestin.¹⁰⁹

As in the case of DPSCs, a diverse array of multi- and single-step protocols for the in vitro neural differentiation of PDLSCs have also been reported (Table 2).^110–117 Multistep protocols may involve neurosphere formation,^110–112 preinduction treatment with β-mercaptoethanol,^112,113 or epigenetic reprogramming with 5-azacytidine,¹¹⁴ before neural induction per se, as has been discussed earlier for DPSCs.^14,60–68 As in the case of DPSCs, the majority of studies utilizing multistaged protocols for the neural induction/differentiation of PDLSCs carried out analyses of neural marker expression only at the protocol endpoint, except for the studies of Sawangmake et al.,¹¹⁰ Osathanon et al.,¹¹¹ and Kadar et al.¹¹⁴ (summarized in Table 2).

Table 2.

In Vitro Neural Induction Protocols Devised for PDLSCs

					Culture milieu
Study	Spheroid/neurosphere formation	Single/multiple stages (duration)	Initial seeding density	Substrata/surface coating	Basal medium	Culture supplements	Growth factors	Small molecules	Neural markers analyzed	Application/functional assessment
Sawangmake et al.¹¹⁰Osathanonet al.¹¹¹	Yes	Neurosphere formation (7 days)	Unspecified	Nonadherent tissue culture dishes	Neurobasal medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	l-glutamine (2 mM) + penicillin (100 U/mL) + streptomycin (100 mg/mL) + amphotericin-B (5 mg/mL)	Immunocytochemistry for detection of βIII-tubulin, Nestin and NF.Semi-quantitative and quantitative RT-PCR for detection of SOX2, SOX9, and βIII-tubulin, NF, GABARβ3, Hes1 and Hey1.	Fluorescent detection of intracellular Ca²⁺ flux upon stimulation with NMDA and DPH
		Neurogenic maturation (7 days)	Unspecified (neurospheres were dissociated and seeded as a monolayer)	Collagen type IV	Neurobasal medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	Retinoic acid (10 nM) + l-glutamine (2 mM) + penicillin (100 U/mL) + streptomycin (100 mg/mL) + amphotericin-B (5 mg/mL)	Immunocytochemistry for detection of βIII-tubulin.Semi-quantitative and quantitative RT-PCR for detection of SOX2, SOX9, and βIII-tubulin, NF, GABARβ3, Hes1 and Hey1.	—
Huang et al.¹¹²	Yes	Neurosphere formation (4 days)	Unspecified	Agarose (nonadherent)	GMEM	FBS (10% v/v) + NEAA (1% v/v)	—	β-mercaptoethanol (0.1 mM) + sodium pyruvate (1 mM) + l-glutamine (2 mM) + retinoic acid (1 μM) + penicillin-streptomycin solution (0.1 mg/mL)	—	—
		Neural differentiation (7 days)	Intact neurospheres were replated	Gelatin	GMEM	FBS (10% v/v) + NEAA (1% v/v)	—	β-mercaptoethanol (0.1 mM) + sodium pyruvate (1 mM) + l-glutamine (2 mM) + retinoic acid (1 μM) + penicillin-streptomycin solution (0.1 mg/mL)	Immuno-cytochemistry for detection of βIII-tubulin.Semi-quantitative RT-PCR for detection of βIII-tubulin, MAP2, NF-M and GFAP.	—
Li et al.¹¹³ Protocol A	No	Preinduction (24 h)	Unspecified—subconfluent monolayer	TCPS	α-MEM	FBS (20% v/v)	—	β-mercaptoethanol (1 mM) + penicillin-streptomycin solution (1% v/v)	—	—
		Neural induction with DMSO (5 h)	—	TCPS	α-MEM	—	—	DMSO (2% v/v)	Immunocytochemistry for detection of S100, Nestin and GFAP.Semi-quantitative and quantitative RT-PCR for detection of S100, Nestin and GFAP.	—
Li et al.¹¹³ Protocol B	No	Preinduction (24 h)	Unspecified—subconfluent monolayer	TCPS	α-MEM	FBS (20% v/v)	—	β-mercaptoethanol (1 mM) + penicillin-streptomycin solution (1% v/v)	—	—
		Neural induction with DMSO (5 h)	—	TCPS	α-MEM	—	—	DMSO (2% v/v)	—	—
		Neural differentiation (14 days)	—	TCPS	α-MEM	FBS (20% v/v)	PDGF (10 ng/mL) + bFGF (10 ng/mL) + BDNF (10 ng/mL)	Forskolin (14 μM) + penicillin-streptomycin solution (1% v/v)	Immunocytochemistry for detection of S100, Nestin and GFAP.Semi-quantitative and quantitative RT-PCR for detection of S100, Nestin and GFAP.	—
Li et al.¹¹³ Protocol C	No	Preinduction (24 h)	Unspecified—subconfluent monolayer	TCPS	α-MEM	FBS (20% v/v)	—	β-mercaptoethanol (1 mM) + penicillin-streptomycin solution (1% v/v)	—	—
		Neural induction with DMSO (5 h)	—	TCPS	α-MEM	—	—	DMSO (2% v/v)	—	—
		Neural differentiation (14 days)	—	TCPS	α-MEM	FBS (20% v/v)	NGF (10 ng/mL) + bFGF (10 ng/mL) + BDNF (10 ng/mL)	Forskolin (14 μM) + penicillin–streptomycin solution (1% v/v)	Immunocytochemistry for detection of S100, Nestin and GFAP.Semiquantitative and quantitative RT-PCR for detection of S100, Nestin, and GFAP.	—
Li et al.¹¹³ Protocol D	No	Preinduction (24 h)	Unspecified—subconfluent monolayer	TCPS	α-MEM	FBS (20% v/v)	—	β-mercaptoethanol (1 mM) + penicillin–streptomycin solution (1% v/v)	—	—
		Neural induction with all-trans retinoic acid (3 days)	—	TCPS	α-MEM	FBS (20% v/v)	—	All-trans retinoic acid (35 ng/mL) + penicillinstreptomycin solution (1% v/v)	—	—
		Neural differentiation (14 days)	—	TCPS	α-MEM	FBS (20% v/v)	NGF (10 ng/mL) + bFGF (10 ng/mL) + BDNF (10 ng/mL)	Forskolin (14 μM) + penicillin–streptomycin solution (1% v/v)	Immunocytochemistry for detection of S100, Nestin, and GFAP.Semiquantitative and quantitative RT-PCR for detection of S100, Nestin, and GFAP.	—
Kadar et al.¹¹⁴	No	Epigenetic reprogramming (2 days)	20,000 cells per well of 24-well plate	Poly-l-lysine	DMEM/F12 (1:1)	FBS (2.5% v/v)	bFGF (10 ng/mL)	5-azacytidine (10 mM)	—	—
		Neural induction (3 days)	—	Poly-l-lysine	DMEM/F12 (1:1)	ITS (1% v/v)	NGF (10 ng/mL) + bFGF (10 ng/mL) + NT-3 (30 ng/mL)	IBM X (250 μM) + Forskolin (50 μM) + TPA (200 nM) + dbcAMP (1 mM)	Immunocytochemistry for detection of NF-M on day 1 of induction.Quantitative RT-PCR for detection of Vimentin and NSE on day 3 and 5 of induction.	—
		Neural maturation (3–8 days)	—	Poly-l-lysine	Neurobasal-A	B27 (1% v/v) + N2 (1% v/v)	NT-3 (30 ng/mL)	dbcAMP (1 mM)	Immunocytochemistry for detection of NF-M on day 8 of maturation.Quantitative RT-PCR for detection of Vimentin and NSE on day 8 of maturation.	—
Fortino et al.¹¹⁵	No	Single stage (8 days)	4000 cells per well of six-well plate	TCPS	DMEM (high-glucose)	FBS (10% v/v)	EGF (50 ng/mL) + bFGF (50 ng/mL)	Penicillin (100 U/mL) + streptomycin (100 mg/mL) + amphotericin-B (0.25 μg/mL)	Immunocytochemistry for detection of βIII-tubulin, GFAP, and Synaptophysin.Quantitative RT-PCR for detection of CD133, SOX1, Nestin, NOG, βIII-tubulin, and NF-M.	Patch-clamp analysis of voltage-gated Na⁺ channels
Bueno et al.¹¹⁶	No	Single stage (21 days)	Unspecified	TCPS	DMEM/F12 (1:1)	—	bFGF (20 ng/mL) + EGF (20 ng/mL) + transferrin (100 μg/mL) + insulin (25 μg/mL)	Glucose (0.8 mg/mL) + putrescine (60 nM) + progesterone (20 nM) + sodium selenate (30 nM) + l-glutamine (2 mM) + penicillin-streptomycin (100 U/mL)	Immuno-cytochemistry for detection of Nestin, TUJ1, Map1b, MAP2, Synaptophyson, NeuN, GABA, GAD65/67, GAT1, ChAT, GFAP, O4, and NG2.	Transplantation of differentiated PDLSC into the hippocampus of adult mice.Immunohistochemistry for detection of FGFr, DCX, GFAP, NG2, NF-M, and GABA expression by engrafted cells.
Coura et al.¹¹⁷	No	Single stage (15 days)	100 cells per well of 24-well plate	TCPS	α-MEM	FBS (10% v/v) + chicken embryo extract (2% v/v)	Transferrin (10 mg/mL) + glucagon (0.01 ng/mL) + insulin (1 ng/mL) + EGF (0.1 ng/mL) + bFGF (1 ng/mL)	Hydrocortisone (0.1 mg/mL) + triiodothyronine (0.4 ng/mL) + penicillin (100 U/mL) + streptomycin (100 mg/mL)	Immunocytochemistry for detection of βIII-tubulin, Nestin, p75, and HNK1.Semiquantitative RT-PCR for detection of Nestin βIII-tubulin, NF-M, MAP2, Peripherin, Protein zero, and GFAP.	—

ChAT, choline O-acetyltransferase; DPH, dopamine hydrochloride; GABARβ3, gamma-aminobutyric acid receptor β3; GAD65/67, Glutamate decarboxylase 65/67; GAT1, GABA transporter 1; GMEM, Glasgow modified Eagle's medium; Hes1, hairy/enhancer of split 1; Hey1, hairy/enhancer-of-split related with YRPW motif 1; Map1b, microtubule-associated protein 1B; NMDA, N-methyl-D-aspartate; NOG, noggin; O4, oligodendrocyte marker 4; PDGF, platelet-derived growth factor; PDLSCs, periodontal ligament stem cells.

As seen in Table 2, the variety of cell culture substrata/coating, basal culture media, supplements, growth factors, and small molecules utilized for neural induction of PDLSCs is similar to that utilized for DPSCs (Table 1), with a few exceptions. For example, the study of Huang et al. utilized agarose-coated culture dishes for nonadherent culture of neurospheres formed from PDLSCs, followed by gelatin-coated dishes for subsequent adherent culture of the neurospheres.¹¹² In addition, Huang et al. also utilized the Glasgow modified Eagle's medium, instead of the more commonly used Neurobasal medium or DMEM/F12.¹¹² The study of Fortino et al. utilized high-glucose DMEM,¹¹⁵ even though Sawangmake et al. showed that high-glucose culture conditions suppressed neurosphere formation by PDLSCs.¹¹⁰

As with DPSCs, bFGF and EGF are the most commonly utilized growth factors for promoting neural differentiation of PDLSCs (Table 2). Indeed, the study of Fortino et al. confirmed the potent neuroinductive effects of bFGF and EGF on PDLSCs.¹¹⁵ Other less commonly utilized growth factors for neural induction of PDLSCs that have not been reported for DPSCs include platelet-derived growth factor (PDGF) and glucagon. Li et al. utilized PDGF for neural induction of PDLSCs, but found that NGF was more effective than PDGF in promoting neurogenesis of PDLSCs.¹¹³ Coura et al. utilized glucagon for neural induction of PDLSCs, in addition to other growth factors.¹¹⁷ Glucagon has previously been reported to exert a neuroprotective effect.¹¹⁸

The most commonly utilized supplements for neural induction of PDLSCs are FBS and B27 (Table 1), even though one study by Coura et al. utilized chicken embryo extract. As in the case of DPSCs, retinoic acid and DMSO are the most commonly utilized small molecules for neural induction of PDLSCs.¹¹⁷ The study of Li et al. reported that DMSO was more effective than retinoic acid for promoting neurogenic differentiation of PDLSCs.¹¹³ Other less commonly utilized small molecules for neural induction of PDLSCs that have not been reported for DPSCs include sodium pyruvate¹¹² and triiodothyronine (T3).¹¹⁷ Like l-glutamine, sodium pyruvate is an energy supplement, being an intermediate molecule of the glycolytic pathway. Thyroid hormone T3 has been previously reported to promote the neural differentiation of other nondental and nonoral stem cells.^118–120 It must be noted that small molecules such as putrescine, progesterone, and sodium selenate, which were utilized in the protocol of Bueno et al. for the neural induction of PDLSCs,¹¹⁶ are in fact constituents of either the ITS or N2 supplements utilized for the neural induction of DPSCs (Table 1).

As in the case of DPSCs, functional assessment of neural lineages derived from PDLSCs was performed in only a few studies through fluorescent detection of intracellular Ca²⁺ flux upon stimulation with neurotransmitters,^110,111 patch-clamp analysis of ion (K⁺ or Na⁺) channels,¹¹⁵ and transplantation into live animal models followed by immunohistochemical detection of neural marker expression by the engrafted cells.¹¹⁶

Stem Cells from Exfoliated Deciduous Teeth: Neurogenesis In Vitro

Compared to DPSCs, there are fewer reported studies on the neurogenic differentiation of SHED.^{8,35,121–127} Both single- and multistep protocols have been devised for the neural induction of SHED (Table 3), as in the case of DPSCs and PDLSCs (Tables 1 and 2). Nevertheless to date, none of these reported protocols involved preinduction treatment with β-mercaptoethanol or epigenetic reprogramming with 5-azacytidine before neural differentiation per se, as discussed earlier for DPSCs and PDLSCs (Tables 1 and 2). Only one study by Wang et al.¹²¹ carried out neurosphere formation, although with superhydrophilic culture plates (Costar, Cambridge, MA) instead of the more commonly utilized hydrophobic nonadherent culture plates. Previously, the study by Sasaki et al. generated neurospheres from dental pulp of adult rat incisors by utilizing similar superhydrophilic culture plates.¹²⁸ Except for the study by Wang et al.,¹²¹ all other studies utilizing multistaged protocols for the neural induction/differentiation of SHED carried out analyses of neural marker expression only at the protocol endpoint (Table 3).

Table 3.

In Vitro Neural Induction Protocols Devised for SHED

					Culture milieu
Study	Spheroid/neurosphere formation	Single/multiple stages (duration)	Initial seeding density	Substrata/surface coating	Basal medium	Culture supplements	Growth factors	Small molecules	Neural markers analyzed	Application/functional assessment
Wang et al.¹²¹	Yes	Neurosphere formation (7–9 days)	1.0 × 10⁶ cells per well of six-well plate	Superhydrophilic culture plates (Costar, Cambridge, MA)	Neurobasal-A medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	—	Immunocytochemistry for detection of Nestin, βIII-tubulin, and TH.Western blot for detection of TH.Quantitative RT-PCR for detection of Nestin, MAP2, βIII-tubulin, and TH.	Transplantation into rat model of Parkinson's disease, followed by behavioral assessment
		Neural differentiation (7–14 days)	Intact neurospheres were replated	Poly-l-lysine	Neurobasal-A medium	B27 (2% v/v)	SHH (200 ng/mL) + FGF-8 (100 ng/mL) + GDNF (10 ng/mL)	Forskolin (10 μM)	Immunocytochemistry for detection of MAP2, βIII-tubulin, and TH.Western blot for detection of TH.Quantitative RT-PCR for detection of Nestin, MAP2, βIII-tubulin, and TH.	Transplantation into rat model of Parkinson's disease, followed by behavioral assessment
Esmaeili et al.¹²²	No	Stage 1 (7 days)	5000 cells/cm²	Poly-l-lysine	Neurobasal medium	ITS (1% v/v)	bFGF (100 ng/mL)	—	—	—
		Stage 2 (7 days)	—	Poly-l-lysine	Neurobasal medium	ITS (1% v/v)	bFGF (100 ng/mL) + FGF-8 (10 ng/mL) + SHH (100 ng/mL)	—	Quantitative RT-PCR for detection of BDNF, NGF, Neurotrophin3, and Neurotrophin4.	—
Jarmalavičiūtė et al.¹²³	No	Stage 1 (7–10 days)	Unspecified—confluent monolayer	Poly-l-lysine+laminin	Neurobasal-A medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	—	—	—
		Stage 2 (14–21 days)	—	Poly-l-lysine+laminin	Neurobasal-A medium	B27 (2% v/v)	NGF (10 ng/mL) + GDNF (10 ng/mL) + BDNF (10 ng/mL)	dbcAMP (1.5 mM)	Immunocytochemistry for detection of βIII-tubulin, MBP, Brn3a, and peripherin.Microarray analysis of markers of sensory (MAF), sympathetic (Midkine, Pleiotrophin) and dopaminergic (TH, Nurr1) neurons.	—
Nourbakhsh et al.¹²⁴	No	Stage 1 (5 days)	5000 cells/cm²	Poly-l-lysine	Neurobasal medium	ITS (1% v/v)	bFGF (100 ng/mL)	—	—	—
		Stage 2 (5 days)	—	Poly-l-lysine	Neurobasal medium	ITS (1% v/v)	bFGF (100 ng/mL) + FGF-8 (10 ng/mL) + SHH (100 ng/mL)	—	Immunocytochemistry and flow cytometry for detection of Nestin, PSA-NCAM, βIII-tubulin, NeuN, Tau, TH, and GFAP.Semiquantitative RT-PCR for detection of Nestin, NCAM, βIII-tubulin, MAP2, and NSE.Western blot for detection of Nestin, βIII-tubulin, NeuN, Tau, and TH.	—
Isobe et al.¹²⁵	No	Single stage (14 days)	Unspecified (confluent monolayer)	TCPS	Neurobasal-A medium	B27 (1% v/v)	EGF (20 ng/mL) + bFGF (40 ng/mL)	Penicillin (100 U/mL) + streptomycin (100 μg/mL)	Immunocytochemistry for detection of Nestin.Semiquantitative RT-PCR for detection of βIII-tubulin and MAP2.	—
Miura et al.⁸	No	Single stage (28 days)	Unspecified	0.1% Gelatin	Neurobasal-A medium	B27 (1% v/v)	EGF (20 ng/mL) + bFGF (40 ng/mL)	Penicillin–streptomycin solution (1% v/v)	Immunocytochemistry and western blot for detection of βIII-tubulin, GAD, NeuN, Nestin, GFAP, NF-M, and CNP, MAP2, and Tau.	Transplantation into the dentae gyrus of the hippocampus of immunocompromised mice, followed by immunohistochemistry for detection of NF-M expression by engrafted cells
Feng et al.³⁵	No	Single stage (7 days	1000 cells per well of 24-well plate	TCPS	DMEM/F12 (1:1)	B27 (2% v/v) + N2 (2% v/v)	Wnt1 (100 ng/mL) + BDNF (25 ng/mL) + NGF (40 ng/mL) + bFGF (25 ng/mL)	—	Western blot and immunocytochemistry for detection of βIII-tubulin, MAP2, Nestin, and TH.	—
Taghipour et al.¹²⁶	No	Single stage (7 days)	5000 cells/cm²	Poly-l-ornithine+fibronectin	Neurobasal medium	B27 (1% v/v) + ITS (1% v/v)	bFGF (100 ng/mL)	—	Immunocytochemistry and flow cytometry for detection of Nestin.Quantitative RT-PCR for detection of BDNF, GDNF, and Neurotrophin3.	Transplantation into rat spinal cord injury model followed by assessment of hind limb motor function.Immunohistochemistry for detection of MAP2, NCAM, GFAP, MBP, NG2, Nestin, and Ki67 by engrafted cells.
Morsczeck et al.¹²⁷	No	Single stage (7 days)	25,000 cells/cm²	TCPS with Nunclon Delta surface modification (Nunc, Wiesbaden, Germany)	Neurobasal medium	G5 (PAA Laboratories, Pasching, Austria)+neural stem cell supplement (PAA Laboratories)	—	—	Quantitative RT-PCR for detection of Nestin, βIII-tubulin, NF-M, MAP2, and GFAP.	—
Morsczeck et al.¹²⁷	No	Single stage (7 days)	25,000 cells/cm²	TCPS with Nunclon Delta surface modification (Nunc)	Neurobasal medium	N2 (1% v/v)	EGF (20 μg/mL) + bFGF (20 μg/mL)	—	Quantitative RT-PCR for detection of Nestin, βIII-tubulin, NF-M, MAP2, and GFAP.	—
Morsczeck et al.¹²⁷	No	Single stage (7 days)	25,000 cells/cm²	TCPS with Nunclon Delta surface modification (Nunc)	Neurobasal medium	B27 (2% v/v)	EGF (20 μg/mL) + bFGF (20 μg/mL)	—	Quantitative RT-PCR for detection of Nestin, βIII-tubulin, NF-M, MAP2, and GFAP.	—
Morsczeck et al.¹²⁷	No	Single stage (7 days)	25,000 cells/cm²	TCPS with Nunclon Delta surface modification (Nunc)	Neurobasal medium	B27 (2% v/v)	—	—	Quantitative RT-PCR for detection of Nestin, βIII-tubulin, NF-M, MAP2, and GFAP.	—

Brn3a, brain-specific homeobox/POU domain protein 3A; GAD, glutamic acid decarboxylase; MBP, myelin basic protein; SHED, stem cells from human exfoliated deciduous teeth.

As seen in Table 3, the variety of cell culture substrata/coating, basal culture media, supplements, growth factors, and small molecules utilized for neural induction of SHED is similar to that utilized for DPSCs and PDLSCs (Tables 1 and 2), with a few exceptions. For example, the study of Taghipour et al. utilized a combination of poly-l-ornithine and fibronectin as the culture substrata.¹²⁶ In the case of DPSCs, Gervois et al. utilized a combination of poly-l-ornithine and laminin.⁶⁰ Fibronectin had previously been reported to induce differentiation of neuroblastomas¹²⁹ and neural progenitors¹³⁰ and is known to be expressed during neurogenesis.¹³¹

The overwhelming majority of protocols for neural induction of SHED utilized the Neurobasal medium together with B27 supplement (Table 3). Nevertheless, one protocol reported by the study of Morsczeck et al. added commercially available G5 and neural stem cell supplements from PAA Laboratories (Pasching, Austria) into the Neurobasal medium, instead of utilizing the B27 supplement.¹²⁷ The G5 supplement was developed by Bottenstein and colleagues¹³² and is composed of 100 μg/mL biotin, 1 μg/mL EGF, 500 ng/mL bFGF, 500 μg/mL human transferrin, 360 ng/mL hydrocortisone, 500 μg/mL insulin, and 520 ng/mL sodium selenite. The exact composition of neural stem cell supplement is, however, a proprietary trade secret of PAA Laboratories. Because Morsczeck et al. reported that neurosphere-like clusters were formed in the presence of B27 but were absent in the presence of the G5 and neural stem cell supplements,¹²⁷ it is likely that B27 could be superior to these two supplements for inducing the neural differentiation of SHED. Furthermore, Morsczeck et al. also observed neurosphere formation by SHED in the presence of N2 supplement with bFGF and EGF.¹²⁷ Nevertheless, it must be noted that the major limitation of the study of Morsczeck et al. was the relatively short culture duration of 1 week for all four single-stage neural induction protocols described (Table 3),¹²⁷ which made it inconclusive as to which of these protocols is most optimal for neural induction of SHED.

Functional assessment of neural lineages derived from SHED was carried out in a few of the aforementioned studies through transplantation into live animal models.^8,121,126 These included an immunocompromised mouse model,⁸ a rat Parkinson's disease model,¹²¹ and a rat spinal cord injury model.¹²⁶ Besides immunohistochemistry to detect neural marker expression by the engrafted cells,^8,126 other analyses that were carried out included behavioral assessment in the case of the rat Parkinson's disease model¹²¹ and assessment of hind limb locomotor function in the case of the rat spinal cord injury model.¹²⁶

DFSC: Neurogenesis In Vitro

To date, there have been relatively few studies on the neural induction of human DFSCs (Table 4).^127,133,134 Völlner et al. found that poly-l-lysine coating was superior to gelatin, laminin, and poly-ornthine for inducing neurosphere formation from DFSCs, and that the combination of commercially available G5 and neural stem cell supplements (PAA Laboratories) was superior to B27 supplement in inducing DFSCs to form neurospheres.¹³³ Subsequently, Völlner et al. developed an optimized two-stage protocol for neural induction of DFSCs that utilized poly-l-lysine coating together with the G5 and neural stem cell supplements for neurosphere formation (Table 1).¹³³ Yang et al. also compared different culture protocols for neural induction of DFSCs and found that a single-stage differentiation protocol (7 days) utilizing the growth factors bFGF and EGF, together with B27 and l-glutamine in Neurobasal media, was superior to a two-stage protocol involving 4-h pretreatment with various chemical inducers such as DMSO, BHA, valproic acid, forskolin, and hydroxycortisone.¹³⁴ The study of Morsczeck et al. also compared a number of different protocols for neural induction of DFSCs, but obtained highly variable results in the expression of various neural markers,¹²⁷ which made it inconclusive as to which of these protocols is most optimal for promoting neural differentiation of DFSCs. Unlike SHED, none of the neural induction protocols investigated by Morsczeck et al. yielded any neurosphere formation.¹²⁷ To date, only two studies reported multistaged protocols for the neural induction/differentiation of DFSCs.^133,134 Of these, only the study of Völlner et al.¹³³ carried out analyses of neural marker expression at different stages of their reported protocol, while the study of Yang et al.¹³⁴ carried out analyses of neural marker expression only at the protocol endpoint. A major deficiency of all these studies on the neural induction/differentiation of DFSCs is that there was no functional assessment of the derived neural lineages through the various techniques discussed earlier for DPSCs, PDLSCs, and SHED.

Table 4.

In Vitro Neural Induction Protocols Devised for DFSCs

					Culture milieu
Study	Spheroid/neurosphere formation	Single/multiple stages (duration)	Initial seeding density	Substrata/surface coating	Basal medium	Culture supplements	Growth factors	Small molecules	Neural markers analyzed	Application/functional assessment
Völlner et al.¹³³NSCM II	Yes	Neurosphere formation (4 days)	25,000 cells/cm²	Poly-l-lysine (2 μg/cm²) or poly-l-ornithine (10 μg/cm²)	Neurobasal medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	l-glutamine (2 mM)	Immunocytochemistry for detection of βIII-tubulin and Nestin.Quantitative RT-PCR for detection of Nestin, NF-H, and βIII-tubulin.	—
		Neural differentiation (7 days)	—	Poly-l-lysine (2 μg/cm²) or poly-l-ornithine (10 μg/cm²)	Neurobasal medium	B27 (2% v/v)	—	l-glutamine (2 mM) + retinoic acid (0.5 μM)	Immunocytochemistry for detection of βIII-tubulin and Nestin, NSE and Pan-NF.Quantitative RT-PCR for detection of Nestin, NF-H, βIII-tubulin, NSE, MAP2, SLC6A4, GAD1, GAL, TAC1, and CALB2.	—
Völlner et al.¹³³ NSCM III- Optimized Protocol	Yes	Neurosphere formation (7 days)	25,000 cells/cm²	Poly-l-lysine (2 μg/cm²)	Neurobasal medium	G5 (PAA Laboratories)+neural stem cell supplement (PAA Laboratories)	—	l-glutamine (2 mM)	Immuno-cytochemistry for detection of βIII-tubulin and Nestin.Quantitative RT-PCR for detection of Nestin, NF-H and βIII-tubulin.	—
		Neural differentiation (14 days)	—	Poly-l-ornithine (10 μg/cm²) + laminin (5 μg/cm²)	DMEM/F12 (1:1)	ITS (1% v/v)	bFGF (40 ng/mL)	Penicillin (100 U/mL) + streptomycin (100 μg/mL) + retinoic acid (0.5 μM) (last 7 days)	Immunocytochemistry for detection of βIII-tubulin and Nestin, NSE and Pan-NF.Quantitative RT-PCR for detection of Nestin, NF-H, βIII-tubulin, NSE, MAP2, SLC6A4, GAD1, GAL, TAC1, and CALB2.	—
Yang et al.¹³⁴	No	Chemical induction (4 h)	Unspecified	TCPS	α-MEM	—	Insulin (5 μg/mL)	DMSO (2% v/v) + BHA (200 μM) + KCl (25 mM) + valproic acid (2 mM) + forskolin (10 mM) + hydroxycortisone (1 mM)	—	—
		Neural differentiation (7 days)	—	TCPS	Neurobasal-A medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	l-glutamine (2 mM)	Quantitative RT-PCR for detection of CNP, DCX, MAP2, NF-M, Nestin, CSPG4, NGFR, and βIII-tubulin.	—
Yang et al.¹³⁴Optimized protocol	No	Single stage (7 days)	—	TCPS	Neurobasal-A medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	l-glutamine (2 mM)	Quantitative RT-PCR for detection of CNP, DCX, MAP2, NF-M, Nestin, CSPG4, NGFR, and βIII-tubulin.	—
Morsczecket al.¹²⁷	No	Single stage (7 days)	25,000 cells/cm²	TCPS with Nunclon Delta surface modification (Nunc)	Neurobasal medium	G5 (PAA Laboratories)+neural stem cell supplement (PAA Laboratories)	—	—	Quantitative RT-PCR for detection of Nestin, βIII-tubulin, NF-M, MAP2, and GFAP.	—
Morsczeck et al.¹²⁷	No	Single stage (7 days)	25,000 cells/cm²	TCPS with Nunclon Delta surface modification (Nunc)	Neurobasal medium	N2 (1% v/v)	EGF (20 μg/mL) + bFGF (20 μg/mL)	—	Quantitative RT-PCR for detection of Nestin, βIII-tubulin, NF-M, MAP2, and GFAP.	—
Morsczecket al.¹²⁷	No	Single stage (7 days)	25,000 cells/cm²	TCPS with Nunclon Delta surface modification (Nunc)	Neurobasal medium	B27 (2% v/v)	EGF (20 μg/mL) + bFGF (20 μg/mL)	—	Quantitative RT-PCR for detection of Nestin, βIII-tubulin, NF-M, MAP2, and GFAP.	—
Morsczeck et al.¹²⁷	No	Single stage (7 days)	25,000 cells/cm²	TCPS with Nunclon Delta surface modification (Nunc)	Neurobasal medium	B27 (2% v/v)	—	—	Quantitative RT-PCR for detection of Nestin, βIII-tubulin, NF-M, MAP2, and GFAP.	—

CALB2, calbindin 2; CSPG4, chondroitin sulfate proteoglycan 4; DCX, doublecortin; DFSCs, dental follicle stem cells; GAD1, glutamate decarboxylase 1; GAL, galanin; NGFR, nerve growth factor receptor; NSCM, neural stem cell medium; SLC6A4, neurotransmitter transporter serotonin; TAC1, tachykinin.

SCAP: Neurogenesis In Vitro

There have also been relatively few studies on the neural induction of SCAP.^134–137 The only multistaged protocol for the neural induction/differentiation of SCAP was reported by Yang et al.,¹³⁴ which carried analyses of neural marker expression only at the protocol endpoint. As previously discussed for DFSCs, the study of Yang et al. demonstrated that there was no added beneficial effect in having a short chemical induction step of 4-h duration before neural induction with EGF and bFGF in the Neurobasal medium supplemented with B27 and l-glutamine.¹³⁴ Vanacker et al. demonstrated that hypoxic conditions (1% O₂) could further augment neural differentiation of SCAP cultured in the Neurobasal medium supplemented with EGF, bFGF, NGF, IBMX, retinoic acid, and dbcAMP (Table 5), as demonstrated by upregulated expression of the neuronal markers NSE, PanF, and NeuN.¹³⁵ The neural induction protocols utilized by Bakopoulou et al.¹³⁶ and Sonoyama et al.¹³⁷ were similar and utilized 0.1% gelatin as the substrata, together with the growth factors EGF (20 ng/mL) and bFGF (40 ng/mL) in the Neurobasal medium supplemented with B27 and antibiotics (Table 5). To date, none of the few studies on the neural induction/differentiation of SCAP has performed any functional assessment of the derived neural lineages through the various techniques discussed earlier for DPSCs, PDLSCs, and SHED.

Table 5.

In Vitro Neural Induction Protocols Devised for SCAP

					Culture milieu
Study	Spheroid/neurosphere formation	Single/multiple stages (duration)	Initial seeding density	Substrata/surface coating	Basal medium	Culture supplements	Growth factors	Small molecules	Neural markers analyzed	Application/functional assessment
Yang et al.¹³⁴	No	Chemical induction (4 h)	Unspecified	TCPS	α-MEM	—	Insulin (5 μg/mL)	DMSO (2% v/v) + BHA (200 μM) + KCl (25 mM) + valproic acid (2 mM) + forskolin (10 mM) + hydroxycortisone (1 mM)	—	—
		Neural differentiation (7 days)	—	TCPS	Neurobasal-A medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	l-glutamine (2 mM)	Quantitative RT-PCR for detection of CNP, DCX, MAP2, NF-M, Nestin, CSPG4, NGFR, and βIII-tubulin.	—
Yang et al.¹³⁴ Optimized Protocol	No	Neural differentiation (7 days)	—	TCPS	Neurobasal-A medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL)	l-glutamine (2 mM)	Quantitative RT-PCR for detection of CNP, DCX, MAP2, NF-M, Nestin, CSPG4, NGFR, and βIII-tubulin.	—
Vanacker et al.¹³⁵	No	Single stage (7 days)	Not specified (50% confluent)	TCPS	Neurobasal-A medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (20 ng/mL) + NGF (50 ng/mL)	dbcAMP (1 mM) + IBMX (0.5 mM) + retinoic acid (2 μM) + penicillin–streptomycin solution (1% v/v)	Quantitative RT-PCR for detection of CNP, SNAIL, NSE, GDNF, and Neurotrophin3.Immunocytochemistry for detection of Pan-NF and NeuN.	—
Bakopoulou et al.¹³⁶	No	Single stage (21 days)	1000 cells/cm²	0.1% Gelatin	Neurobasal-A medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (40 ng/mL)	Antibiotic–antimycotic solution (1% v/v)	Semiquantitative RT-PCR for detection of NF-L, NCAM, Nestin, and βIII-tubulin.	—
Sonoyama et al.¹³⁷	No	Single stage (28 days)	Not specified	0.1% Gelatin	Neurobasal-A medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (40 ng/mL)	Penicillin–streptomycin solution (1% v/v)	Immunocytochemistry for detection of βIII-tubulin, GAD, NeuN, Nestin, GFAP, NF-M, NSE, and CNP.	—

NF-L, neurofilament light polypeptide; SCAP, stem cells from apical papilla.

GMSCs: Neurogenesis In Vitro

As with DFSCs and SCAP, there have only been a handful of studies on the neural induction of GMSCs (Table 6).^138–140 To date, there has been no reported multistaged protocol for the neural induction/differentiation of GMSCs. The study of El-Bialy et al. demonstrated that low-intensity pulsed ultrasound (LIPUS) could enhance the neurogenic differentiation of GMSCs cultured in α-MEM supplemented with bFGF, insulin, DMSO, BHA, KCl, valproic acid, and forskolin.¹³⁸ The studies of Xu et al.¹³⁹ and Hsu et al.¹⁴⁰ utilized EGF and bFGF for the neural induction of GMSCs in the absence of any chemical inducers. Nevertheless, Xu et al.¹³⁹ utilized DMEM/F12 as the basal medium together with N2 supplement and FBS, whereas Hsu et al.¹⁴⁰ utilized the Neurobasal-A medium together with B27 supplement. Moreover, 0.1% gelatin coating was utilized as the culture substrata by Hsu et al.,¹⁴⁰ whereas Xu et al.¹³⁹ utilized bare TCPS surface as the culture substrata. To date, none of the few studies on the neural induction/differentiation of GMSCs has performed any functional assessment of the derived neural lineages through the various techniques discussed earlier for DPSCs, PDLSCs, and SHED.

Table 6.

In Vitro Neural Induction Protocols Devised for GMSCs

					Culture milieu
Study	Spheroid/neurosphere formation	Single/multiple stages (duration)	Initial seeding density	Substrata/surface coating	Basal medium	Culture supplements	Growth factors	Small molecules	Neural markers analyzed	Application/functional assessment
El Bialy et al.¹³⁸Protocol utilized together with ultrasound stimulation	No	Single stage (4 days)	2.5 × 10³ cells per well of 48-well plate	TCPS	α-MEM	—	bFGF (10 ng/mL) + insulin (5 μg/mL)	DMSO (2% v/v) + BHA (100 μM) + KCl (25 mM) + valproic acid (2 mM) + forskolin (10 μM)	Immunocytochemistry for detection of βIII-tubulin, GFAP, MAPK2, nucleostemin, neurofilament, and synaptophysin quantitative RT-PCR for detection of neurofilament, nucleostemin, and Vimentin.	—
Xu et al.¹³⁹	No	Single stage (14–21 days)	Not specified	TCPS	DMEM/F12 (1:1)	FBS (10% v/v) + N2 (1% v/v)	EGF (10 ng/mL) + bFGF (10 ng/mL)	Penicillin (100 U/mL) + streptomycin (100 μg/mL)	Immunocytochemistry and western blot for detection of Nestin, βIII-tubulin, and NF-M.	—
Hsu et al.¹⁴⁰	No	Single stage (21 days)	3.0 × 10⁴ cells/cm²	0.1% Gelatin	Neurobasal-A medium	B27 (2% v/v)	EGF (20 ng/mL) + bFGF (40 ng/mL)	Penicillin–streptomycin solution (1% v/v)	Immunocytochemistry for detection of Nestin.Quantitative RT-PCR for detection of Nestin, GFAP, and βIII-tubulin.	—

GMSCs, gingival mesenchymal stem cells; MAPK2, MAP kinase-activated protein kinase 2.

Conclusions and Future Outlook

Currently within the scientific literature, there have only been a few studies that directly compared the neurogenic potential of the various dental and oral-derived stem cells. In the study of Kadar et al.,¹¹⁴ in which the neurogenesis of PDLSCs was compared to DPSCs, it was demonstrated that DPSCs consistently displayed higher expression levels of NSE compared to PDLSCs throughout the entire period of neural induction. Contrary results were, however, reported by the study of Lee et al.,¹⁴¹ which showed that PDLSCs and SCAP exhibited higher expression levels of various other neural markers compared to DPSCs under neural differentiation culture conditions. Feng et al.³⁵ reported that DPSCs possessed lower neurogenic potential than SHED due to age-related impairment of WnT/β-catenin signaling, as SHED is derived from a developmentally less mature stage of dental pulp compared to DPSCs. The study of Morsczeck et al.¹²⁷ compared the neurogenesis of DFCs versus SHED, but could not find any conclusive evidence as to which of these cell types possessed greater neurogenic potential due to differential expression of neural markers after neural induction. For example, the mature neural marker MAP2 was more highly expressed in DFCs versus SHED, whereas a reverse trend was observed for GFAP expression.¹²⁷ Moreover, SHED could form neurosphere-like clusters in the presence of EGF and FGF2, unlike DFCs.¹²⁷ The study of Yang et al.¹³⁴ reported that DFCs possessed higher neurogenic potential than either dental papilla stem cells or SCAP, as evidenced by higher neural marker expression levels after neural induction. Hence, the available evidence from these few studies may suggest that other dental and oral-derived stem cells such as SHED, PDLSCs, and SCAP may possess higher neurogenic potential than DPSCs, but more studies are needed to validate this.

The current trajectory in the development of in vitro differentiation protocols for neural induction of dental and oral-derived stem cells is gravitating toward a chemically defined serum-free and xeno-free culture milieu together with utilization of synthetic substrata and the replacement of high-molecular-weight protein growth factors with small-molecule chemical inducers. Furthermore, as previously mentioned, multistage rather than single-stage protocols are currently in vogue, as these would better recapitulate the dynamic changes within the physiological microenvironment of the adult stem cell niche during neurogenesis in vivo.

The elimination of serum and animal products from the in vitro culture milieu would not only avoid pathogenic transmission but also prevent adhesion of potentially antigenic proteins on the surface of the cultured stem cells, which could in turn provoke an immunological reaction upon transplantation in vivo within the human body. As such, serum-free and xeno-free culture of transplanted stem cells is currently a mandatory requirement for compliance with FDA (US Food and Drug Administration) regulations and current good manufacturing practice standards for cell therapy.

Both the N2 and B27 supplements that are commonly utilized for neural induction of dental and oral-derived stem cells (Tables 1 –6) are chemically defined. While the N2 supplement is free of xenogenic products,⁸² the same cannot be said of the B27 supplement, which contains bovine serum albumin.⁸² Hence, it is imperative to replace the bovine serum albumin in B27 supplement with an appropriate substitute such as recombinant human serum albumin,¹⁴¹ so as to achieve a completely xeno-free culture milieu for the neural induction of human dental and oral-derived stem cells.

Another potential future development could be the fabrication of synthetic substrata for neural induction of dental and oral-derived stem cells, in place of the more commonly utilized poly-l-lysine, laminin, and poly-l-ornithine (Tables 1 –6). Previously, Lewandowska et al. demonstrated that chemically derivatized substrata could influence fibronectin-mediated adhesion of neural cells,¹⁴² while Saha et al. developed a synthetic hydrogel system for culturing neural stem cells and demonstrated that variation in the elasticity (modulus) of the substrate could profoundly influence cellular behavior.¹⁴³ Nevertheless, to date, there has not yet been any studies on the use of specially fabricated synthetic substrata for neural induction of dental and oral-derived stem cells. This is anticipated to materialize in the near future.

As can be seen in Tables 1 –6, the vast majority of neural induction protocols devised for dental and oral-derived stem cells utilized high-molecular-weight protein growth factors, most commonly bFGF and EGF. This has the disadvantage of much higher costs, as well as shorter half-life compared to small-molecule chemical inducers. The most commonly utilized small molecules for neural induction of dental and oral-derived stem cells are retinoic acid, DMSO, BHA, and forskolin (Tables 1 –6). It is imperative that future studies should also investigate other small molecules such as SB431542,¹⁴⁴ CHIR99021,¹⁴⁵ purmorphamine,¹⁴⁶ dorsomorphin,¹⁴⁷ and the SMO agonist SAG,¹⁴⁸ which have been utilized for neural induction of other stem cell types.

Future in vitro neural induction protocols could also incorporate the use of physical and environmental stimuli to further enhance the neurogenesis of dental and oral-derived stem cells. As mentioned previously, low O₂ tension has been utilized for neural induction from SCAP,¹³⁵ while ultrasound stimulation has been utilized for neural induction of GMSCs.¹³⁸ What has not yet been investigated is electrical stimulation, which has been previously reported to enhance the differentiation of neural progenitor cells.¹⁴⁹

It is anticipated that the gradual transition toward chemically defined culture milieu together with the replacement of protein growth factors with small-molecule inducers would reduce interbatch variability, improve replicability, and facilitate production scale-up of in vitro differentiated neurons from dental and oral-derived stem cells for various therapeutic and nontherapeutic applications.

Large numbers of neural lineage cells are certainly needed for various therapeutic and nontherapeutic applications. Hence, a major challenge and bottleneck is that relatively few dental and oral-derived stem cells can be isolated from each individual patient, which in turn necessitates extensive ex vivo proliferation of these cells. Nevertheless, extensive ex vivo proliferation is all too often associated with cellular senescence, which limits proliferative capacity. It is possible that optimization of the culture milieu through inclusion of appropriate small molecules that can mimic the effects of various growth factors and extracellular matrix components could mitigate cellular senescence during extensive ex vivo proliferation. Because mature neurons are mitotically quiescent, it is only possible to carry out ex vivo proliferation at either the undifferentiated stage before neural induction or after limited neural induction to the neural stem/progenitor stage. Currently, it is unclear which of these two stages is more appropriate for extensive ex vivo proliferation, which in turn needs to be addressed in future studies.

Another pertinent issue that has to be addressed in the development of neural induction protocols for dental and oral-derived stem cells is the appropriate differentiation stage required for various therapeutic and nontherapeutic applications. Stem cell-derived neural lineages transplanted into the human body can exert their therapeutic effects by anatomically replacing damaged/lost cells or by secreting various neurotrophic factors that induce migration/proliferation and differentiation of endogenous cells that participate in neural regeneration. In either case, the less mature neural stem/progenitor stage may be more appropriate for transplantation therapy; since mature neurons are mitotically quiescent with less regenerative capacity, as well as less likely to secrete neurotrophic factors due to their primary function in maintaining synaptic connectivity within neural circuits. For nontherapeutic applications in pharmacology, toxicology, and disease modeling, both mature neurons and neural stem/progenitor cells would likely be required to recapitulate different stages of neural development in vitro.

Footnotes

Acknowledgments

This work was supported by a grant from the National Nature Science Foundation of China (81470735) and the General Research Fund (GRF) grants from the Research Grants Council of Hong Kong (grant no. HKU17126914) to C. Zhang.

Disclosure Statement

No competing financial interests exist.

References

Hemmat

, Lieberman

D.M.

, and Most

S.P.

An introduction to stem cell biology. Facial Plast Surg, 26, 343, 2010.

Ding

, Niu

, and Wei

Current understanding of orofacial tissue derived mesenchymal stem cells: an immunological perspective. Histol Histopathol, 30, 255, 2015.

Xiao

, and Nasu

From regenerative dentistry to regenerative medicine: progress, challenges, and potential applications of oral stem cells. Stem Cells Cloning, 7, 89, 2014.

Zhao

, and Chai

Stem cells in teeth and craniofacial bones. J Dent Res, 94, 1495–1501, 2015.

Egusa

, Sonoyama

, Nishimura

, Atsuta

, and Akiyama

Stem cells in dentistry—part I: stem cell sources. J Prosthodont Res, 56, 151, 2012.

Sedgley

C.M.

, and Botero

T.M.

Dental stem cells and their sources. Dent Clin North Am, 35, 549, 2012.

Potdar

P.D.

, and Jethmalani

Y.D.

Human dental pulp stem cells: applications in future regenerative medicine. World J Stem Cells, 7, 839, 2015.

Miura

, Gronthos

, Zhao

, Lu

, Fisher

L.W.

, Robey

P.G.

, and Shi

SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A, 100, 5807, 2003.

Huang

G.T.

, Sonoyama

, Liu

, Wang

, and Shi

The hidden treasure in apical papilla: the potential role in pulp/dentin regeneration and bioroot engineering. J Endod, 34, 645, 2008.

10.

Honda

M.J.

, Imaizumi

, Tsuchiya

, and Morsczeck

Dental follicle stem cells and tissue engineering. J Oral Sci, 52, 541, 2010.

11.

Zhu

, and Liang

Periodontal ligament stem cells: current status, concerns, and future prospects. Stem Cells Int, 2015, 972313, 2015.

12.

Fournier

B.P.

, Larjava

, and Häkkinen

Gingiva as a source of stem cells with therapeutic potential. Stem Cells Dev, 22, 3157, 2013.

13.

Dominici

, Le Blanc

, Mueller

, Slaper-Cortenbach

, Marini

, Krause

, Deans

, Keating

, Prockop

D.J.

, and Horwitz

Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8, 315, 2006.

14.

Yalvac

M.E.

, Ramazanoglu

, Rizvanov

A.A.

, Sahin

, Bayrak

O.F.

, Salli

, Palotás

, and Kose

G.T.

Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo- and neurogenesis. Pharmacogenomics J, 10, 105, 2010.

15.

Bruder

S.P.

, Jaiswal

, and Haynesworth

S.E.

Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem, 64, 278, 1997.

16.

Zhao

, Wang

X.Y.

, Yu

X.X.

, Zhai

Y.X.

, He

, Wu

, and Shi

Y.A.

Expression of human telomerase reverse transcriptase mediates the senescence of mesenchymal stem cells through the PI3K/AKT signaling pathway. Int J Mol Med, 36, 857, 2015.

17.

Wang

, Sha

X.J.

, Li

G.H.

, Yang

F.S.

, Ji

, Wen

L.Y.

, Liu

S.Y.

, Chen

, Ding

, and Xuan

Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells. Arch Oral Biol, 57, 1231, 2012.

18.

Abdullah

M.F.

, Abdullah

S.F.

, Omar

N.S.

, Mahmood

, Fazliah Mohd Noor

S.N.

, Kannan

T.P.

, and Mokhtar

K.I.

Proliferation rate of stem cells derived from human dental pulp and identification of differentially expressed genes. Cell Biol Int, 38, 582, 2014.

19.

Aimetti

, Ferrarotti

, Cricenti

, Mariani

G.M.

, and Romano

Autologous dental pulp stem cells in periodontal regeneration: a case report. Int J Periodontics Restorative Dent, 34, s27, 2014.

20.

Alkaisi

, Ismail

A.R.

, Mutum

S.S.

, Ahmad

Z.A.

, Masudi

, and Abd Razak

N.H.

Transplantation of human dental pulp stem cells: enhance bone consolidation in mandibular distraction osteogenesis. J Oral Maxillofac Surg, 71, 1758, 2013.

21.

Nakamura

, Saruwatari

, Aita

, Takeuchi

, and Ogawa

Molecular and biomechanical characterization of mineralized tissue by dental pulp cells on titanium. J Dent Res, 84, 515, 2005.

22.

Zhu

, Wang

, Liu

, Huang

G.T.

, and Zhang

Immunohistochemical and histochemical analysis of newly formed tissues in root canal space transplanted with dental pulp stem cells plus platelet-rich plasma. J Endod, 40, 1573, 2014.

23.

Achilleos

, and Trainor

P.A.

Neural crest stem cells: discovery, properties and potential for therapy. Cell Res, 22, 288, 2012.

24.

La Noce

, Mele

, Tirino

, Paino

, De Rosa

, Naddeo

, Papagerakis

, Papaccio

, and Desiderio

Neural crest stem cell population in craniomaxillofacial development and tissue repair. Eur Cell Mater, 28, 348, 2014.

25.

Mayanil

C.S.

Transcriptional and epigenetic regulation of neural crest induction during neurulation. Dev Neurosci, 35, 361, 2013.

26.

Mitsiadis

T.A.

, and Graf

Cell fate determination during tooth development and regeneration. Birth Defects Res C Embryo Today, 87, 199, 2009.

27.

Miletich

, and Sharpe

P.T.

Neural crest contribution to mammalian tooth formation. Birth Defects Res C Embryo Today, 72, 200, 2004.

28.

Hall

B.K.

The neural crest and neural crest cells: discovery and significance for theories of embryonic organization. J Biosci, 33, 781, 2008.

29.

Rinon

, Molchadsky

, Nathan

, Yovel

, Rotter

, Sarig

, and Tzahor

p53 Coordinates cranial neural crest cell growth and epithelial-mesenchymal transition/delamination processes. Development, 138, 1827, 2011.

30.

Huang

G.T.

, Gronthos

, and Shi

Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res, 88, 792, 2009.

31.

Harada

, and Ohshima

New perspectives on tooth development and the dental stem cell niche. Arch Histol Cytol, 67, 1, 2004.

32.

Chai

, Jiang

, Ito

, Bringas

, Han

, Rowitch

D.H.

, Soriano

, McMahon

A.P.

, and Sucov

H.M.

Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development, 127, 1671, 2000.

33.

Yamazaki

, Tsuneto

, Yoshino

, Yamamura

, and Hayashi

Potential of dental mesenchymal cells in developing teeth. Stem Cells, 25, 78, 2007.

34.

Foudah

, Monfrini

, Donzelli

, Niada

, Brini

A.T.

, Orciani

, Tredici

, and Miloso

Expression of neural markers by undifferentiated mesenchymal-like stem cells from different sources. J Immunol Res, 2014, 987678, 2014.

35.

Feng

, Xing

, Feng

, Sang

, Shen

, Xu

, Jiang

, Liu

, Tan

, Gu

, and Li

Age-dependent impaired neurogenic differentiation capacity of dental stem cell is associated with Wnt/β-catenin signaling. Cell Mol Neurobiol, 33, 1023, 2013.

36.

Govindasamy

, Abdullah

A.N.

, Ronald

V.S.

, Musa

, Ab Aziz

Z.A.

, Zain

R.B.

, Totey

, Bhonde

R.R.

, and Abu Kasim

N.H.

Inherent differential propensity of dental pulp stem cells derived from human deciduous and permanent teeth. J Endod, 36, 1504, 2010.

37.

Martens

, Wolfs

, Struys

, Politis

, Bronckaers

, and Lambrichts

Expression pattern of basal markers in human dental pulp stem cells and tissue. Cells Tissues Organs, 196, 490, 2012.

38.

Tamaki

, Nakahara

, Ishikawa

, and Sato

In vitro analysis of mesenchymal stem cells derived from human teeth and bone marrow. Odontology, 101, 121, 2013.

39.

Karaöz

, Doğan

B.N.

, Aksoy

, Gacar

, Akyüz

, Ayhan

, Genç

Z.S.

, Yürüker

, Duruksu

, Demircan

P.C.

, and Sariboyaci

A.E.

Isolation and in vitro characterisation of dental pulp stem cells from natal teeth. Histochem Cell Biol, 133, 95, 2010.

40.

Davidson

R.M.

Neural form of voltage-dependent sodium current in human cultured dental pulp cells. Arch Oral Biol, 39, 613, 1994.

41.

Yang

K.L.

, Chen

M.F.

, Liao

C.H.

, Pang

C.Y.

, and Lin

P.Y.

A simple and efficient method for generating Nurr1-positive neuronal stem cells from human wisdom teeth (tNSC) and the potential of tNSC for stroke therapy. Cytotherapy, 11, 606, 2009.

42.

Sasaki

, Aoki

, Yamato

, Uchiyama

, Wada

, Ogiuchi

, Okano

, and Ando

PLGA artificial nerve conduits with dental pulp cells promote facial nerve regeneration. J Tissue Eng Regen Med, 5, 823, 2011.

43.

Spyridopoulos

, Lambropoulou

, Pagonopoulou

, Birbilis

, Tsaroucha

A.K.

, Kouzi-Koliakou

, Botaitis

, Deftereou

T.E.

, Gaitanidis

, and Pitiakoudis

Regenerated nerve defects with a nerve conduit containing dental pulp stem cells in pigs: an immunohistochemical and electrophysiological evaluation. J Reconstr Microsurg, 31, 516, 2015.

44.

Mead

, Logan

, Berry

, Leadbeater

, and Scheven

B.A.

Intravitreally transplanted dental pulp stem cells promote neuroprotection and axon regeneration of retinal ganglion cells after optic nerve injury. Invest Ophthalmol Vis Sci, 54, 7544, 2013.

45.

Huang

A.H.

, Snyder

B.R.

, Cheng

P.H.

, and Chan

A.W.

Putative dental pulp-derived stem/stromal cells promote proliferation and differentiation of endogenous neural cells in the hippocampus of mice. Stem Cells, 26, 2654, 2008.

46.

Leong

W.K.

, Henshall

T.L.

, Arthur

, Kremer

K.L.

, Lewis

M.D.

, Helps

S.C.

, Field

, Hamilton-Bruce

M.A.

, Warming

, Manavis

, Vink

, Gronthos

, and Koblar

S.A.

Human adult dental pulp stem cells enhance poststroke functional recovery through non-neural replacement mechanisms. Stem Cells Transl Med, 1, 177, 2012.

47.

Arthur

, Shi

, Zannettino

A.C.

, Fujii

, Gronthos

, and Koblar

S.A.

Implanted adult human dental pulp stem cells induce endogenous axon guidance. Stem Cells, 27, 2229, 2009.

48.

Beigi

M.H.

, Ghasemi-Mobarakeh

, Prabhakaran

M.P.

, Karbalaie

, Azadeh

, Ramakrishna

, Baharvand

, and Nasr-Esfahani

M.H.

In vivo integration of poly(ɛ-caprolactone)/gelatin nanofibrous nerve guide seeded with teeth derived stem cells for peripheral nerve regeneration. J Biomed Mater Res A, 102, 4554, 2014.

49.

, Jung

H.J.

, Kim

S.M.

, Kim

M.J.

, Jahng

J.W.

, and Lee

J.H.

Human periodontal ligament stem cells repair mental nerve injury. Neural Regen Res, 8, 2827, 2013.

50.

, Yang

, Li

, Xiong

, Xie

, Yang

, Yu

, Feng

, Jiang

, Guo

, and Tian

A therapeutic strategy for spinal cord defect: human dental follicle cells combined with aligned PCL/PLGA electrospun material. Biomed Res Int, 2015, 197183, 2015.

51.

Sugaya

, Alvarez

, Marutle

, Kwak

Y.D.

, and Choumkina

Stem cell strategies for Alzheimer's disease therapy. Panminerva Med, 48, 87, 2006.

52.

Siniscalco

, Bradstreet

J.J.

, Sych

, and Antonucci

Perspectives on the use of stem cells for autism treatment. Stem Cells Int, 2013, 262438, 2013.

53.

Wilems

T.S.

, Pardieck

, Iyer

, and Sakiyama-Elbert

S.E.

Combination therapy of stem cell derived neural progenitors and drug delivery of anti-inhibitory molecules for spinal cord injury. Acta Biomater, 15, 30112, 2015.

54.

Pavlova

, Lopatina

, Kalinina

, Rybalkina

, Parfyonova

, Tkachuk

, and Revishchin

In vitro neuronal induction of adipose-derived stem cells and their fate after transplantation into injured mouse brain. Curr Med Chem, 19, 5170, 2012.

55.

Abeysinghe

H.C.

, Bokhari

, Quigley

, Choolani

, Chan

, Dusting

G.J.

, Crook

J.M.

, Kobayashi

N.R.

, and Roulston

C.L.

Pre-differentiation of human neural stem cells into GABAergic neurons prior to transplant results in greater repopulation of the damaged brain and accelerates functional recovery after transient ischemic stroke. Stem Cell Res Ther, 6, 186, 2015.

56.

Luo

, Zhu

, Zhang

, and Jin

Tissue-engineered nerve constructs under a microgravity system for peripheral nerve regeneration. Tissue Eng Part A, 21, 267, 2015.

57.

Yap

M.S.

, Nathan

K.R.

, Yeo

, Lim

L.W.

, Poh

C.L.

, Richards

, Lim

W.L.

, Othman

, and Heng

B.C.

Neural differentiation of human pluripotent stem cells for nontherapeutic applications: toxicology, pharmacology, and in vitro disease modeling. Stem Cells Int, 2015, 105172, 2015.

58.

Srikanth

, and Young-Pearse

T.L.

Stem cells on the brain: modeling neurodevelopmental and neurodegenerative diseases using human induced pluripotent stem cells. J Neurogenet, 28, 5, 2014.

59.

Dai

J.W.

, Yuan

, Shen

S.Y.

, Lu

J.T.

, Zhu

X.F.

, Yang

, Zhang

J.F.

, and Shen

G.F.

p75 neurotrophin receptor positive dental pulp stem cells: new hope for patients with neurodegenerative disease and neural injury. Shanghai Kou Qiang Yi Xue, 22, 469, 2013.

60.

Gervois

, Struys

, Hilkens

, Bronckaers

, Ratajczak

, Politis

, Brône

, Lambrichts

, and Martens

Neurogenic maturation of human dental pulp stem cells following neurosphere generation induces morphological and electrophysiological characteristics of functional neurons. Stem Cells Dev, 24, 296, 2015.

61.

Osathanon

, Sawangmake

, Nowwarote

, and Pavasant

Neurogenic differentiation of human dental pulp stem cells using different induction protocols. Oral Dis, 20, 352, 2014.

62.

Karbanová

, Soukup

, Suchánek

, Pytlík

, Corbeil

, and Mokrý

Characterization of dental pulp stem cells from impacted third molars cultured in low serum-containing medium. Cells Tissues Organs, 193, 244, 2011.

63.

Xiao

, and Tsutsui

Characterization of human dental pulp cells-derived spheroids in serum-free medium: stem cells in the core. J Cell Biochem, 114, 2624, 2013.

64.

Pisciotta

, Carnevale

, Meloni

, Riccio

, De Biasi

, Gibellini

, Ferrari

, Bruzzesi

, and De Pol

Human dental pulp stem cells (hDPSCs): isolation, enrichment and comparative differentiation of two sub-populations. BMC Dev Biol, 15, 14, 2015.

65.

Ryu

J.S.

, Ko

, Lee

J.W.

, Park

S.B.

, Byun

S.J.

, Jeong

E.J.

, Ko

, and Choo

Y.K.

Gangliosides are involved in neural differentiation of human dental pulp-derived stem cells. Biochem Biophys Res Commun, 387, 266, 2009.

66.

Zhang

, Walboomers

X.F.

, Shi

, Fan

, and Jansen

J.A.

Multilineage differentiation potential of stem cells derived from human dental pulp after cryopreservation. Tissue Eng, 12, 2813, 2006.

67.

Király

, Kádár

, Horváthy

D.B.

, Nardai

, Rácz

G.Z.

, Lacza

, Varga

, and Gerber

Integration of neuronally predifferentiated human dental pulp stem cells into rat brain in vivo. Neurochem Int, 59, 371, 2011.

68.

Király

, Porcsalmy

, Pataki

, Kádár

, Jelitai

, Molnár

, Hermann

, Gera

, Grimm

W.D.

, Ganss

, Zsembery

, and Varga

Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons. Neurochem Int, 55, 323, 2009.

69.

, Wen

, Peng

, and Jonakait

G.M.

Antioxidants N-acetylcysteine (NAC) and 2-mercaptoethanol (2-ME) affect the survival and differentiative potential of cholinergic precursors from the embryonic septal nuclei and basal forebrain: involvement of ras signaling. Brain Res Dev Brain Res, 130, 207, 2001.

70.

, Yuan

, Ou

, Cai

, Wang

, Sun

, and Zhang

Autophagy and apoptosis during adult adipose-derived stromal cells differentiation into neuron-like cells in vitro. Neural Regen Res, 7, 1205, 2012.

71.

Chang

C.C.

, Chang

K.C.

, Tsai

S.J.

, Chang

H.H.

, and Lin

C.P.

Neurogenic differentiation of dental pulp stem cells to neuron-like cells in dopaminergic and motor neuronal inductive media. J Formos Med Assoc, 113, 956, 2014.

72.

Kanafi

, Majumdar

, Bhonde

, Gupta

, and Datta

Midbrain cues dictate differentiation of human dental pulp stem cells towards functional dopaminergic neurons. J Cell Physiol, 229, 1369, 2014.

73.

Arthur

, Rychkov

, Shi

, Koblar

S.A.

, and Gronthos

Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells, 26, 1787, 2008.

74.

Feng

, Lu

, Huang

, Xing

, Feng

, Jin

, Yi

, Li

, Lu

, Nie

, Chen

, Zhang

, Gu

, and Zhang

3D porous chitosan scaffolds suit survival and neural differentiation of dental pulp stem cells. Cell Mol Neurobiol, 34, 859, 2014.

75.

Osathanon

, Nowwarote

, and Pavasant

Basic fibroblast growth factor inhibits mineralization but induces neuronal differentiation by human dental pulp stem cells through a FGFR and PLCγ signaling pathway. J Cell Biochem, 112, 1807, 2011.

76.

Bressan

, Ferroni

, Gardin

, Pinton

, Stellini

, Botticelli

, Sivolella

, and Zavan

Donor age-related biological properties of human dental pulp stem cells change in nanostructured scaffolds. PLoS One, 7, e49146, 2012.

77.

Karaöz

, Demircan

P.C.

, Sağlam

, Aksoy

, Kaymaz

, and Duruksu

Human dental pulp stem cells demonstrate better neural and epithelial stem cell properties than bone marrow-derived mesenchymal stem cells. Histochem Cell Biol, 136, 455, 2011.

78.

H.X.

, Jin

, Shi

J.N.

, Luo

Y.Q.

, Zhou

Y.N.

, Peng

, and Xu

Y.H.

Experiment on inducing human dental pulp stem cells into neural-like cells. Hua Xi Kou Qiang Yi Xue Za Zhi, 25, 331, 2007.

79.

van Kooten

T.G.

, Spijker

H.T.

, and Busscher

H.J.

Plasma-treated polystyrene surfaces: model surfaces for studying cell-biomaterial interactions. Biomaterials, 25, 1735, 2004.

80.

, Yu

, Davidsion

M.C.

, and Silva

G.A.

Comparison of standard surface chemistries for culturing mesenchymal stem cells prior to neural differentiation. Biomaterials, 24, 4333, 2006.

81.

, Tan

, Wu

, Yin

, Liu

, Meng

, Cui

, Wu

, Lin

, Hu

, and Feng

Poly-L-ornithine promotes preferred differentiation of neural stem/progenitor cells via ERK signalling pathway. Sci Rep, 5, 15535, 2015.

82.

Brewer

G.J.

, Torricelli

J.R.

, Evege

E.K.

, and Price

P.J.

Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res, 35, 567, 1993.

83.

Neurobasal^®-A Medium. Manufacturer (ThermoFisher Scientific, Inc.) website. Available at: www.thermofisher.com/order/catalog/product/10888022 (accessed October 19, 2015).

84.

Bottenstein

J.E.

, and Sato

G.H.

Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci U S A, 76, 514, 1979.

85.

Mainzer

, Barrichello

, Debret

, Remoué

, Sigaudo-Roussel

, and Sommer

Insulin-transferrin-selenium as an alternative to foetal serum for epidermal equivalents. Int J Cosmet Sci, 36, 427, 2014.

86.

Dhara

S.K.

, Hasneen

, Machacek

D.W.

, Boyd

N.L.

, Rao

R.R.

, and Stice

S.L.

Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures. Differentiation, 76, 454, 2008.

87.

Santa-Olalla

, and Covarrubias

Epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and basic fibroblast growth factor (bFGF) differentially influence neural precursor cells of mouse embryonic mesencephalon. J Neurosci Res, 42, 172, 1995.

88.

Aigner

, and Bogdahn

TGF-beta in neural stem cells and in tumors of the central nervous system. Cell Tissue Res, 331, 225, 2008.

89.

, Sommer

, and Anderson

D.J.

MASH1 maintains competence for BMP2-induced neuronal differentiation in post-migratory neural crest cells. Curr Biol, 7, 440, 1997.

90.

Dhandapani

K.M.

, and Brann

D.W.

Transforming growth factor-beta: a neuroprotective factor in cerebral ischemia. Cell Biochem Biophys, 39, 13, 2003.

91.

Zhao

, Haney

M.J.

, Gupta

, Bohnsack

J.P.

, He

, Kabanov

A.V.

, and Batrakova

E.V.

GDNF-transfected macrophages produce potent neuroprotective effects in Parkinson's disease mouse model. PLoS One, 9, e106867, 2014.

92.

Barde

Y.A.

Neurotrophins: a family of proteins supporting the survival of neurons. Prog Clin Biol Res, 390, 45, 1994.

93.

, Li

, and Jiang

Neurotrophine-3 may contribute to neuronal differentiation of mesenchymal stem cells through the activation of the bone morphogenetic protein pathway. Cell Mol Biol Lett, 20, 385, 2015.

94.

Yuan

, Huang

, Xiao

, Lin

, and Han

Overexpression of β-NGF promotes differentiation of bone marrow mesenchymal stem cells into neurons through regulation of AKT and MAPK pathway. Mol Cell Biochem, 383, 201, 2013.

95.

Lim

J.Y.

, Park

S.I.

, Kim

S.M.

, Jun

J.A.

, Oh

J.H.

, Ryu

C.H.

, Jeong

C.H.

, Park

S.H.

, Park

S.A.

, Oh

, Chang

J.W.

, and Jeun

S.S.

Neural differentiation of brain-derived neurotrophic factor-expressing human umbilical cord blood-derived mesenchymal stem cells in culture via TrkB-mediated ERK and β-catenin phosphorylation and following transplantation into the developing brain. Cell Transplant, 20, 1855, 2011.

96.

Dutton

, Yamada

, Turnley

, Bartlett

P.F.

, and Murphy

Regulation of spinal motoneuron differentiation by the combined action of Sonic hedgehog and neurotrophin 3. Clin Exp Pharmacol Physiol, 26, 746, 1999.

97.

Hynes

, Porter

J.A.

, Chiang

, Chang

, Tessier-Lavigne

, Beachy

P.A.

, and Rosenthal

Induction of midbrain dopaminergic neurons by Sonic hedgehog. Neuron, 15, 35, 1995.

98.

Tsai

H.L.

, Deng

W.P.

, Lai

W.F.

, Chiu

W.T.

, Yang

C.B.

, Tsai

Y.H.

, and Hwang

S.M.

, Renshaw

P.F.

Wnts enhance neurotrophin-induced neuronal differentiation in adult bone-marrow-derived mesenchymal stem cells via canonical and noncanonical signaling pathways. PLoS One, 9, e104937, 2014.

99.

Trzaska

K.A.

, and Rameshwar

Dopaminergic neuronal differentiation protocol for human mesenchymal stem cells. Methods Mol Biol, 698, 295, 2011.

100.

Huat

T.J.

, Khan

A.A.

, Pati

, Mustafa

, Abdullah

J.M.

, and Jaafar

IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells. BMC Neurosci, 15, 91, 2014.

101.

Tao

Y.X.

, Xu

H.W.

, Zheng

Q.Y.

, and FitzGibbon

Noggin induces human bone marrow-derived mesenchymal stem cells to differentiate into neural and photoreceptor cells. Indian J Exp Biol, 48, 444, 2010.

102.

Zhang

, Gao

, Yu

, Wu

, Ai

, Wu

, Liu

, Du

, Guo

, and Zhang

Retinoic acid induces embryonic stem cell differentiation by altering both encoding RNA and microRNA expression. PLoS One, 10, e0132566, 2015.

103.

Kim

, Zahir

, Tator

C.H.

, and Shoichet

M.S.

Effects of dibutyryl cyclic-AMP on survival and neuronal differentiation of neural stem/progenitor cells transplanted into spinal cord injured rats. PLoS One, 6, e21744, 2011.

104.

Deng

, Obrocka

, Fischer

, and Prockop

D.J.

In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun, 282, 148, 2001.

105.

Mitchell

K.E.

, Weiss

M.L.

, Mitchell

B.M.

, Martin

, Davis

, Morales

, Helwig

, Beerenstrauch

, Abou-Easa

, Hildreth

, Troyer

, and Medicetty

Matrix cells from Wharton's jelly form neurons and glia. Stem Cells, 21, 50, 2003.

106.

Kilmer

S.L.

, and Carlsen

R.C.

Forskolin activation of adenylate cyclase in vivo stimulates nerve regeneration. Nature, 307, 455, 1984.

107.

Hammerling

, Bjelfman

, and Påhlman

Different regulation of N- and c-myc expression during phorbol ester-induced maturation of human SH-SY5Y neuroblastoma cells. Oncogene, 2, 73, 1987.

108.

Ved

H.S.

, and Pieringer

R.A.

Regulation of neuronal differentiation by retinoic acid alone and in cooperation with thyroid hormone or hydrocortisone. Dev Neurosci, 15, 49, 1993.

109.

Pelaez

, Huang

C.Y.

, and Cheung

H.S.

Isolation of pluripotent neural crest-derived stem cells from adult human tissues by connexin-43 enrichment. Stem Cells Dev, 22, 2906, 2013.

110.

Sawangmake

, Pavasant

, Chansiripornchai

, and Osathanon

High glucose condition suppresses neurosphere formation by human periodontal ligament-derived mesenchymal stem cells. J Cell Biochem, 115, 928, 2014.

111.

Osathanon

, Manokawinchoke

, Nowwarote

, Aguilar

, Palaga

, and Pavasant

Notch signaling is involved in neurogenic commitment of human periodontal ligament-derived mesenchymal stem cells. Stem Cells Dev, 22, 1220, 2013.

112.

Huang

C.Y.

, Pelaez

, Dominguez-Bendala

, Garcia-Godoy

, and Cheung

H.S.

Plasticity of stem cells derived from adult periodontal ligament. Regen Med, 4, 809, 2009.

113.

, Gong

, and Liao

In vitro neural/glial differentiation potential of periodontal ligament stem cells. Arch Med Sci, 6, 678, 2010.

114.

Kadar

, Kiraly

, Porcsalmy

, Molnar

, Racz

G.Z.

, Blazsek

, Kallo

, Szabo

E.L.

, Gera

, Gerber

, and Varga

Differentiation potential of stem cells from human dental origin – promise for tissue engineering. J Physiol Pharmacol, 60, 167, 2009.

115.

Fortino

V.R.

, Chen

R.S.

, Pelaez

, and Cheung

H.S.

Neurogenesis of neural crest-derived periodontal ligament stem cells by EGF and bFGF. J Cell Physiol, 229, 479, 2014.

116.

Bueno

, Ramirez

, Rodríguez-Lozano

F.J.

, Tabarés-Seisdedos

, Rodenas

, Moraleda

J.M.

, Jones

J.R.

, and Martinez

Human adult periodontal ligament-derived cells integrate and differentiate after implantation into the adult mammalian brain. Cell Transplant, 22, 2017, 2013.

117.

Coura

G.S.

, Garcez

R.C.

, de Aguiar

C.B.

, Alvarez-Silva

, Magini

R.S.

, and Trentin

A.G.

Human periodontal ligament: a niche of neural crest stem cells. J Periodontal Res, 43, 531, 2008.

118.

Razavi

, Mostafavi

F.S.

, Mardani

, Zarkesh Esfahani

, Kazemi

, and Esfandiari

Effect of T3 hormone on neural differentiation of human adipose derived stem cells. Cell Biochem Funct, 32, 702, 2014.

119.

Chen

, Zhou

, Zhong

, Zhang

, Li

, Zhang

, Qu

, Yang

, Wang

, and Yu

Thyroid hormone promotes neuronal differentiation of embryonic neural stem cells by inhibiting STAT3 signaling through TRα1. Stem Cells Dev, 21, 2667, 2012.

120.

König

, and Moura Neto

Thyroid hormone actions on neural cells. Cell Mol Neurobiol, 22, 517, 2002.

121.

Wang

, Wang

, Sun

, Wang

, Yang

, Shi

, and Wang

Stem cells from human-exfoliated deciduous teeth can differentiate into dopaminergic neuron-like cells. Stem Cells Dev, 19, 1375, 2010.

122.

Esmaeili

, Alifarja

, Nourbakhsh

, and Talebi

Messenger RNA expression patterns of neurotrophins during transdifferentiation of stem cells from human-exfoliated deciduous teeth into neural-like cells. Avicenna J Med Biotechnol, 6, 21, 2014.

123.

Jarmalavičiūtė

, Tunaitis

, Strainienė

, Aldonytė

, Ramanavičius

, Venalis

, Magnusson

K.E.

, and Pivoriūnas

A new experimental model for neuronal and glial differentiation using stem cells derived from human exfoliated deciduous teeth. J Mol Neurosci, 51, 307, 2013.

124.

Nourbakhsh

, Soleimani

, Taghipour

, Karbalaie

, Mousavi

S.B.

, Talebi

, Nadali

, Tanhaei

, Kiyani

G.A.

, Nematollahi

, Rabiei

, Mardani

, Bahramiyan

, Torabinejad

, Nasr-Esfahani

M.H.

, and Baharvand

Induced in vitro differentiation of neural-like cells from human exfoliated deciduous teeth-derived stem cells. Int J Dev Biol, 55, 189, 2011.

125.

Isobe

, Koyama

, Nakao

, Osawa

, Ikeno

, Yamanaka

, Okubo

, Fujimura

, and Bessho

Comparison of human mesenchymal stem cells derived from bone marrow, synovial fluid, adult dental pulp, and exfoliated deciduous tooth pulp. Int J Oral Maxillofac Surg, 45, 124, 2016.

126.

Taghipour

, Karbalaie

, Kiani

, Niapour

, Bahramian

, Nasr-Esfahani

M.H.

, and Baharvand

Transplantation of undifferentiated and induced human exfoliated deciduous teeth-derived stem cells promote functional recovery of rat spinal cord contusion injury model. Stem Cells Dev, 21, 1794, 2012.

127.

Morsczeck

, Völlner

, Saugspier

, Brandl

, Reichert

T.E.

, Driemel

, and Schmalz

Comparison of human dental follicle cells (DFCs) and stem cells from human exfoliated deciduous teeth (SHED) after neural differentiation in vitro. Clin Oral Investig, 14, 433, 2010.

128.

Sasaki

, Aoki

, Yamato

, Uchiyama

, Wada

, Okano

, and Ogiuchi

Neurosphere generation from dental pulp of adult rat incisor. Eur J Neurosci, 27, 538, 2008.

129.

Kidowaki

, Thiele

C.J.

, Kleinman

H.K.

, and Israel

M.A.

Matrix proteins induce neuroblastoma cell differentiation without altering cell growth. Pathobiology, 59, 316, 1991.

130.

Y.C.

, Lin

Y.C.

, and Young

T.H.

Combination of media, biomaterials and extracellular matrix proteins to enhance the differentiation of neural stem/precursor cells into neurons. Acta Biomater, 8, 3035, 2012.

131.

Sheppard

A.M.

, Brunstrom

J.E.

, Thornton

T.N.

, Gerfen

R.W.

, Broekelmann

T.J.

, McDonald

J.A.

, and Pearlman

A.L.

Neuronal production of fibronectin in the cerebral cortex during migration and layer formation is unique to specific cortical domains. Dev Biol, 172, 504, 1995.

132.

Michler-Stuke

, Wolff

J.R.

, and Bottenstein

J.E.

Factors influencing astrocyte growth and development in defined media. Int J Dev Neurosci, 2, 575, 1984.

133.

Völlner

, Ernst

, Driemel

, and Morsczeck

A two-step strategy for neuronal differentiation in vitro of human dental follicle cells. Differentiation, 77, 433, 2009.

134.

Yang

, Sun

, Li

, Xie

, Yu

, Feng

, Jiang

, Guo

, and Tian

The potential of dental stem cells differentiating into neurogenic cell lineage after cultivation in different modes in vitro. Cell Reprogram, 16, 379, 2014.

135.

Vanacker

, Viswanath

, De Berdt

, Everard

, Cani

P.D.

, Bouzin

, Feron

, Diogenes

, Leprince

J.G.

, and des Rieux

Hypoxia modulates the differentiation potential of stem cells of the apical papilla. J Endod, 40, 1410, 2014.

136.

Bakopoulou

, Leyhausen

, Volk

, Koidis

, and Geurtsen

Comparative characterization of STRO-1(neg)/CD146(pos) and STRO-1(pos)/CD146(pos) apical papilla stem cells enriched with flow cytometry. Arch Oral Biol, 58, 1556, 2013.

137.

Sonoyama

, Liu

, Yamaza

, Tuan

R.S.

, Wang

, Shi

, and Huang

G.T.

Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod, 34, 166, 2008.

138.

El-Bialy

, Alhadlaq

, Wong

, and Kucharski

Ultrasound effect on neural differentiation of gingival stem/progenitor cells. Ann Biomed Eng, 42, 1406, 2014.

139.

, Chen

, Akiyama

, Chai

, Le

A.D.

, Wang

, and Shi

Gingivae contain neural-crest- and mesoderm-derived mesenchymal stem cells. J Dent Res, 92, 825, 2013.

140.

Hsu

S.H.

, Huang

G.S.

, and Feng

Isolation of the multipotent MSC subpopulation from human gingival fibroblasts by culturing on chitosan membranes. Biomaterials, 33, 2642, 2012.

141.

Lee

J.H.

, Um

, Song

I.S.

, Kim

H.Y.

, and Seo

B.M.

Neurogenic differentiation of human dental stem cells in vitro. J Korean Assoc Oral Maxillofac Surg, 40, 173, 2014.

142.

Lewandowska

, Balachander

, Sukenik

C.N.

, and Culp

L.A.

Modulation of fibronectin adhesive functions for fibroblasts and neural cells by chemically derivatized substrata. J Cell Physiol, 141, 334, 1989.

143.

Saha

, Keung

A.J.

, Irwin

E.F.

, Li

, Little

, Schaffer

D.V.

, and Healy

K.E.

Substrate modulus directs neural stem cell behavior. Biophys J, 95, 4426, 2008.

144.

Chambers

S.M.

, Fasano

C.A.

, Papapetrou

E.P.

, Tomishima

, Sadelain

, and Studer

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol, 27, 275, 2009.

145.

Manceur

A.P.

, Tseng

, Holowacz

, Witterick

, Weksberg

, McCurdy

R.D.

, Warsh

J.J.

, and Audet

Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors. Exp Cell Res, 317, 2086, 2011.

146.

X.J.

, Hu

B.Y.

, Jones

S.A.

, Zhang

Y.S.

, Lavaute

, Du

Z.W.

, and Zhang

S.C.

Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules. Stem Cells, 26, 886, 2008.

147.

Zhou

, Su

, Li

, Tsang

, Duan

, and Wang

High-efficiency induction of neural conversion in human ESCs and human induced pluripotent stem cells with a single chemical inhibitor of transforming growth factor beta superfamily receptors. Stem Cells, 10, 1741, 2010.

148.

Bragina

, Sergejeva

, Serg

, Zarkovsky

, Maloverjan

, Kogerman

, and Zarkovsky

Smoothened agonist augments proliferation and survival of neural cells. Neurosci Lett, 482, 81, 2010.

149.

Qun

Electrical stimulation promote proliferation and differentiation of endogenous neural stem cells in normal and injured spinal cord. Zhen Ci Yan Jiu, 33, 34, 2008.