Abstract Neuronal regeneration remains a major challenge in the treatment of neurological disorders because of the limited regenerative capacity of neurons. Human dental pulp stem cells (hDPSCs) represent a promising cell source for neural regeneration because of their neurogenic differentiation potential. Rho-associated kinase (ROCK), a key regulator of cytoskeletal dynamics, influences neuronal morphogenesis. However, the morphological and functional consequences of ROCK, Y-27632 inhibition in hDPSCs remain incompletely characterized. This study investigated the neurogenic effects of ROCK inhibition in hDPSCs. Cells were characterized for mesenchymal stem cell properties and treated with 30 μM Y-27632. Neuronal differentiation was evaluated by Cresyl violet staining, scanning electron microscopy, βIII-tubulin immunofluorescence, and intracellular Ca²⁺ imaging. Y-27632 treatment induced pronounced morphological remodeling, resulting in distinct bipolar and multipolar neuron-like phenotypes accompanied by βIII-tubulin expression. Importantly, Y-27632-treated cells exhibited a rapid increase in intracellular Ca²⁺ fluorescence following KCl stimulation, supporting the acquisition of depolarization-responsive properties. These findings demonstrate that ROCK inhibition promotes morphological and functional neuronal-like differentiation of hDPSCs, supporting their potential utility in neural regeneration research.

Keywords: Morphological differentiation, Dental pulp stem cells, ROCK inhibitor, Neuronal differentiation, Neuronal-like cells

Funding: The funding of this research was supported by Kasetsart University Research and Development Institute, KURDI (Research ID: YF(KU)11.67).

Citation:  Srikawnawan, W., Piromrat, P., Lumbikananda, S., Thonabulsombat, C., Ruangsawasdi, N., and Thongklam Songsaad, A. 2027. Morphological neuronal-like differentiation of human dental pulp stem cells induced by ROCK inhibitor. Natural and Life Sciences Communications. 26(1): e2027005.

Graphical Abstract:

INTRODUCTION

Neurons of the nervous system exhibit diverse morphological characteristics that are closely linked to their functional roles (Goorha et al., 2022). Neuronal cell types differ in the shape, number, and branching patterns of their processes, which determine how they receive, integrate, and transmit signals (Peng et al., 2021; Zehtabian et al., 2024). Based on the organization of their processes extending from the soma, neurons are commonly classified as pseudounipolar, unipolar, bipolar, or multipolar neurons (Fan et al., 2025). Pseudounipolar neurons, which are typical sensory neurons located in the dorsal root ganglia (DRG), mediate proprioception, touch, temperature, and pain (Sun et al., 2024; Costa et al., 2025). Bipolar neurons are involved in special sensory systems, including olfactory, auditory, gustatory, and visual pathways (McClintock et al., 2020; Afanasyeva et al., 2021; Lao-Rodríguez et al., 2024; Muluh et al., 2024), whereas multipolar neurons, the most abundant neuronal type in the brain and spinal cord, primarily control motor functions (Fan et al., 2025; Kussick et al., 2025). Disruption of these neuronal populations or their morphology contributes to impaired neural signaling and is associated with the development of neurodegenerative and neurological disorders, as mature neurons have a limited capacity for self-repair (Wu et al., 2024).

Stem cell-based strategies have therefore been proposed as potential approaches to replace or support damaged neurons (Lee et al., 2022; Chang and Lee, 2025). Oral-derived cells have emerged as valuable experimental models for studying cellular responses and regenerative mechanisms in dentistry (Kantawong et al., 2022). Among candidate cell sources, human dental pulp stem cells (hDPSCs) represent an accessible population of mesenchymal stem cells (MSCs) with plastic adherence, self-renewal capacity, and multilineage differentiation potential (Srikawnawan et al., 2022). Owing to their neural crest origin, hDPSCs exhibit neurogenic competence and have been reported to differentiate toward neuronal and glial lineages, as well as to secrete neurotrophic factors that promote neurite outgrowth and neuroprotection (Al-Maswary et al., 2022; Kwack and Lee, 2022; Fu et al., 2023). In addition, hDPSCs have been shown to express regeneration-related genes and neurotrophic molecules that may support endogenous neural repair mechanisms (Candelise et al., 2023; Xiong et al., 2024; Lopes et al., 2025). These properties highlight the potential of hDPSCs as a cellular model and candidate cell source for studies related to neuroregeneration and neurodegenerative diseases.

The Rho/Rho-associated coiled-coil containing kinase (Rho/ROCK) signaling pathway is a key regulator of cytoskeletal dynamics, cell contraction, migration, proliferation, and differentiation (Guan et al., 2023; Melzer et al., 2023). In the nervous system, ROCK signaling plays an important role in neuronal morphology, including dendritic spine remodeling, synaptic plasticity, and axonal growth during development and after injury (Benarroch, 2023). Dysregulation or overactivation of the ROCK pathway has been associated with impaired neuronal structure and function. Suppression of ROCK activity has been reported to promote the differentiation of stem cells toward multiple lineages, including neurons (Srikawnawan et al., 2022), hepatocytes (Choi et al., 2024), cardiomyocytes (Jiang et al., 2022), neuroglia (Nizamudeen et al., 2018), and endothelial cells (Joo et al., 2012). Although hDPSCs possess intrinsic neurogenic potential, their mesenchymal lineage bias may limit spontaneous neuronal differentiation, and modulation of ROCK signaling may specifically promote morphological features characteristic of a neuronal-like phenotype.

Therefore, the objective of this study was to investigate the effects of ROCK inhibition by Y-27632 on neuronal differentiation of hDPSCs. Following Y-27632 treatment, neuronal differentiation was evaluated through morphological analysis, Nissl substance identification using Cresyl violet staining, ultrastructural analysis using SEM, neuronal-associated protein (βIII-tubulin) expression, and depolarization-evoked intracellular Ca²⁺ responses.

MATERIALS AND METHODS

Collection of teeth

Impacted third molars (n = 3) were obtained from Thai patients at the Dental Hospital, Faculty of Dentistry, Mahidol University, Bangkok, Thailand. The study was approved by the Kasetsart University Research Ethics Committee (KUREC-HSR67/037), and written informed consent was obtained from all donors. All procedures were conducted in accordance with the Declaration of Helsinki (1964). Only healthy teeth were included. Teeth showing pulp necrosis, caries, trauma, or other pathological changes were excluded.

Isolation and culture of hDPSCs

To establish the hDPSCs, the human dental pulp tissue from impacted third molars was performed enzymatic digestion with previously described (Thongsuk et al., 2025). Briefly, the teeth were maintained in the proliferative medium containing Alpha Minimum Essential Medium (αMEM, Gibco, Life Technologies, NY, USA) containing 10% Fetal Bovine Serum (FBS, Gibco, Life Technologies), and 1% Penicillin/Streptomycin (Gibco, Life Technologies). The teeth were rinsed with phosphate-buffered saline (PBS, Sigma-Aldrich, MO, USA), and dental pulp tissues were carefully removed using tooth-cutting pliers to break the dentin. The pulp tissues were digested in a solution of 3 mg/mL collagenase type I (Worthington, NJ, USA) and 4 mg/mL dispase II (Sigma-Aldrich) at 37°C. After digestion, the cell suspension was filtered through a 70 μm cell strainer (Falcon™, Thermo Fisher Scientific, MA, USA). The filtered cells were then cultured in a culture flask (Nunc™, Thermo Fisher Scientific) using proliferative medium and maintained at 37°C in a humidified incubator with 5% CO₂. Cells from passages 2 to 5 were used for subsequent experiments.

Characterization of hDPSCs

The hDPSCs were characterized based on typical MSCs characteristics: colony-forming ability, derivative of migratory neural crest stem cells, and differentiation potential. To assess colony-forming units (CFU), cells were seeded at a low density (500 cells/well) in 6-well plates (Nunc™, Thermo Fisher Scientific) and cultured in proliferation medium for 12 days. Formed colonies were stained with Giemsa (Sigma-Aldrich) and visualized under an inverted microscope (CKX3, Olympus, Hamburg, Germany).

To indicate their derivative of migratory neural crest stem cells, cells were cultured in four-chamber culture slides (Corning, NY, USA) at a density of 2,000 cells/well with the proliferation medium for 3 days, and Nestin immunofluorescent staining was performed. The samples were subsequently fixed with 4% paraformaldehyde (Sigma-Aldrich) for 1 hour. After fixation, the cells were dehydrated with 20% cold methanol (Sigma-Aldrich) in PBS for 5 minutes and rinsed with PBS. Permeabilization was performed using 0.5% Triton X-100 (Sigma-Aldrich) in PBS at 4°C for 24 hours. To block nonspecific binding, cells were incubated with 15% bovine serum albumin (BSA, Sigma-Aldrich) at 4°C for 12 hours. The cells were then incubated at 4°C for 24 hours with the primary antibody, anti-mouse Nestin (1:500, Biolegend, CA, USA). Subsequently, they were incubated with the secondary antibody, goat anti-mouse Alexa Fluor Plus 488 (1:1,000, Invitrogen, NY, USA), for 4 hours at room temperature. The omission control was performed by adding only the secondary antibody. Finally, the slides were washed and mounted using Prolong Diamond Antifade Mountant containing 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen), and coverslips were applied. Fluorescent images were acquired using a confocal laser scanning microscope (FV10i-DOC, Olympus).

To demonstrate differentiation abilities, the isolated cells were induced toward adipogenic and osteogenic lineages. The cells were cultured in 24-well plates (Nunc™, Thermo Fisher Scientific) at a density of 2,000 cells/well with an adipogenic induction medium, which consisted of αMEM, 10% FBS, 1% Penicillin/Streptomycin, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 10 μg/mL insulin (Sigma-Aldrich), 10 μM dexamethasone (Sigma-Aldrich), and 100 μM indomethacin (Sigma-Aldrich) for 6 weeks. The adipogenic differentiation ability was visualized by Oil Red O staining (Sigma-Aldrich) to reveal the accumulation of lipid droplets.

Furthermore, cells were cultured in an osteogenic induction medium, which consisted of αMEM, 10% FBS, 1% Penicillin/Streptomycin, 0.1 μM dexamethasone, 50 μg/mL ascorbate-2-phosphate (Sigma-Aldrich), and 10 mM β-glycerophosphate (Sigma-Aldrich) for 4 weeks to investigate their osteogenic differentiation ability. The mineralized calcium nodules were detected by Alizarin Red staining (Sigma-Aldrich).

Morphological differentiation of hDPSCs

The hDPSCs were seeded in 4-chamber culture slides at a density of 2,000 cells/well in the proliferation medium for 24 hours and treated with or without 30 μM of the ROCK inhibitor (Y-27632, Sigma-Aldrich) in the basal medium (αMEM and 1% Penicillin/Streptomycin). Cultures were incubated at 37°C in a humidified atmosphere with 5% CO₂ for 72 hours, and their cell morphology was observed under a phase-contrast microscope.

To visualize neuronal-like morphological features, cells were subjected to Cresyl violet staining. Briefly, cells were fixed with 4% paraformaldehyde in PBS for 60 minutes, washed with PBS, and subsequently stained with Cresyl violet acetate working solution (Electron Microscopy Sciences, PA, USA) for 1 hour in the dark. After staining, cells were washed with double-distilled water and serially dehydrated with 90%, 95%, and 100% ethanol, respectively. Neuronal-like cells were identified by dark purple Nissl-like cytoplasmic staining. Sensory-like cells were defined by a round soma with two processes, whereas motor-like cells exhibited pyramidal or multipolar morphologies with multiple processes. Cresyl violet-positive neuronal-like cells were counted in three randomly selected microscopic fields per specimen. Data were obtained from three independent experiments, and the percentage of neuronal-like cells was calculated as:

To reveal the ultrastructure of neuronal-like morphologies, the investigation through SEM was performed. Firstly, cells were fixed in 4% paraformaldehyde for 60 minutes. The fixed cells were subjected to serial dehydration in ethanol (30%, 50%, 70%, 90%, and 100% ethanol, respectively) for 15 minutes in each step. The specimens were mounted onto aluminum stubs using carbon adhesive tape and subsequently coated with a thin layer of gold using a sputter coater. Cell imaging was then performed, and images were acquired for analysis (SEM, JEOL, Tokyo, Japan).

Immunofluorescence staining

hDPSCs cultured in four-chamber culture slides were treated with 30 μM of ROCK inhibitor in the culture medium for 72 hours. Untreated hDPSCs served as the control.

To demonstrate the neuronal differentiation ability, the samples were investigated with a neuronal-associated protein marker (βIII-tubulin, Biolegend) at a dilution of 1:1,000. The omission control was performed by adding only the secondary antibody. Fluorescence images were randomly acquired from five areas per specimen using an FV10i-DOC confocal laser scanning microscope. Fluorescence intensity was quantified using ImageJ. Three independent experiments were performed, and the resulting values were averaged.

Intracellular calcium activity

To demonstrate the potential of functional neuronal activity, the depolarization-induced Ca²⁺-evoked response was assessed by intracellular calcium oscillations. The hDPSCs cultured in 24-well plates were treated with/without 30 μM of ROCK inhibitor in the culture medium for 72 hours. The samples were washed with PBS and subsequently incubated with 3 μM Fluo-3 AM (Invitrogen) and 0.08% Pluronic acid (Invitrogen) in αMEM at 37°C for 1 hour. The cells were then washed with PBS and maintained in Tyrode’s solution containing 5 mM KCl, 129 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 30 mM glucose, and 25 mM HEPES (Sigma-Aldrich) until analysis. The undifferentiated cells (hDPSCs) were used as the non-neuronal reference cells. The fluorescent intensity at an excitation wavelength of 506 nm was recorded for 120 seconds after stimulation with 50 mM KCl (Sigma-Aldrich) using the confocal microscope platform STELLARIS 5 (Leica Microsystems, Wetzlar, Germany), and the fluorescent intensity was interpreted with the Leica Application Suite X software (Leica Microsystems).

Statistical analysis

Experiments were replicated three times, and data are shown as mean ± SEM. Comparisons between two groups (untreated and treated hDPSCs or the percentage of motor-like neuronal cells and sensory-like neuronal cells) were assessed by independent-samples t-tests using GraphPad Prism (San Diego, CA, USA). P < 0.05 was regarded as statistically significant.

RESULTS

Characterization of hDPSCs

The isolated hDPSCs were comprehensively characterized to confirm their MSCs properties, including clonogenicity, lineage-specific marker expression, and multilineage differentiation potential. Giemsa staining demonstrated the presence of multiple well-defined colony clusters (Figure 1a). These colonies exhibited a dense, fibroblast-like morphology, indicative of colony-forming unit (CFU) and their self-renewal capacity. To further verify their cellular origin, immunofluorescence staining was performed. The omission control revealed the nuclear localization of hDPSCs (Figure 1b). The hDPSCs showed positive expression of Nestin (green fluorescence) (Figure 1c). The multipotent differentiation capacity of hDPSCs was subsequently assessed under lineage-specific induction conditions. Upon osteogenic induction, the cells exhibited clear evidence of mineralized matrix deposition, as indicated by the formation of calcified nodules (Figure 1d). In parallel, adipogenic differentiation was confirmed by the accumulation of intracellular lipid droplets within the cytoplasm of induced cells (Figure 1e).

Figure 1. Characterization of MSCs properties of isolated hDPSCs. Macroscopic view of Giemsa staining revealed multiple clusters of colonies, indicating their self-renewal ability (black arrows) (a). Negative control without primary antibody, nuclei were counterstained with DAPI (blue) (b). Nestin expression (green) indicated neural crest origin. Nuclei were counterstained with DAPI (blue) (c). Osteogenic differentiation was confirmed by mineralized nodule formation (black arrows) (d). Adipogenic differentiation was demonstrated by the presence of lipid droplets (black arrow) (e). Scale bar: a = 10 mm, b, c, d, and e = 100 μm.

Morphological differentiation towards neuronal-like cells of hDPSCs

To evaluate the inductive effects of ROCK inhibition on morphological differentiation, hDPSCs were treated with 30 μM Y-27632 for 72 hours. The morphological assessment revealed clear differences between untreated and treated cells. Undifferentiated hDPSCs displayed a typical fibroblast-like morphology, characterized by elongated, spindle-shaped cells with pale cytoplasm and minimal cytoplasmic complexity (Figure 2a). In contrast, ROCK inhibitor-treated cells exhibited marked morphological transformation, acquiring features reminiscent of neuronal-like cells (Figures 2b-2d). Specifically, a subset of cells developed a rounded or oval soma with two elongated cytoplasmic processes extending in opposite directions, consistent with bipolar, sensory-like neuronal morphology (Figures 2c-2d, white arrows). Another population displayed more complex morphologies, including pyramidal or multipolar shapes with multiple cytoplasmic extensions, resembling motor-like neuronal phenotypes (Figures 2b-2d, black arrows). These differentiated cells also demonstrated increased cytoplasmic density and structural organization compared to untreated controls.

Figure 2. Phase contrast images showing morphological changes of hDPSCs following ROCK inhibition. Untreated cells exhibited fibroblast-like morphology (black asterisks) (a). Treated cells progressively differentiated into motor-like neuronal cells (black arrows) and sensory-like neuronal cells (white arrows) (b-d). Scale bar: a, b, c, and d = 50 μm.

Cresyl violet staining further supported neuronal-like differentiation. While undifferentiated hDPSCs showed weak or negligible staining, maintained a stellate, fibroblast-like appearance with small cell bodies and elongated processes (Figure 3a). Treated cells exhibited intense purple staining within the cytoplasm, indicative of Nissl substance accumulation. The cells displayed neuronal-like characteristics, multipolar cells exhibited several branching projections, consistent with motor-like neurons. Whereas bipolar cells showed two distinct processes extending from a central soma, characteristic of sensory-like neurons (Figures 3b-3c). The quantitative analysis demonstrated that motor-like neuronal cells accounted for 11.93 ± 0.43% of the population, while sensory-like neuronal cells comprised 10.39 ± 0.81%. Although the proportion of motor-like cells was slightly higher, the difference was not statistically significant (not statistically significant, t-test, n = 3) (Figure 3d). 

Figure 3.  Identification of Nissl substance in neuronal-like cells revealed by Cresyl violet staining. Phase-contrast images show morphological features of undifferentiated hDPSCs (black asterisks) (a). The treated hDPSCs were positively stained with intense purple staining and presented sensory-like neuronal morphology (white arrows) (b) and motor-like neuronal morphology (black arrows) (c). Quantitative analysis of neuronal subtypes is shown as the percentage of sensory and motor neuron-like cells (d). The data are presented as mean ± SEM (ns: not statistically significant, t-test, n = 3). Scale bar: a, b, and c = 100 μm.

Ultrastructure of neuronal-like cells          

SEM was performed to examine the surface morphology and three-dimensional ultrastructural features of hDPSC-derived neuronal-like cells following ROCK inhibition.

Untreated hDPSCs exhibited a flattened, fibroblast-like morphology with multiple thin cytoplasmic extensions that were closely adhered to the culture substrate (Figure 4a). These cells displayed a relatively irregular surface topology, consistent with undifferentiated mesenchymal cells. In contrast, hDPSCs treated with the ROCK inhibitor demonstrated marked ultrastructural alterations (Figures 4b-4d). Treated cells exhibited pronounced soma elevation, smoother plasma membranes, and a transition toward rounded or pyramidal cell bodies, resembling motor neuron-like characteristics. In addition, cells developed elongated processes of variable length and thickness, some of which displayed fine, dendrite-like branching patterns. Notably, a subset of cells adopted bipolar morphologies characterized by two extended processes emerging from opposite poles of the soma, consistent with sensory-like neuronal phenotypes.

Figure 4. SEM reveals three-dimensional morphological changes of hDPSCs following ROCK-induced neuronal-like differentiation. Untreated hDPSCs exhibit fibroblast-like, stellate morphology (black asterisks) (a). Treated hDPSCs display neuronal-like morphology. Black arrows indicate motor-like neuronal cells, and white arrows indicate sensory-like neuronal cells (b-d). Scale bar: a, b = 100 μm; c, d = 50 μm.

Immunofluorescence imaging

After 72 hours of culture in the presence of the ROCK inhibitor Y-27632, hDPSCs exhibited βIII-tubulin immunoreactivity. The omission control presented nuclear localization (Figure 5a). Compared with untreated cells (Figure 5b), hDPSCs treated with 30 μM Y-27632 showed more prominent elongated, βIII-tubulin-positive neurite-like processes (Figure 5c). Quantitative analysis demonstrated a significant increase in βIII-tubulin fluorescence intensity in the Y-27632-treated group compared with the untreated group (Figure 5d).

Figure 5. Immunofluorescence analysis of βIII-tubulin in hDPSCs following Y-27632 treatment. Negative control processed without primary antibody showed no detectable βIII-tubulin immunoreactivity (a). Representative images of untreated (b) and 30 μM Y-27632-treated hDPSCs (c) after 72 hours, respectively. βIII-tubulin immunoreactivity is shown in green, and nuclei were counterstained with DAPI (blue). Quantitative analysis demonstrated significantly increased βIII-tubulin fluorescence intensity following Y-27632 treatment (d). Data are presented as mean ± SEM (***P <0.001, t-test, n = 3). Scale bars: a, b, and c = 100 μm.

The potential functional neuronal activity

To assess the potential functional activity of differentiated cells, intracellular Ca²⁺ responses to KCl stimulation were examined. After treatment with 30 μM Y-27632 for 72 hours, Y-27632-treated hDPSCs exhibited higher Ca²⁺ fluorescence intensity than untreated cells and exhibited a rapid increase in fluorescence intensity, indicating Ca²⁺ influx following KCl stimulation. In contrast, untreated hDPSCs showed relatively modest dynamic changes in Ca²⁺ fluorescence (Figure 6).

Figure 6. Intracellular Ca²⁺ oscillations in differentiated cells following treatment with ROCK inhibitors. Cells treated with 30 μM Y-27632 for 72 hours showed a rapid and sustained increase in Ca²⁺ fluorescence intensity (pink lines), whereas untreated cells exhibited lower responses with modest fluctuations (blue lines). Representative traces from three measurements per group are shown.

DISCUSSION

Neuronal morphology is closely associated with functional specialization, and alterations in neuronal structure can contribute to neurological dysfunction. In this context, hDPSCs represent a promising cell source for neural regenerative applications because of their capacity to differentiate into neuronal cells and secrete neurotrophic factors. Previous studies have demonstrated that the ROCK inhibitor, Y-27632, promotes neuronal differentiation in various stem cell types, as evidenced by changes in the expression of neurogenesis-associated proteins and genes. In addition, the ROCK inhibitor has been reported to exert no significant cytotoxic effects on hDPSCs at concentrations of 10-50 μM (Srikawnawan et al., 2022). Accordingly, hDPSCs in the present study were treated with 30 μM Y-27632 for 72 hours, representing an intermediate concentration within this previously evaluated range. However, despite evidence of neuronal differentiation at the molecular level, the specific morphological phenotypes associated with ROCK inhibition remain incompletely characterized. The present study extends these molecular findings by demonstrating that Y-27632 induces distinct bipolar and multipolar neuron-like phenotypes in hDPSCs, thereby providing more specific morphological evidence of ROCK inhibitor-mediated neuronal differentiation. To our knowledge, this is the first study to characterize these distinct neuronal-like phenotypes in Y-27632-treated hDPSCs.

According to the International Society for Cellular Therapy (ISCT) criteria for defining MSCs, which include plastic adherence, tissue origin, and the ability to differentiate into multiple lineages (Wright et al., 2021), the isolated hDPSCs exhibited typical MSCs properties. Giemsa staining revealed multiple well-defined colony clusters displaying a dense, fibroblast-like morphology, consistent with colony-forming unit characteristics, a hallmark of MSCs. The presence of discrete colonies indicates successful isolation and expansion of progenitor cells with self-renewal capacity (Pilbauerova et al., 2019). Immunofluorescence analysis was performed to further assess cellular origin. The hDPSCs showed positive Nestin expression, a marker associated with neural crest–derived cells, supporting their ectomesenchymal origin (Songsaad et al., 2020). The multilineage differentiation potential of hDPSCs was evaluated under lineage-specific induction conditions. Osteogenic induction resulted in the formation of mineralized nodules, indicating matrix mineralization. Similarly, adipogenic differentiation was confirmed by the presence of intracellular lipid droplets within the cytoplasm, reflecting adipocyte-like cell development and demonstrating mesodermal differentiation capacity (Lei et al., 2014). Collectively, these findings confirm that the isolated hDPSCs exhibit key MSCs characteristics.

Our study demonstrated substantial morphological and ultrastructural remodeling of hDPSCs following ROCK inhibitor. SEM revealed prominent soma protrusion, smoother plasma membranes, and elongated neurite-like processes with dendrite-like branching, resembling morphological changes reported during neuronal differentiation of embryonic and induced pluripotent stem cells (Mumaw et al., 2010; Harberts et al., 2021). Cresyl violet staining revealed Nissl-like cytoplasmic granules, reflecting increased rough endoplasmic reticulum and ribosomal activity associated with the protein-synthetic demands of neuronal cells (Darvishi et al., 2021). Notably, Y-27632-treated hDPSCs exhibited distinct bipolar and multipolar neuron-like morphologies, which are commonly associated with sensory neurons and motor neurons or interneurons, respectively (Fan et al., 2025; Kussick et al., 2025). Collectively, these structural features support Y-27632-induced cytoskeletal remodeling and the acquisition of neuronal-like phenotypes. However, morphological characteristics alone are insufficient to establish specific neuronal subtype identities, which require confirmation using subtype-specific molecular markers and neuronal function analysis.

Neuronal differentiation was further assessed by immunofluorescence staining of βIII-tubulin, a neuron-enriched cytoskeletal protein and neuronal marker (Thongsuk et al., 2025). Following ROCK inhibitor treatment, hDPSCs exhibited βIII-tubulin-positive staining along elongated neurite-like processes. Mechanistically, ROCK inhibitor can reduce actomyosin contractility, thereby promoting cytoskeletal reorganization and neurite extension. (Amano et al., 2010; Gonmanee et al., 2020). ROCK inhibitor also involves PI3K/Akt signaling, which supports neuronal survival and neurite extension (Kamishibahara et al., 2016). Together, these mechanisms may contribute to the acquisition of neuronal-like phenotypes in hDPSCs. However, further investigation is required to confirm the underlying signaling pathways.

Importantly, the morphological and molecular changes induced by ROCK inhibition were accompanied by altered intracellular Ca²⁺ dynamics, providing additional evidence of neuronal-like functional responsiveness. Intracellular Ca²⁺ signaling is essential for neuronal excitability, signal transduction, and neurotransmitter release (Lee et al., 2012; Gonmanee et al., 2018), and KCl-induced depolarization is commonly used to assess Ca²⁺ responsiveness (Radotić et al., 2018). In the present study, KCl stimulation elicited a rapid increase in Ca²⁺ intensity in Y-27632-treated hDPSCs, whereas untreated cells showed lower responses with relatively modest fluctuations. Similar increases in intracellular Ca²⁺ have been reported in neuronally differentiated hDPSCs following co-culture with auditory brainstem slices and ergothioneine-associated induction (Gonmanee et al., 2020; Thongsuk et al., 2025). These findings suggest that enhanced Ca²⁺ responsiveness represents a functional feature accompanying neuronal-like differentiation of hDPSCs.

Nevertheless, several limitations of the present study should be acknowledged. Although the combination of neuronal-like morphology, βIII-tubulin expression, and KCl-evoked intracellular Ca²⁺ responses provides complementary evidence supporting neuronal-like differentiation, the functional maturity of the differentiated cells was not directly assessed by electrophysiological recordings, such as membrane potential or action potential measurements. In addition, the classification of bipolar sensory-like and multipolar motor-like cells was based primarily on morphological characteristics and was not confirmed using neuronal subtype-specific molecular markers. Therefore, these classifications should be interpreted as morphological phenotypes rather than definitive neuronal subtype identities. Future studies incorporating electrophysiological recordings and subtype-specific neuronal markers are required to further determine the functional maturity and neuronal identity of Y-27632-treated hDPSCs.

The concurrent appearance of neuronal morphology, neuronal-associated proteins, and enhanced KCl-responsive Ca²⁺ signals further suggests that ROCK inhibition promotes coordinated structural and functional remodeling rather than morphological changes alone. These findings may reflect an interplay between ROCK-dependent cytoskeletal reorganization and Ca²⁺ signaling, both of which contribute to neuronal differentiation and function.

CONCLUSION

This study demonstrates that ROCK inhibition by Y-27632 promotes neuronal-like differentiation of hDPSCs, as evidenced by distinct bipolar and multipolar neuron-like morphologies and βIII-tubulin expression. Furthermore, the enhanced intracellular Ca²⁺ response to KCl stimulation provides functional evidence supporting the acquisition of neuronal-like properties. Collectively, these findings indicate that ROCK inhibition promotes both morphological and functional changes associated with neuronal differentiation in hDPSCs. Y-27632-treated hDPSCs may therefore provide a useful in vitro model for investigating neuronal differentiation, cytoskeletal remodeling, and intracellular Ca²⁺ dynamics, with potential relevance to future neural regeneration research.

ACKNOWLEDGEMENTS

The authors would like to thank the Faculty of Dentistry, Mahidol University, and the Faculty of Medicine, Kasetsart University, Thailand, for providing laboratory facilities and technical support essential to this study.

AUTHOR CONTRIBUTIONS

Wittawas Srikawnawan: Conceptualization (Equal), Data Curation (Lead), Formal Analysis (Equal), Investigation (Lead), Methodology (Lead), Visualization (Lead), Funding Acquisition (Lead), Validation (Equal), Writing – Original Draft (Lead), Writing – Review & Editing (Lead); Parichaya Piromrat: Validation (Equal), Writing – Review & Editing (Supporting); Supanat Lumbikananda: Validation (Equal), Writing– Review & Editing (Supporting); Charoensri Thonabulsombat: Resources (Equal), Supervision (Supporting); Nisarat Ruangsawasdi: Resources (Equal), Supervision (Supporting); Anupong Thongklam Songsaad: Conceptualization (Equal), Formal Analysis (Equal), Funding Acquisition (Supporting), Project Administration (Lead), Resources (Equal), Supervision (Lead), Validation (Equal), Writing – Review & Editing (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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