Research ArticleExperimental Studies
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5-Azacytidine Enhances Differentiation of Human Placenta-derived Mesenchymal Stem Cells Towards Schwann-like Cells

AREECHUN SOTTHIBUNDHU, WILASINEE PROMJANTUEK, PHONGSAKORN KUNHORM, SITAKAN NATPHOPSUK, NIPHA CHAICHAROENAUDOMRUNG and PARINYA NOISA
In Vivo January 2026, 40 (1) 235-248; DOI: https://doi.org/10.21873/invivo.14187

Abstract

Background/Aim: Mesenchymal stem cells (MSCs) can be used for regenerative medicine, particularly in the treatment of neurodegenerative diseases and peripheral nerve injuries. Among these, human placenta-derived mesenchymal stem cells (hPMSCs) possess a high potential for differentiation into various cell types, including those of the neural lineage under appropriate conditions. The primary aim of this study was to isolate and induce the differentiation of hPMSCs into Schwann cell-like cells (SC-like cells) that function to support and play a role in the peripheral nervous system.

Materials and Methods: Human placental tissues were isolated, and hPMSCs were cultured and characterized. The isolated hPMSCs were positive for mesenchymal stem cell surface markers CD73, CD90, and CD105. The multipotency of hPMSCs was confirmed by their ability to differentiate into osteocytes, chondrocytes, and adipocytes. To induce Schwann cell differentiation, hPMSCs were cultured in Schwann cell differentiation medium with or without the addition of 5-azacytidine (5-aza). Gene and protein expression analyses were performed to assess Schwann cell-specific markers.

Results: After 14 days of induction, hPMSCs differentiated in Schwann cell differentiation medium showed significant upregulation of Schwann cell-specific genes S100β, P75, GFAP, and PMP22 (p<0.01). Interestingly, the group treated with 5-aza exhibited even higher expression levels of these genes compared to the Schwann cell differentiation medium alone and the control group (p<0.01). Furthermore, protein expression analysis demonstrated that glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were highly expressed in the 5-aza-treated condition, confirming enhanced Schwann cell-like differentiation.

Conclusion: This study demonstrates that hPMSCs can be successfully differentiated into Schwann cell-like cells. The addition of 5-aza significantly promoted this differentiation, leading to higher expression of Schwann cell-specific genes and proteins. These findings suggest that 5-aza plays a supportive role in enhancing Schwann cell differentiation from hPMSCs and may provide a promising approach for future clinical applications in peripheral nerve regeneration.

Keywords:

Introduction

Mesenchymal stem cells (MSCs) possess the capability to differentiate into various cell types, including neurons and glial cells within the nervous system (1). Consequently, understanding the mechanisms that drive MSC differentiation into specific neuronal lineages is crucial for developing cell-based therapies for neurological disorders. This is particularly relevant in conditions involving inflammation of the spinal cord and peripheral nerves, where current treatment options remain inadequate for restoring normal function (2).

Schwann cells play a critical role in the regeneration of peripheral nerve injuries (PNI). As a type of glial cell, they are responsible for producing myelin, which insulates nerve fibers and creates an environment conducive to nerve repair. Beyond their regenerative capacity, Schwann cells are essential for maintaining the function of peripheral nerves and supporting the normal development of the nervous system (3, 4). In contrast, oligodendrocytes perform a similar role in the central nervous system (CNS) by myelinating axons within the brain and spinal cord (5). Given their therapeutic potential, strategies for Schwann cell acquisition have been explored as a means to enhance nerve regeneration (6). Human placental mesenchymal stem cells (hPMSCs) have emerged as promising precursor cells capable of differentiating into Schwann cells, offering a potential avenue for treating nerve-related conditions (7).

PNI are primarily caused by genetic factors or accidental trauma, often leading to significant pain and social complications for affected individuals (8). The current gold-standard surgical treatment for such injuries is nerve autografting, which involves the autologous transplantation of the patient’s own nerve tissue to bridge defects in damaged nerves (9). However, this approach presents several limitations, including the potential need for additional surgical interventions, the risk of neuroma formation, and the challenge of identifying suitable donor nerves (10). Moreover, nerve autografting does not fully restore neuronal function (11). Emerging research underscores the critical role of cellular processes in peripheral nerve regeneration. Effective rehabilitation of PNI relies on cellular mechanisms that support neuronal function (12). Numerous studies have demonstrated that Schwann cell transplantation can significantly enhance motor neuron recovery following spinal cord injury (13-16). Schwann cells are pivotal in promoting axonal regeneration by remyelinating damaged neurons and preserving their electrical conductivity, ultimately contributing to improved clinical outcomes (17). Building upon previous research, various sources of MSCs have been identified as potential precursors for Schwann cell differentiation, including MSCs derived from the spinal cord, amniotic membrane, adipose tissue, and other organ-specific stem cells from the patient (18, 19). This study aims to develop a more efficient protocol for differentiating human placental mesenchymal stem cells (hPMSCs) into Schwann cells and to establish a method for evaluating their functional capacity in myelin formation. The findings will provide crucial insights for future preclinical trials exploring the therapeutic potential of Schwann cell transplantation, particularly for patients with spinal cord inflammation and related neurodegenerative conditions.

Furthermore, this study aims to examine the regulatory mechanisms of transcription factors involved in protein expression, as well as the key signaling pathways driving the transformation of mesenchymal stem cells into Schwann cells. These insights will enhance the understanding of Schwann cell development and its applications in regenerative medicine.

Materials and Methods

Methodology and study site. Human placental tissues were collected from healthy donors with written informed consent from the Suranaree University of Technology Hospital (SUTH, Nakhon Ratchasima, Thailand). The protocol was approved by the ethics committee of Suranaree University of Technology. The experimental process including isolated stem cells from human placenta tissues and differentiation into Schwann cells, as well as the analysis of cellular and molecular biological properties was performed by The Cells Base Assay and Innovation Laboratory (CBAI) at Suranaree University of Technology.

Isolation and cell culture of mesenchymal stem cells derived from placental tissues. The human placental tissues underwent isolation and culturing following the protocols outlined in a previous study, with some adjustments (20). In brief, the placental tissue was minced into small fragments and incubated in a dish containing 4 mg/ml collagenase/dispase (Roche, Germany) for digestion at 37°C for 1 hour. After digestion, the tissues were cultured in a medium composed of Dulbecco’s Modified Eagle Medium high glucose (DMEM/HG; Hyclone, Logan, UT, USA), supplemented with 20% (v/v) fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA), 1 mM L-glutamine, 1 mM Minimal essential medium (MEM; Sigma-Aldrich, St. Louis, MO, USA), 100 U/ml Penicillin, and 100 g/ml Streptomycin (Sigma-Aldrich). The human placental mesenchymal stem cells (hPMSCs) were maintained at 37°C in a 5% CO2 environment for 7 to 14 days. The medium was refreshed every 3 days until fibroblast-like cells migrated out from the placental tissues. Upon reaching 90-100% confluence, the hPMSCs were passaged, and three independent lines of hPMSCs were utilized for all experiments. To determine whether the cultured cells obtained were mesenchymal stem cells, immunocytochemistry was performed and the ability to differentiate into various cell types was assessed.

Expression of specific genes and proteins in hPMSCs. hPMSCs can be identified by the expression of specific proteins, such as CD73, CD90, and CD105, which are specific proteins on the cell surface of mesenchymal stem cells derived from placental tissues. The protein expression was confirmed by immunocytochemistry. First, cells were cultured on glass coverslips until they grew to approximately 80-90% confluence. Then, cells were washed with PBS three times and fixed with 4% PFA for 15-20 min. After that, cells were incubated in blocking solution containing 2% bovine serum albumin (BSA) and 1% TritonX100 in PBS for 30 minutes. Primary antibodies against CD73 (#MABD122), CD90 (#ZRB1285), and CD105 (#MABT117) from MERK, Millipore were added at a dilution of 1:1000, and cells were incubated at 4°C overnight. They were then incubated with secondary antibodies, donkey anti-rabbit and goat anti-mouse (#A-31572 and #A-11005), from Thermo Fisher Scientific for 1.5 h, washed with PBS and stained with DAPI, and then examined under a fluorescence microscope.

Capability of mesenchymal stem cells derived from placental tissues to differentiate into different cell types. hPMSCs have the ability to differentiate into various cell types, particularly cells in the middle layer. To test this characteristic, stem cells derived from placental tissues were cultured in a medium containing a specific inducer for the development of adipose cells, bone cells and cartilage cells for 14 days as follows. For the induction of hPMSCs into bone cells, hPMSCs were cultured in an induction solution containing DMEM high glucose, 10% FBS, 100 nM dexamethasone, 0.2 mM L-ascorbate-2-phosphate, and 10 mM β-glycerophosphate (Sigma-Aldrich). The solution was changed every 3 days for a total of 21 days. At maturity, cells were fixed in 4% paraformaldehyde (PFA) for 15 min and stained with Alizarin Red (Sigma-Aldrich) to detect bone mineralization.

To induce of hPMSCs into cartilage cells, hPMSCs stem cells were cultured with an induction solution containing the premix ITS-plus (BD Biosciences, San Jose, CA, USA) at a concentration of 6.25 μg/ml transferrin and 6.25 ng/ml selenious acid, supplemented with 50 mg ascorbate 2-phosphate (A8960), 40 μg/ml L-proline (P0380), 100 μg/ml sodium pyruvate (P5280), 100 nM dexamethasone (D4902), and 10 ng/ml of TGF-β3 (939250) (all from Sigma-Aldrich, Burlington, MA, USA). After 21 days, cells were fixed with 4% paraformaldehyde (PFA) for 15 min, and the cartilage cells were stained with Alcian Blue. For the induction of hPMSCs into adipose cells, hPMSCs were cultured in 10 μg/ml insulin, 60 μM indomethacin, 0.5 μM hydrocortisone and 0.5 mM isobutyl methylxanthine (IBMX) for 21 days. The lipid oil droplet cells were then stained with Alizarin red (all from Sigma-Aldrich).

Transformation of hPMSCs into Schwann-like cells. hPMSCs were cultured in a solution known to effectively induce differentiation into Schwann-like cells. Additionally, 5-azacytidine (5-aza), a chemical that induces epigenetic modifications affecting DNA, was introduced to enhance the efficiency of the differentiation process.

Development of a method for induction of mesenchymal stem cells derived from placental tissue into Schwann-like cells. Placenta-derived mesenchymal stem cells can be induced to differentiate into Schwann cells, depending on three key factors: (1) the induction method—chemical, recombinant, or genetic; (2) the culture surface coating that supports cell adhesion and growth; and (3) the medium formulation that promotes Schwann cell differentiation. hPMSCs with 50-60% cell densities were grown in DMEM High-glucose, 10% FBS, and 2 mM L-glutamine as base components. After the cell confluence reached 70-80%, pre-induction was performed with 1 mM beta mercaptoethanol (BME) in DMEM high-glucose, 10% FBS, and L-glutamine 2 mM for 24 h. The medium was then changed to be 10 μM RA in DMEM high-glucose, 10% FBS, L-glutamine 2 mM. After pre-induction, the induction of mesenchymal stem cells derived from placental tissue into Schwann-like cells was initiated, by rinsing the cells with PBS and changing the culture medium solution to DMEM high-glucose (11965092), 2% FBS (A5670701), L-glutamine 2 mM 25030081, Platelet-derived Growth factor (PDGF-AA) 5 ng/ml (100-13A-10UG), basic fibroblast growth factor (BFGF) 10 ng/ml (100-18B-50UG) (all from Thermo Fisher Scientific), Forskolin 10 μM (F6886) and human Heregulin β-1 200 ng/ml (SRP3055) (all from Sigma-Aldrich), changing the medium solution every 3 days. Additionally, we tested the effectiveness of using epigenetic agents, such as 5-aza, at a concentration of 10 μM, to enhance the differentiation of mesenchymal stem cells derived from placental tissue into Schwann cells.

Evaluation of Schwann cell gene expression using qRT-PCR. The induced Schwann cells can be verified by the expression of specific genes. To prove that these experimental cells have similar properties to real Schwann cells, then the expression of specific genes and proteins was examined both before and after induction. The cells obtained after induction were examined for the expression of Schwann cell-related genes and proteins to confirm the efficiency of the induction method. The quantitative real-time polymerase chain reaction (q-RT PCR) technique was used to verify the expression levels of specific genes in Schwann cells, such as GFAP, NESTIN, S-100β, P75, and SOX10. RNA from cells at different stages of induction was separated and converted into complementary DNA (cDNA) using reverse transcription. The obtained cDNA was then examined for the expression level of the specific genes by PCR using gene-specific primers as shown in Table I. The protein expression levels were confirmed by immunocytochemistry, staining cells with specific antibodies like GFAP and MBP, and examining them under a microscope.

View this table:
Table I.

Primers used in this study for gene expression analysis.

Verification of Schwann cell-specific protein expression and myelin production. The formation of myelin proteins from mesenchymal stem cells was being studied from placental tissue at a density of 5×102 were cultured on a nanofiber chamber slide for 14 days. At maturity, the ability to wrap nanofiber was observed under a SEM microscope.

Statistical analysis. The experimental data were analyzed using GraphPad Prism v5.0 (GraphPad Software, San Diego, CA, USA). Results are presented as mean ± standard deviation (SD). Statistical comparisons were performed using one-way ANOVA, followed by Tukey’s post hoc test. A p-value of <0.05 was considered statistically significant, while p<0.01 indicated a highly significant difference.

Results

Cell separation and examination of hPMSCs. After collecting the placental samples, extracting the stem cells and incubating them in a CO2 cabinet at 37°C, it was found that on the third day, The hPMSCs were observed to migrate out of the tissue and proliferate over time, reaching a cell density of approximately 90-100% by the fourteenth day. The hPMSCs were then subcultured with 0.025% trypsin in DPBS. After subculture, the hPMSCs were transferred to a new cell culture dish at a density of 104 cells per cm2 as passage 1. Afterwards, the cells were expanded and cryopreserved for subsequent characterization of mesenchymal properties and induction into Schwann cells.

To study hPMSCs properties, the expression of specific proteins on the cell surface was investigated. hPMSCs can be identified by the expression of the specific cell surface proteins CD73, CD90 and CD105 (Figure 1). These proteins were tested using immunocytochemistry. It was found that hPMSCs can express three mesenchymal progenitor-specific proteins, i.e., CD73, CD90 and CD105 (Figure 1A). Another feature used to confirm they were MSCs was the ability to differentiate into different types of cells, especially cells in the middle tissue layer. Therefore, hPMSCs from placenta were cultured with specific induction conditions for the development of fat, bone and cartilage cells for 14 days. Oil Red O was used to stain fat cells, Alizarin Red to stain bone cells, and Alcian Blue to stain cartilage cells (Figure 1B). The hPMSCs were able to exhibit mesenchymal properties and differentiate into the above (Figure 1B). From the above results, it can be shown that the hPMSCs in this experiment have mesenchymal properties.

Figure 1.
Figure 1.

Properties of human placenta-derived mesenchymal stem cells (hPMSCs) obtained from human placenta tissue. (A) Staining for cell surface proteins specific to mesenchymal cells, namely CD73, CD90, and CD105. (B) Human placenta-derived mesenchymal stem cells differentiated towards bone cells (stained with Alizarin red), cartilage cells (stained with Alcian Blue), and fat cells (stained with Oil Red O).

Transformation of hPMSCs into Schwann cell-like cells. hPMSCs at 60-80% density were grown in DMEM high glucose solution containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml Streptomycin. The culture medium was then replaced with an induction medium previously described by Dezawa et al. (21) to promote Schwann cell differentiation. The hPMSCs exhibited morphological and phenotypic characteristics of Schwann-like cells compared to the control group (Figure 2). Furthermore, to enhance the efficiency of Schwann cell differentiation, the study investigated the effects of 5-aza, a compound known to induce epigenetic modifications. The induction efficiency was evaluated by comparing hPMSCs treated with 5-aza to those subjected to standard induction conditions and the control group in DMEM supplemented with 10% FBS (Figure 2).

Figure 2.
Figure 2.

Induction of human placenta-derived mesenchymal stem cells (hPMSCs) into Schwann cells. (A-C) Human placenta-derived mesenchymal stem cells after pre-induction with retinoic acid (RA) for 3 days. (D) Human placenta-derived mesenchymal stem cells after 14 days of induction in the control media. (E) Human placenta-derived mesenchymal stem cells induced towards Schwann cell-like cells with the inducing agent without 5-azacytidine (5-aza), and (F) the morphology of Schwann cells after induction with the inducing agent supplemented with 5-aza. (H-J) For the process length, the percentage of cells, and the bipolar index of Schwann cell-like cells, we compared 5-aza-treated group with the control (Student’s t-test, *significantly different at p=0.01).

Enhancement of induction of hPMSCs into Schwann-like cells by addition of 5-aza. After 14 days of induction with Schwann cell induction solution, Schwann-like cells from hPMSCs were found. After being induced with a Schwann cell induction reagent kit with the addition of 10 μM 5-aza, more cells were found to have transformed into a Schwann cell-like shape. The nucleus was enlarged and exhibited bipolar fusiform, characteristic of a Schwann cell shape. The same was found for the normal Schwann cell induction kit without 5-aza addition. Controls within DMEM high glucose (10% FBS) retained normal spindle-shaped cells without transformation into Schwann cells. After 14 days of induction, qRT-PCR was used to determine the expression of Schwann cell-related genes: S100β, P75, GFAP and PMP22. In the Schwann cell induction group with the epigenetic additive 5-aza, the expression of the Schwann cell-related genes S100β and P75 was significantly higher than the normal Schwann cell induction group and the control group (p<0.001). In addition, it was found that the glial fibrillary acidic protein (GFAP)-associated cells of the 5-aza-treated group had significantly higher expression (p <0.001) than the control group (p<0.001). Peripheral myelin protein (PMP22) gene was higher than the normal Schwann cell induction group (p<0.01) and the control group (p<0.001) (Figure 3).

Figure 3.
Figure 3.

Expression of neuronal and Schwann cell-related genes after 14 days of the induction of human placental-derived mesenchymal stem cells (hPMSCs) (one-way ANOVA, *significantly different at p=0.01, **significantly different at p=0.05).

To test whether the induced cells were neurons, the expression of neuronal related genes, SOX2 and NESTIN, were measured by qRT-PCR. The expression of both SOX2 and NESTIN genes in the normal Schwann cell induction group was significantly decreased compared to the control group (p<0.001). When 5-aza was added to the Schwann cell induction solution, the expression of NESTIN, a pro-neural gene, was significantly lower compared with the normal Schwann cell induction group (p<0.001) (Figure 3).

Expression of Schwann cell-associated proteins by immunocytochemistry. After 14 days of induction, coverslips with cultured cells were stained with Schwann cell-specific antibodies to determine the expression of GFAP and MBP. Schwann cell inducers with the epigenetic addition 5-aza had higher expression of Schwann cell-related proteins GFAP and MBP than normal and control Schwann cells. The experimental results demonstrated that the expression of Schwann cell-related genes and proteins indicated that hPMSCs can differentiate into Schwann cells when cultured in an induction medium under optimal conditions. In addition, the group with 5-aza added to the Schwann cell induction solution had higher expression of both Schwann cell-related genes and proteins than the induction group (Figure 4).

Figure 4.
Figure 4.
Figure 4.

Expression of neuronal and Schwann cell-related markers GFAP and MBP was observed 14 days after inducing human placental-derived mesenchymal stem cells (hPMSCs) to differentiate into Schwann-like cells.

In addition, Schwann cells were induced from hPMSCs using conventional 2D and 3D culture in a nanofiber chamber slide for 14 days, after which they were examined under a scanning electron microscope (SEM). Based on the SEM imaging results, the hPMSCs induced with 5-aza exhibited morphological characteristics of Schwann cells. hPMSCs after induction into Schwann-like cells were large and had protruding nuclei that resembled a reticular protrusion from the cell body. The induced Schwann cells in the 3D culture demonstrated Schwann cell-like phenotype through wrapping with nanofibers (Figure 5).

Figure 5.
Figure 5.

Scanning electron microscope (SEM) images in a group of stem cell induction solutions from human placental-derived mesenchymal stem cells (hPMSCs) to Schwann-like cells by adding 5-azacytidine and culturing in (A) a 2D culture system and (B) a 3D culture format on a nanofiber chamber slide.

Discussion

hPMSCs exhibit strong potential to differentiate into various cell types, including glial cells, similar to dental pulp-derived stem cells, which express neural crest markers and can form osteogenic, melanocytic, and Schwann cell lineages under defined induction conditions (22). Several studies have demonstrated successful differentiation of hPMSCs into the neural and lineage under specific conditions (23, 24). Consequently, the neural differentiation of hPMSCs holds promise as a cell-based therapy for neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s disease, and peripheral nerve injury. In our study, hPMSCs cultured in our laboratory demonstrated a robust expression of MSC markers, including CD73, CD90, and CD105, along with multipotent differentiation capabilities such as adipogenesis, osteogenesis, and chondrogenesis, consistent with findings reported for other MSCs. A significant discovery in this investigation is the myelin-forming ability observed in Schwann cell-like cells derived from hPMSCs, which represents a crucial function of Schwann cells.

Notably, the addition of 5-aza significantly enhanced the differentiation and myelination processes, aligning with previous findings that hPMSCs upregulate neuronal genes such as S100β, p75, and GFAP, with even higher expression observed when 5-aza is co-supplemented in the differentiation culture medium (25). Peripheral nerve injury poses a widespread challenge globally, leading to long-term morbidity, disability, and economic burden. Various factors, including trauma, surgery, cancer, and anesthesia injections, can contribute to peripheral nerve injury (26). Schwann cells, a type of glial cell in the peripheral nervous system, hold promise as agents for promoting peripheral nerve regeneration and functional recovery. These cells, which encompass nerve fiber axons in the peripheral nervous system, produce neurotrophic factors, extracellular matrix molecules, and integrins, offering trophic guidance and structural support essential for axon regeneration (27).

The hPMSCs we obtained had typical characteristic immunophenotyping expressions which were positive for markers common to MSCs and negative for the hematopoietic surface markers (Figure 1). Following induction, the undifferentiated hPMSCs gradually transformed into morphologically distinct Schwann-like cells over time (Figure 2). Upon induction using the Schwann cell induction kit, noteworthy morphological transformations were observed, including a shift from a spindle shape to one characterized by a larger nucleus and a branched or reticular cytoplasm. Additionally, the incorporation of the 5-aza DNA demethylating agent, aimed at mitigating DNA methylation, yielded a further augmentation in the expression of Schwann cell-related genes, including S100β, P75, GFAP, PMP22, and Krox-20. Moreover, the expression levels of the neuronal-associated proteins NESTIN and SOX2 were notably diminished in the inducible normal Schwann cell group in comparison to the control group.

Furthermore, there was a significant elevation in the expression levels of Schwann cell-related proteins including S100β, GFAP, P75, PMP22, and Krox-20, which are expressed at both the transcriptional and translational levels (Figure 3). Remarkably, the highest expression of all these markers was observed on day 14. Transcription factors such as Krox-20, known to be essential during myelination, served as markers for identifying Schwann-like cells in recent studies (28, 29). Notably, our analysis of the inducing reagents used indicated a low probability of inducing neural lineage cells. In addition to glial markers, we also detected the expression of NESTIN and SOX2 in differentiated hPMSCs. The expression of PMP22 and Krox-20 revealed that hPMSCs began to develop into Schwann-like cells 14 days after induction. The expression of S100, GFAP, and P75, on day 14, suggested that the hPMSCs were induced into Schwann-like cells (30).

Although immunofluorescence and qRT-PCR results indicated that hPMSCs differentiated into Schwann-like cells, they did not imply that these cells had the same biological functions as mature Schwann cells. The induction of Schwann cells in a 3D culture system can facilitate the myelination process through their interaction and wrapping around nanofibers. Significantly, induction of hPMSCs into Schwann cells in both the Schwann cell induction and 5-aza addition groups results in a discernible reduction in neural activity, congruent with the upregulation of Schwann cell-specific proteins GFAP, PMP22 and Krox-20, which are indicative of myelin encapsulation proteins (30). These findings underscore the pivotal role of 5-aza in enhancing the induction of hPMSCs into Schwann-like cells, as underscored by the observed patterns in gene and protein expression.

Conclusion

The results of this study demonstrate that hPMSCs isolated from placental tissue possess mesenchymal stem cell characteristics, including proliferation, multilineage differentiation, and expression of CD73, CD90, and CD105. When induced toward Schwann cells, hPMSCs exhibited morphological changes consistent with Schwann cell features and significantly increased expression of Schwann cell-related markers (S100β, P75, GFAP, and PMP22). The addition of the DNA demethylating agent 5-aza further enhanced the expression of these Schwann cell-specific genes and proteins (GFAP and MBP), indicating its key role in promoting Schwann cell differentiation. Overall, 5-aza effectively facilitates the conversion of hPMSCs into Schwann cell-like cells, providing a potential strategy for peripheral nerve regeneration.

Acknowledgements

This research was funded by the Suranaree University of Technology (SUT) Research and Development Fund.

Footnotes

  • Authors’ Contributions

    Areechun Sotthibundhu: provided the concept and design of the study, performed experiments, analyzed and interpreted data, and prepared the article for publication Wilasinee Promjantuek: performed experiments, collected and analyzed data. Phongsakorn Kunhorm: performed experiments, collected and analyzed data. Sitakan Natphopsuk: analyzed and interpreted data. Nipha Chaicharoenaudomrung: analyzed and interpreted the data and prepared the article for publication. Parinya Noisa: provided the concept and design of the study, analyzed and interpreted data, wrote the article, prepared the article for publication, and gave the final publication approval of the article.

  • Conflicts of Interest

    There are no conflicts of interest.

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received September 18, 2025.
  • Revision received October 24, 2025.
  • Accepted November 20, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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5-Azacytidine Enhances Differentiation of Human Placenta-derived Mesenchymal Stem Cells Towards Schwann-like Cells
AREECHUN SOTTHIBUNDHU, WILASINEE PROMJANTUEK, PHONGSAKORN KUNHORM, SITAKAN NATPHOPSUK, NIPHA CHAICHAROENAUDOMRUNG, PARINYA NOISA
In Vivo Jan 2026, 40 (1) 235-248; DOI: 10.21873/invivo.14187
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