Neural crest-derived mesenchymal stem cells: Fates and perspectives

Abstract

Neural crest-derived mesenchymal stem cells (NC-MSCs) represent a unique population with remarkable regenerative potential, owing to their embryonic origin and exceptional differentiation capacity. These cells demonstrate superior performance in neural and craniofacial tissue regeneration compared to conventional mesenchymal stem cells, with dental stem cells emerging as particularly promising candidates for clinical applications in periodontics and endodontics. Despite their therapeutic promise, adult NC-MSCs face significant challenges including donor site limitations, cellular heterogeneity, and scalability issues. Recent advances in pluripotent stem cell offer potential solutions through the generation of NC-MSCs in vitro, though safety concerns regarding tumorigenicity and long-term stability remain to be addressed through comprehensive preclinical studies. This review provides a comprehensive analysis of NC-MSC biology, highlighting their developmental origins, molecular characteristics, and current applications in regenerative medicine. We critically evaluate existing challenges and future directions, emphasizing the need for standardized protocols, improved characterization methods, and rigorous preclinical evaluation to facilitate clinical translation and therapeutic implementation.

Key Words: Neural crest; Mesenchymal stem cell; Development; Pluripotent stem cell; Clinical trials

Core Tip: Recent studies have revealed the distribution and functions of neural crest (NC)-derived mesenchymal stem cells (MSCs) in various tissues, with regenerative therapies based on these cells showing positive results in research and clinical practice. Future research focuses on optimizing therapeutic strategies (timing, dosage), expanding clinical applications, and addressing current challenges. Several approaches aim to solve issues like insufficient cell numbers, heterogeneity, and trauma. Notably, MSCs differentiated from pluripotent stem cells via NC pathways show promise as a stable NC-derived MSC source, potentially overcoming current limitations in clinical applications.

INTRODUCTION

The neural crest (NC) is a highly migratory cell population derived from the neuroectoderm during embryonic development, capable of acquiring diverse cell fates[1-3]. NC cells (NCCs) undergo epithelial-mesenchymal transition (EMT), migrate to various tissues throughout the body, and differentiate into multiple cell types, including neurons, Schwann cells, and melanocytes, as well as specific mesenchymal derivatives such as craniofacial osteoblasts, chondrocytes, dental mesenchymal cells, and peripheral nerve-associated mesenchymal cells[4-6]. The mesenchymal stem cell (MSC)-like populations derived from the NC are collectively referred to as NC-derived MSCs (NC-MSCs).

In recent years, NC-MSCs have garnered increasing attention in the field of regenerative medicine due to their remarkable differentiation potential and regenerative capacity. In particular, studies have demonstrated the unique advantages of dental stem cells (DSCs), craniofacial bone stem cells, and peripheral nerve-associated MSCs in osteogenesis, neuroregeneration, and immunomodulation[7-10]. For example, dental pulp stem cells (DPSCs) have been extensively studied for their roles in bone tissue repair and neuroprotection, while Schwann cell-like cells derived from peripheral nerves have been shown to effectively promote peripheral nerve regeneration[11-14]. However, similar to conventional mesoderm-derived MSCs, such as bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (ADSCs), NC-MSCs still face several challenges, including limited cell sources, high heterogeneity, and the invasive nature of their extraction procedures[7,15-18].

To overcome these limitations, pluripotent stem cells (PSCs) have emerged as a promising alternative source for NC-MSCs. PSCs, primarily including embryonic stem cells (ESCs) and induced PSCs (iPSCs), possess self-renewal capacity and multilineage differentiation potential[19-23]. In recent years, various methods have been developed to differentiate PSCs into NC-MSCs and explore their applications in tissue engineering and cell therapy[24-26]. Moreover, the advancement of extended PSCs (EPSCs) has provided a novel candidate cell source for NC-MSC research due to their enhanced genetic and epigenetic stability and their ability to circumvent ethical and regulatory concerns[27-29]. Despite rapid progress in PSCs-derived NC-MSCs research, significant challenges remain in clinical translation. A critical concern is the risk of teratoma formation, as residual undifferentiated PSCs may lead to tumor-like overgrowth post-transplantation[30]. Therefore, developing efficient strategies to eliminate residual PSCs and ensuring the safety of PSC-derived NC-MSCs remain key challenges in the field.

This review comprehensively discusses the developmental origins, cellular properties, and clinical applications of NC-MSCs. First, we provide an overview of NC-MSC development, migration pathways, and differentiation characteristics, systematically summarizing their functional roles across different tissues. Second, we review the latest clinical research progress on NC-MSCs, including DSCs and other NC-MSCs in the treatment of oral diseases, neurological disorders, and other systemic conditions. Subsequently, we highlight PSCs-derived NC-MSCs, detailing their directed differentiation strategies and evaluating their potential in overcoming the limitations of adult NC-MSCs. Finally, we discuss the safety concerns associated with PSC-derived NC-MSCs and propose future research directions. Through this review, we aim to provide a theoretical foundation for NC-MSC applications in regenerative medicine and cell therapy, facilitating their transition toward clinical applications.

DEVELOPMENTAL PROCESS AND DISTRIBUTION OF NC-MSCS

The NC originates from the region at the edge of the neural plate, situated between the neural plate and the adjacent non-neural ectoderm. The NC is a group of progenitor cells that plays a crucial role in the embryonic development of vertebrates[31,32]. During embryogenesis, NCCs undergo EMT, subsequently migrating extensively to various regions of the embryo[33,34] (Figure 1). These multipotent cells can differentiate into a variety of cell types, including chondrocytes, osteocytes, melanocytes, neurons of the peripheral nervous system (PNS), Schwann cells, chromaffin cells, and cardiomyocytes, among others, contributing to the formation of multiple tissues and organs in the embryo (Figure 1)[35,36]. However, the definition of their stem cell properties was not clearly established until 1992, when the research by Stemple and Anderson[37] provided clarity. They first proposed the concept of “neural crest stem cells” and, through in vitro experiments, confirmed that NCCs derived from mice not only possess multipotent differentiation ability but also exhibit self-renewal characteristics, which are core features of stem cells[37]. Although these adult stem cells originate from the ectoderm, under the regulation of specific signaling pathways and epigenetic memory, they have the capacity to differentiate into both ectodermal and mesodermal cells[38], opening new therapeutic targets for regenerative medicine. The following section will systematically summarize different types of MSCs derived from the NC, categorizing and discussing their developmental origins, migration paths, and related molecular markers based on tissue origin differences.

Figure 1 The development of the neural crest and the organization formed by its differentiated cells. Image was created using Biorender.com. PNS: Peripheral nervous system.

DSCs

DSCs are the most extensively studied subgroup of NC-MSCs, receiving considerable attention due to their accessibility, high proliferation potential, and multipotent differentiation capabilities[39]. These cells play a crucial role in the formation of teeth and their surrounding periodontal tissues. Among DSCs, DPSCs, stem cells from human exfoliated deciduous teeth (SHED), and periodontal ligament stem cells (PDLSCs) are all types of NC-derived stem cells. These cells express MSC markers such as Stro-1, CD146, CD106, CD90, CD73, CD29, and CD13, and consistently test negative for hematopoietic stem cell markers such as CD34, CD45, CD14, and CD19, as well as class II major histocompatibility complex antigen[40].

These DSCs originate from the cranial NC (CNC), acquiring a mesenchymal fate as they migrate to the first pharyngeal arch and differentiate along various developmental programs, ultimately contributing to the formation of craniofacial structures, including teeth[41,42]. During the bud stage of tooth development, a special homogeneous population of cells emerges within the condensed CNC-derived mesenchyme. These cells are marked as Tfap2b+/Lhx6+/Pax9+, and this NCC cell population is considered to be progenitor cells that aid in tooth development[43]. Cells at this stage provide the fundamental cell source for subsequent tooth tissue formation and further differentiate into specific lineages of the dental papilla and dental sac. The dental papilla cell lineage is marked by genes such as Crym, Egr3, and fibroblast growth factor 3 (Fgf3), while the dental sac lineage is marked by Epha3, Foxf1, and Fxyd7[43]. As tooth development progresses, dental mesenchymal cells undergo partitioning and differentiate into various structures and cell types. The dental mesenchymal cells in the dental papilla region gradually differentiate into dental pulp tissue and odontoblasts that secrete dentin, while the dental sac region surrounding the papilla differentiates into periodontal-related structures, including cementum, periodontal ligament (PDL), and part of the alveolar bone[44].

Specifically, DPSCs migrate from the CNC to the first pharyngeal arch, where they gather to form the dental papilla during the cap stage of tooth development[39]. These cells are then enveloped by the enamel organ, and during the bell stage, they differentiate into odontoblasts, ultimately forming the dental pulp mesenchyme[45]. Apical papilla stem cells (SCAP) are primarily located in the apical papilla region of immature permanent teeth. After tooth eruption, NCCs continue to migrate to the apical region and interact with Hertwig’s epithelial root sheath, jointly promoting root development[46]. Due to SCAP’s high telomerase activity, these cells have strong proliferative potential, providing an important cellular source for root formation. Their self-renewal ability also ensures their vitality during the process of root growth[47]. The biological behavior of SCAP, including differentiation, maturation, and mineralization, is finely regulated by various signaling pathways such as transforming growth factor (TGF)-β, WNT, and mitogen-activated protein kinases, ensuring a continuous supply of odontoblasts and promoting proper root development[48].

Moreover, NCCs also migrate to the dental follicle and gradually differentiate into mesenchymal cell populations that give rise to the PDL[49]. PDLSCs play a crucial role in the formation and repair of periodontal tissues. Their differentiation is tightly regulated by insulin-like growth factor and the transcription factor Foxp4. Studies have shown that although insulin-like growth factor and Foxp4 do not significantly affect root development, they do result in a marked increase in PDL area and suppress PDL differentiation[43]. PDLSCs are capable of differentiating into osteoblasts and fibroblasts and contribute to the maintenance of PDL function, thereby enhancing the stability and regeneration of periodontal tissues[50].

Dental follicle stem cells (DFSCs), the only cell type obtained during the early stages of tooth development prior to eruption, also originate from the CNC. These cells are derived from ectomesenchyme and form a loose connective tissue[51]. During the bud stage, the dental follicle begins to form and surrounds the dental papilla and enamel organ. By the cap stage of tooth development, it migrates to the dental follicle region and encloses the entire tooth germ[51]. As development progresses, DFSCs gradually differentiate into key components such as cementum, PDL, and alveolar bone[52].

In conclusion, NC-MSCs regulate the lineage commitment of DSCs with spatial and temporal specificity through orchestrated migration and differentiation programs. This process involves coordinated expression of multiple genes and a dynamic balance of microenvironmental signaling networks, forming a crucial theoretical foundation for both tooth development and regenerative medicine research.

Craniofacial BMSCs

Unlike mesoderm-derived MSCs from long bones, craniofacial BMSCs (C-BMSCs) possess distinct NC origins and undergo intramembranous ossification during development, rather than endochondral ossification[53,54]. In the early stages of embryonic development, NCCs migrate along specific pathways from the neural tube to the branchial arch regions of the head and neck. In the anterior region, cranial NCCs contribute to the formation of the frontonasal skeleton; in more posterior regions, they populate the pharyngeal arches, where they give rise to the jawbones, middle ear, and various cartilaginous and skeletal structures of the neck[55-57].

Using transgenic mice (Wnt1-Cre/R26R) to trace NCCs, researchers have discovered that the interface between the frontal and parietal bones is formed through the combined contributions of NCCs and mesodermal cells[8]. Specifically, NCCs predominantly give rise to the frontal bone, while the parietal bone is derived from the mesoderm. Cranial NCCs originate mainly from the forebrain and midbrain regions. Diencephalic and anterior mesencephalic NCCs migrate to the primordia of the frontal, nasal, and parietal bones, while posterior mesencephalic NCCs contribute to the formation of the temporal bones[56]. These cranial NCCs express markers such as Sox9, Pax3, and Runx2[57]. After migrating to cranial regions, they retain the ability to differentiate into multiple cell types of the skeletal system, particularly osteoblasts and chondrocytes, playing essential roles in the formation and remodeling of craniofacial bones.

The development of the maxilla also begins with the migration and differentiation of NCCs. During the early stages of embryogenesis, NCCs aggregate in the frontonasal region and migrate into the frontonasal prominence and the first branchial arch. As development progresses, they contribute to the formation of the maxilla and its associated structures[56]. Mandibular development, on the other hand, is initiated by the formation of Meckel’s cartilage. NCCs migrate into the mandibular arch, where they intermingle with non-NC-derived cells to establish the initial ossification centers of the mandible[56,58]. NC markers such as SNAIL2, TWIST, SOX9, and SOX10 have also been identified in maxillary and mandibular BMSCs cultured in various media[59]. These cells possess the ability to differentiate into osteoblasts and chondrocytes and play critical roles in the development and repair of jawbones[60].

Other NC-MSCs

Among NC-MSCs, corneal stromal stem cells (CSSCs) are one of the most representative types[61-64]. CSSCs have the primary ability to differentiate into corneal epithelial cells, which are responsible for maintaining the corneal stroma[65]. In chick embryos, for instance, corneal development begins around embryonic day 3 (E3), when the optic cup and lens induce the surface ectoderm to synthesize collagen fibers, forming a primary acellular stroma[66,67]. By E4, mesenchymal NCCs begin to migrate into this stroma and form the corneal endothelium near the lens. A second wave of NCCs later enters to further develop the primary stroma[66,68]. Within the stroma, NC-derived mesenchymal cells start differentiating into keratocytes around E6, synthesizing and secreting types I, V, and VI collagen, along with proteoglycans, essential for maintaining corneal transparency and mechanical properties[69-71]. CSSCs express a variety of specific genes, including adult stem cell markers such as ABCG2, BMi1, CD166, cKIT, and Notch1, as well as PAX6 and Six2, which are involved in early corneal development[72]. In addition to exhibiting adult stem cell characteristics, CSSCs also demonstrate immune privilege. Studies have shown that injecting human CSSCs into the corneal stroma of mice does not trigger a T cell-mediated immune rejection[73]. This immune privilege makes CSSCs a promising therapeutic tool in the field of bioengineering.

As multipotent embryonic progenitors, NCCs are the developmental origin of all neurons and glial cells in the PNS[74]. Their development involves three key stages: First, following the establishment of the central nervous system primordium, NCCs delaminate from the dorsal neural tube through EMT[75-77]. This process is tightly regulated by bone morphogentic protein (BMP) and Wnt signaling pathways. BMP promotes NC migration by modulating the cell cycle and inducing EMT, while Wnt signaling activates cyclin D1 to drive the G1/S transition - a crucial step for NC lineage stratification[78,79]. As migration and differentiation proceed, NCCs give rise to various PNS cell types, including neurons and glia. In this context, NC-MSCs are considered vital for PNS development and regeneration.

Peripheral nerve-associated NC-MSCs, such as Schwann cell precursors (SCPs), express surface markers like CD271, CD90, and CD106[80,81]. These MSCs not only differentiate into Schwann cells but also secrete neurotrophic factors that support axonal regeneration[14]. Another example is the hair follicle-derived NC MSCs (HFNCSCs), which are multipotent stem cells located in the bulge region of hair follicles. During early embryogenesis, these cells migrate from the neural tube along the dorsolateral pathway, passing through the dermis into the epidermis and ultimately settling in the follicular bulge[82]. While the epithelial components of hair follicles are typically derived from surface ectoderm, the presence of NC-derived cells in the bulge region suggests the existence of a distinct population retaining NC characteristics[82]. HFNCSCs express CD271, Nestin, Sox10, and other markers associated with immature NCCs[83,84]. Due to their similarity to BMSCs and their ability to differentiate into myogenic, osteogenic, chondrogenic, and adipogenic lineages, HFNCSCs represent a promising source for cell-based therapies[85].

CHARACTERISTICS OF NC-MSCS

NC-MSCs exhibit distinct biological properties compared to mesoderm-derived MSCs. Notably, NC-MSCs demonstrate a higher proliferative capacity, indicating a stronger potential for self-renewal. This proliferative advantage is likely related to the retention of certain NC traits during development[86,87]. Moreover, NC-MSCs continue to express key multipotency markers such as Sox10 and Nestin even after birth, and they retain the ability to differentiate into multiple cell types. These markers are rarely expressed in mesodermal MSCs, further highlighting the unique identity of NC-MSCs[88]. In terms of differentiation potential, NC-MSCs display remarkable versatility under appropriate in vitro and in vivo conditions. They are capable of differentiating into osteoblasts, odontoblasts, and various neural cell types, with particularly strong capacity for generating neurons and glial cells[38,46,89,90].

Given the significant differences between NC-MSCs and mesoderm-derived MSCs (such as those from bone marrow or adipose tissue) in developmental origin, molecular profile, and functional characteristics, comparative studies between these cell types are essential. Such research will help elucidate the unique regenerative advantages of NC-MSCs in specific tissues and provide a theoretical foundation for selecting optimal stem cell sources in precision regenerative medicine.

DSCs vs mesoderm-derived MSCs

DSCs originate from the NC, and their developmental origin distinguishes them significantly from mesoderm-derived MSCs such as BMSCs and ADSCs. These differences in developmental origin lead to notable disparities in their biological properties. While both cell types share basic MSC characteristics, such as self-renewal, multipotent differentiation, and immune modulation, they exhibit critical differences in these aspects[91,92].

Firstly, DSCs demonstrate a significantly higher proliferation rate compared to mesoderm-derived MSCs. Gronthos et al[86] found that DPSCs isolated from a single tooth exhibited higher proliferative potential. The colony-forming efficiency of DPSCs (22-70 colony forming units-fibroblast per 10⁴ cells) was substantially greater than that of BMSCs (only 2.4-3.1 colony forming units-fibroblast per 10⁴ cells). SHED showed an even higher proliferation rate than both DPSCs and BMSCs, with the proliferation ranking as follows: SHED > DPSC > BMSC[93-95]. This difference might be attributed to the early expression of ESC markers in SHED, such as octamer-binding transcription factor 4 (Oct4) and Nanog[94,95].

In terms of differentiation potential, DSCs display a stronger capacity for differentiating into neural cells[96]. This unique differentiation ability is likely linked to the selective retention of early NC developmental characteristics by NCCs[97-99]. Pagella et al[100] used microfluidic organ-on-a-chip technology to compare the effects of DPSCs and BMSCs on neural ganglia and found that DPSCs, after co-culture, induced neurons to express higher levels of neurotrophic factors and form more extensive axonal networks than BMSCs, suggesting the stronger neuroguiding capability of DPSCs. Furthermore, DPSCs have been shown to differentiate into functional neurons in appropriate microenvironments. When cultured in neurogenic media, DPSCs form bipolar and stellate neuron-like phenotypes and are capable of generating sodium currents consistent with functional neurons[101]. SHED also exhibits excellent neurogenic differentiation potential, and upon in vivo transplantation, SHED not only induced bone formation but also survived in the mouse brain and expressed neural markers[102]. Similarly, De Berdt et al[103] assessed the neurorepair potential of SCAP using a rat spinal cord hemi-section model, showing that SCAP implantation significantly reduced local inflammation, improved motor function, and did not induce chronic pain, demonstrating promising neurotherapeutic potential. Therefore, DSCs may outperform other tissue-derived MSCs in promoting myelination of the central nervous system and repairing neural injuries[101].

In addition to their neural potential, DSCs also exhibit stronger osteogenic and odontogenic differentiation capabilities[104-106]. Couble et al[107] cultured human dental pulp cells in Eagle’s basal medium with β-glycerophosphate and observed that the dental pulp cells exhibited typical cellular extensions and polarization, forming a spatial structure similar to odontoblasts, thereby demonstrating odontogenic differentiation potential. Compared to mesoderm-derived MSCs, DSCs show significantly higher alkaline phosphatase activity following osteogenic induction, a result further confirmed by alizarin red staining[108]. Park et al[105] applied decalcified bone matrix and fibrin scaffolds, evaluated the osteogenic capabilities of MSCs isolated from skin, bone marrow, and dental follicle, and found that DFSCs demonstrated stronger osteogenic potential, with higher expression of osteocalcin and calcium deposition. This suggests that DFSCs could serve as a potential alternative source of autologous bone tissue engineering cells[105]. It is important to note that NC-MSCs may have certain limitations in adipogenic differentiation[109,110]. DPSCs show slower adipogenic differentiation compared to ADSCs, with fewer lipid vesicles formed and weaker expression of adipogenic-related genes[111].

In terms of chondrogenic differentiation, several studies have compared the differentiation abilities of DSCs and BMSCs. Both DPSCs and BMSCs show positive staining and expression of cartilage-related genes such as collagen II during chondrogenic induction[112,113]. However, unlike mesoderm-derived MSCs, DPSCs tend to form fibrous cartilage rather than hyaline cartilage during culture, while BMSCs under certain conditions are more likely to form hyaline cartilage[114]. This suggests that the NC origin of DSCs might limit their ability to form hyaline cartilage, but they could potentially serve as a source for the regeneration of fibrous cartilage in joints[114].

From the perspective of angiogenic differentiation, DSCs show strong vascularization capabilities. In a study on bone organ vascularization, it was noted that DPSCs formed a reticular structure earlier (on day 3, compared to day 7 in BMSCs) with longer branching lengths and higher expression of angiogenesis-related markers such as vascular endothelial growth factor A and C-X-C motif ligand 1. In contrast, BMSCs from long bones exhibited almost no significant angiogenic potential under the same conditions, and no vascular-like structures were formed even after 20 days[115].

Beyond their multipotent differentiation capabilities, MSCs influence the immune system through direct cell-to-cell contact and/or intercellular communication, thereby exerting immunomodulatory effects[116]. Compared to mesoderm-derived MSCs, NC-MSCs exhibit stronger anti-inflammatory potential. Studies have shown that NC-derived gingival MSCs are more effective than mesoderm-derived gingival MSCs in ameliorating inflammation-related diseases. Specifically, NC-derived gingival MSCs alleviate symptoms of colitis in mice by inducing T-cell apoptosis[117,118]. Furthermore, Yamaguchi et al[119] found that the conditioned medium from SHED significantly reduced myocardial injury in ischemia-reperfusion mice and decreased local inflammatory factors such as tumor necrosis factor-α and interleukin-1β. The SHED-conditioned medium also demonstrated a significantly stronger anti-apoptotic effect on cardiomyocytes compared to BMSC-conditioned medium and ADSC-conditioned medium[119]. These immunomodulatory advantages offer new strategies for cell-based therapies in treating inflammation-related diseases.

Craniofacial BMSCs vs mesoderm-derived MSCs

Craniofacial bones can be divided into neurocranium (including the calvaria and cranial base) and viscerocranium (including the jawbones and other derivatives of the pharyngeal arches). From the perspective of bone development, the formation of the neurocranium fully depends on NCCs, while the viscerocranium is formed by both mesodermal cells and NCCs[53,120]. Other skeletal structures related to the craniofacial region are believed to originate from the mesoderm[121]. Compared to mesoderm-derived MSCs from long bones, C-BMSCs exhibit similar or even stronger osteogenic and angiogenic abilities, making them potentially more suitable for tissue engineering applications[8].

Recent studies have shown that BMSCs derived from craniofacial bones proliferate faster and exhibit stronger osteogenic potential compared to BMSCs from other anatomical sites[122]. For instance, Yamaza et al[123] isolated BMSCs from mouse mandibles and found that they exhibited greater proliferative potential, faster proliferation rates, and higher colony-forming efficiency compared to BMSCs derived from long bones. In another study comparing osteogenic potential, mandible BMSCs from the same SD rats were able to form more mineralized calcium nodules and significantly upregulate osteogenesis-related genes such as RUNX2 and osteocalcin during culture[124]. Moreover, several studies have also demonstrated the strong osteogenic capacity of NC-derived BMSCs in vivo[125,126]. Aghaloo et al[125] performed subcutaneous implantation experiments in nude mice and found that mandible BMSCs formed significantly larger bone nodules with mineralized bone content three times higher than that of long bone BMSCs. Similar results were further validated in implant-associated bone defect models[126]. However, mandible-derived BMSCs exhibited lower potential for chondrogenesis and adipogenesis compared to iliac bone BMSCs, which might explain the rare occurrence of cartilaginous healing at mandibular fracture sites[127].

The vascular system plays a crucial role in bone tissue repair, as angiogenesis is vital for normal bone development and fracture healing[128]. BMSCs have been shown to promote angiogenesis in the bone microenvironment, enhancing vascular endothelial growth factor expression[129,130]. A study comparing C-BMSCs, DPSCs, and SCAPs for their ability to form capillary-like networks when co-cultured with human umbilical vein endothelial cells in gelatin methacrylate hydrogels found that C-BMSCs exhibited higher levels of pericyte marker expression (such as neural-glial antigen 2 and alpha-smooth muscle actin), showing greater potential for pericyte differentiation[131]. Moreover, C-BMSCs were more suitable for building mature vascular networks compared to DPSCs or SCAPs[131]. Based on these properties, C-BMSCs may be more suitable for craniofacial bone defect repair, such as alveolar ridge augmentation, as they accelerate bone integration of implants, compared to long bone-derived BMSCs.

Additionally, C-BMSCs possess similar immunomodulatory potential to long bone-derived BMSCs, including the ability to inhibit monocyte activation, promote macrophage phagocytic activity, and suppress T-cell proliferation and activation[132]. As C-BMSCs can be obtained with relatively low-invasive methods, they may offer an advantage over long bone-derived BMSCs in clinical applications. However, there is still a need to verify the heterogeneity among different sources of MSCs in larger clinical trials.

Peripheral nerve-associated NC-MSCs vs mesoderm-derived MSC

Peripheral nerve-associated NC-MSCs and MSCs both have significant research value in tissue repair and regenerative medicine. However, comparative studies between the two are limited. SCPs, which originate from the NC, exhibit broad developmental potential and can differentiate into Schwann cells, neurons, and myofibroblasts[133,134]. In contrast, mesoderm-derived MSCs primarily differentiate into osteoblasts, chondrocytes, and adipocytes. While mesoderm-derived MSCs can differentiate into Schwann cell-like cells under specific stimulation by growth factors, this typically requires induced differentiation[135].

Studies have shown that ADSCs have the potential to differentiate into neural cells[136]. However, previous research has indicated that the neurorepair effect of ADSCs in clinical applications often does not meet expectations[137,138]. In comparison, NC-MSCs, due to their unique NC origin, may be more prone to differentiating into neurons or supporting cells such as Schwann cells. For example, De La Garza-Castro et al[9] compared the repair effects of SCPs and BMSCs in a rat spinal cord injury model and found that SCPs outperformed BMSCs in inflammation regulation, myelination remodeling, and motor function recovery. This suggests that NCCs may have better adaptability in the local neuroregenerative microenvironment. Further research showed that chitosan scaffolds loaded with skin-derived SCPs significantly enhanced peripheral nerve regeneration by upregulating repair-related genes (such as nerve growth factor, brain-derived neurotrophic factor) and secreting neurotrophic factors[10].

Regarding immune modulation, both peripheral nerve stem cells and MSCs exhibit good immunomodulatory functions. Studies have shown that both can influence the polarization of macrophages[8]. Skin-derived SCPs, during nerve injury repair, can increase the expression of interleukin-6, promote macrophage activation, and enhance their phagocytosis of myelin debris, thus facilitating sciatic nerve repair in rats[139]. In addition, MSCs (e.g., BMSCs and ADSCs) in co-culture conditions promote the polarization of macrophages toward the M2 phenotype, suppress inflammation, and reduce tumor necrosis factor-α secretion[140]. Based on these findings, SCPs may have greater potential for treating neuroinflammatory diseases due to their NC origin. Although MSCs are relatively easy to obtain and have wide clinical application prospects, their immunomodulatory effects may vary depending on their source[141]. Therefore, further research is needed to verify the specific mechanisms and clinical effects of MSCs from different sources in immune modulation.

CURRENT CLINICAL APPLICATIONS OF NC-MSCS

In 2022, our research group conducted a comprehensive review of the clinical applications of DSCs, summarizing findings from thirty published clinical studies and twenty-eight registered clinical trials [ClinicalTrials.gov and the International Clinical Trials Registry Platform (ICTRP)][7]. These studies explored the potential use of DSCs (including DPSCs, PDLSCs, gingival MSCs, and SHEDs) in treating various conditions, including oral diseases (such as pulp necrosis, irreversible pulpitis, apical periodontitis, periodontitis, alveolar bone/jaw defects, and cleft lip and palate), neurological disorders (such as Huntington’s disease, acute ischemic stroke, and chronic post-stroke disabilities), coronavirus disease 2019, and other diseases or conditions (such as diabetes, liver cirrhosis, hair loss, wrinkles, and erectile dysfunction)[7]. Given recent advancements in clinical therapies based on DSCs over the past three years, this section will first supplement our previous summary with updated information. Additionally, the clinical applications of other NC-MSCs beyond DSCs were collected and analyzed to provide a comprehensive overview of the current landscape of NC-MSC clinical applications.

Clinical trials based on DSCs

Although DSCs have demonstrated immense potential in regenerative medicine, no new clinical studies based on DSCs have been published since 2022 (as per a MEDLINE search). This may be attributed to the extended follow-up periods and significant research investment required for clinical studies. However, during this period, several new clinical trials have been registered to explore the therapeutic potential of DSCs in various diseases (Tables 1 and 2). According to ClinicalTrials.gov, four newly registered clinical trials focus on the treatment of Huntington’s disease, periodontitis, pulp necrosis, and apical periodontitis (Table 1). In addition, studies registered in another platform are primarily investigating the therapeutic potential of DSCs in type 1 diabetes, Hodgkin and non-Hodgkin lymphoma, wrinkles, and pulp necrosis (Table 2).

Table 1 Dental stem cell-based clinical trials registered at ClinicalTrials.gov.

Registration ID	Status	Diseases	Study design	Cell source	Administration route	Number of patients	Interventions		Follow-up period	Phase	Outcomes	Ref.
							Test group	Control group
NCT06097780	Not yet recruiting	Huntington’s disease	Randomized; parallel assignment; single-blind (outcomes assessor)	Dental pulp	Intravenous administrations	120	Intravenous human DPSCs administrations	Intravenous placebo administrations	1 year	Phase 3
NCT05924373	Recruiting	Periodontitis	Randomized; parallel assignment; double (participant investigator)	Dental pulp	Local injection at periodontal defect site	204	Single-dose group (DPSCs, 1.0 × 107); two-dose group (low-dose, DPSCs, 1.0 × 106); two-dose group (high-dose, DPSCs, 1.0 × 107)	Normal saline	90, 180, 360, 720 days	Phase 2
NCT05728346	Not yet recruiting	Pulp necroses	Nonrandomized; single group assignment; open label	Allogeneic deciduous pulp	Transplantation after the preparation and disinfection of the root canal of the affected teeth	30	SHED mixed with hyaluronic acid polymers	None	1, 3, 6, 12, 18, 24 months	Not applicable
NCT06043453	Recruiting	Apical periodontitis; trauma	Observational; multicenter blinded	Remaining dental pulp and apical papilla	Attraction or transplantation after root canal disinfection		With apical periodontitis	Without apical periodontitis	1, 2, 3 years	Not applicable
NCT04983225	Recruiting to active, not recruiting	Periodontitis	Randomized; parallel assignment; double-blind (participant, investigator)	Dental pulp	Injecting into the periodontal defect site	36	DPSCs (1 × 106)/site; DPSCs (5 × 106)/site; DPSCs [(3-4) × 107]/three or four sites; DPSCs (1 × 107)/site; DPSCs (2 × 107)/two sites	Saline solution	90, 180, 360, 720 days	Phase 1
NCT02523651	Unknown	Periodontitis	Randomized; parallel assignment; triple-blind (participant, investigator, outcomes assessor)	Allogeneic dental pulp	Injecting into the periodontal defect site	40	DPSCs (1 × 106)	Saline solution	1 year	Phase 1/2
NCT03386877	Completed	Periodontitis	Randomized; parallel assignment; triple-blind (participant, investigator, outcomes assessor)	Autologous dental pulp	Delivering into intrabony defect via minimally invasive surgical technique	29	Micrografts of DPSCs + collagen sponge	Collagen sponge	6, 12 months	Not applicable
NCT01082822	Unknown	Periodontitis	Nonrandomized; parallel assignment; open label	Periodontal ligament	Implanted into bone defect sites via surgical approach	80	PDLSCs sheet fragment + DBBM (Bio-oss); PDLSCs sheet pellets + DBBM (Bio-oss); DBBM (Bio-oss)	Sham comparator	4, 12, 24 weeks; 1 year	Phase 1/2
NCT03638154	Completed	Periodontitis	Randomized; parallel assignment; double-blind (care provider, outcomes assessor)	Gingival	Implanted into bone defect sites via surgical approach	20	GFs + GMSCs + β-TCP	β-TCP	1, 3, 7, 14 days; 6 months	Not applicable
NCT03137979	Unknown	Periodontitis	Randomized; parallel assignment	Gingival	Implanted into bone defect sites via surgical approach	30	GMSCs + collagen scaffolds; collagen scaffolds	Open flap debridement	1, 3, 6 months	Phase1/2
NCT01357785	Unknown	Periodontitis	Randomized; parallel assignment; open label	Autologous periodontal ligament		35		None	3-12 months	Phase1		Chen et al[174]
NCT04641533	Completed	Post-extraction sockets	Split-mouth; randomized; crossover assignment; double-blind (investigator, outcomes assessor)	Dental pulp	Placing into the extraction socket	13	DPSCs + L-PRF	L-PRF	7 days; 6 months	Not applicable		Cubuk et al[175]
NCT02731586	Unknown	Edentulous alveolar ridge	Single group assignment; open label	Allogeneic dental pulp	Introducing dental pulp-derived mesenchymal stem cells during placement of dental implants	10	Dental pulp-derived MSCs	None	3 months	Early phase 1
NCT03766217	Completed	Cleft lip and palate	Randomized; parallel assignment; single-blind (outcomes assessor)	Autologous deciduous pulp	Placed into the alveolar defect via surgical approach	62	SHED + hydroxyapatite-collagen sponge	Iliac crest autogenous bone graft	15 days; 3, 6, 12 months	Phase 3		Tanikawa et al[176], Pinheiro et al[177]
NCT01932164	Completed; has results	Cleft lip and palate	Single group assignment; open label	Autologous deciduous pulp	Maxillary alveolar graft by tissue engineering	5	SHED + hydroxyapatite-collagen sponge	None	3, 6 months	Not applicable	Percentage of bone filling at 6 months postoperatively: 89.5%	Tanikawa et al[176]
NCT04130100	Unknown	Knee osteoarthritis	Randomized; parallel assignment; open label	Dental pulp	Intraarticular injection	60	Low dose of DPSCs; high dose of DPSCs	Sodium hyaluronate	12 months	Early phase 1
NCT01814436	Unknown	Dental pulp necrosis	Single group assignment; open label	Autologous deciduous pulp		80	Scaffold-free SHED-derived pellet	None	3-12 months	Not applicable
NCT03957655	Unknown	Liver cirrhosis	Randomized; parallel assignment; single-blind (outcomes assessor)	Autologous deciduous pulp	Peripheral vein infusion	40	SHED (1 × 106 cells/kg body weight)	Standard medication for viral hepatitis and cirrhosis	4, 8, 12, 16, 24 weeks	Early phase 1
NCT03912480	Unknown	Type 1 diabetes	Single group assignment; open label	Deciduous pulp	Intravenous drip	24	SHED (0.11I U/kg body weight) + insulin + oral hypoglycemic drugs	None	1, 2, 6 weeks; 2, 3, 6, 9, 12 months	Early phase 1
NCT04608838	Completed	Acute ischemic stroke	Randomized; parallel assignment; quadruple-blind (participant, care provider, investigator, outcomes assessor)	Allogeneic dental pulp	Intravenously infusion	79	DPSCs (JTR-161, 1 × 108 cells); DPSCs (JTR-161, 3 × 108 cells)	Placebo	91, 366 days	Phase 1/2		Suda et al[178]
NCT02728115	Active, not recruiting to unknown		Nonrandomized; parallel assignment; open label	Allogeneic deciduous pulp	Intravenous administration	6	SHED (Cellavita HD, 1 × 106 cells); SHED (Cellavita HD, 2 × 106 cells)	None	1, 4 years	Phase 1
NCT04219241	Active, not recruiting to unknown	Huntington’s disease	Single group assignment; open label	Allogeneic deciduous pulp	Intravenous administration	35	SHED (Cellavita HD, 2 × 106 cells)	None	1, 2 years	Phase 2/3
NCT03252535	Completed	Huntington’s disease	Randomized; parallel assignment; triple-blind (participant, investigator, outcomes assessor)	Allogeneic deciduous pulp	Intravenous administration	35	SHED (Cellavita HD, 1 × 106 cells); SHED (Cellavita HD, 2 × 106 cells)	Physiological solution without cells	Monthly for 14 months	Phase 2		Wenceslau et al[179]
NCT04336254	Recruiting to unknown	COVID-19	Randomized; parallel assignment; triple-blind (participant, investigator, outcomes assessor)	Allogeneic dental pulp	Intravenous injection	20	DPSCs (3 × 107 cells)	Saline	28 days	Phase 1/2		Ye et al[180]
NCT04302519	Unknown	COVID-19	Single group assignment; open label	Dental pulp	Intravenous injection	24	DPSCs (1 × 107 cells/kg body weight)	None	3, 7, 14, 28, 360 days	Early phase 1

DPSCs: Dental pulp stem cells; SHED: Stem cells from human exfoliated deciduous teeth; PDLSCs: Periodontal ligament stem cells; DBBM: Deproteinized bovine bone mineral; GFs: Gingival fibroblast; GMSCs: Gingival mesenchymal stem cells; TCP: Tricalcium phosphate; PRF: Platelet-rich fibrin; MSCs: Mesenchymal stem cells; COVID-19: Coronavirus disease 2019.

Table 2 Dental stem cell-based clinical trials registered on the International Clinical Trials Registry Platform.

Registration ID	Status	Diseases	Study design	Cell Source	Administration route	Number of patients	Interventions		Follow-up period	Phase	Outcomes	Ref.
							Test group	Control group
IRCT20140911019125N12	Pending	Children with type 1 diabetes	Not randomized; no placebo; not blinded	Deciduous pulp	Intravenous injection	10	SHEDs (3 × 106/kg, twice, 3 months apart)	None	3, 6, 12 months	Not applicable
RCT20140911019125N10	Pending	Hodgkin and non-Hodgkin lymphoma	Not randomized; no placebo; not blinded	Deciduous pulp	Intravenous injection	30	SHEDs (1 × 106/kg, 4 hours before the injection of hematopoietic stem cells); SHEDs (1 × 106/kg, on day 0, + 2, + 4, + 6 after hematopoietic stem cell transplantation); no SHEDs injection	None	Daily until discharge from the hospital	Not applicable
JPRN-UMIN000049760	Complete: Follow-up complete	Wrinkles	Randomized; parallel assignment; single-blind	Dental pulp		12	All-in-one gel containing immortalized human DPSCs-CM and various useful ingredients	None	4 weeks	Not applicable
CTRI/2024/10/075535	Not yet recruiting	Necrosis of pulp	Randomized; parallel assignment; double-blind	Deciduous pulp	Transplantation	30	Scaffold of fibrin glue loaded with E-nanoHA seeded with SHED	Blood clot scaffold with MTA	1, 3, 6, 12, 18, 24 months	Phase 3
JPRN-UMIN000042791	Complete: Follow-up complete	Periodontitis	Randomized; parallel assignment; single-blind (participants)	Deciduous pulp	Gargle	30	Mouthwash containing SHED culture supernatant	Mouthwash without SHED culture supernatant	1 month	Not applicable
ChiCTR2100051466	Recruiting	Periodontitis	Randomized; parallel assignment; open label		Bilateral multipoint injection on a single tooth	96	DPSCs (1 × 107 cells) for once; DPSCs (1 × 107 cells) for twice	Saline	90, 180, 360 days	Phage 0
ChiCTR2100049178	Pending	Periodontitis	Randomized; parallel assignment; double-blind	Dental pulp	Local injection	36	DPSCs (1 × 106 cells) for single injection; DPSCs (5 × 106 cells) for single injection; DPSCs (1 × 107 cells) for single injection; DPSCs (1 × 107 cells) for single injection in 2 Locations; DPSCs (1 × 107 cells) for single injection in 3-4 Locations	None		Phage 1
ISRCTN13093912	Completed	Periodontitis	Randomized; parallel assignment; single-blind (patients and examiners)	Dental pulp	Implanted into bone defect sites via surgical approach	20	DPSCs (1 × 107 cells) + hydroxyapatite-collagen scaffold	Hydroxyapatite-collagen scaffold	1, 2, 4, 12, 24, 36 weeks; 12, 24, 36, 48, 60 months	Not applicable		Sánchez et al[181]
JPRN-UMIN000045926	Complete: Follow-up complete	Wrinkles	Randomized; parallel assignment; single-blind (outcomes assessor)	Dental pulp		12	All-in-one gel containing immortalized DPSCs-CM solution and various beauty ingredients	No treatment	4 weeks	Not applicable
JPRN-UMIN000043528	Complete: Follow-up complete	Wrinkles	Randomized; parallel assignment; single-blind (outcomes assessor)	Dental pulp		12	All-in-one gel containing immortalized DPSC-CM solution and the latest peptide raw materials	No treatment	4 weeks	Not applicable
JPRN-UMIN000045897	Complete: Follow-up continuing → Complete: Follow-up complete	Hair loss	Nonrandomized;parallel assignment; open label	Deciduous pulp	Injection	22	SHED-CM; after SHED-CM injection, one dose of micrografts (Rigenera) followed by another SHED-CM injection; SHED-CM injection after one dose of micrografts (Rigenera)	None	6 months	Not applicable

SHED: Stem cells from human exfoliated deciduous teeth; DPSCs: Dental pulp stem cells; CM: Conditioned medium; E-nanoHA: E- nanophased hydroxyapatite; MTA: Mineral trioxide aggregate.

Combining our previous summary[7], a total of 25 clinical trials have been registered in ClinicalTrials.gov to evaluate the potential of DSCs in treating periodontitis (32%, 8/25), post-extraction sockets (4.8%, 1/25), edentulous alveolar ridge (4%, 1/25), cleft lip and palate (8%, 2/25), knee osteoarthritis (4%, 1/25), dental pulp necrosis (8%, 2/25), apical periodontitis (4%, 1/25), liver cirrhosis (4%, 1/25), type 1 diabetes (4%, 1/25), acute ischemic stroke (4%, 1/25), Huntington’s disease (16%, 4/25), and coronavirus disease 2019 (8%, 2/25) (Table 1). Beyond those registered in ClinicalTrials.gov, 11 additional clinical trials related to DSCs have been recorded in the ICTRP, investigating periodontitis (36.4%, 4/11), necrosis of pulp (9.1%, 1/11), wrinkles (27.3%, 3/11), type 1 diabetes (9.1%, 1/11), Hodgkin and non-Hodgkin lymphoma (9.1%, 1/11), and hair loss (9.1%, 1/11) (Table 2).

Additionally, the statuses of several clinical trials have changed (Tables 1 and 2). In ClinicalTrials.gov, a clinical trial investigating the use of DPSCs for periodontitis (NCT04983225) has transitioned from “Recruiting” to “Active, not recruiting”. In addition, other trials (NCT04336254, NCT04219241, and NCT02728115) have changed to “Unknown” status at different trial stages. In ICTRP, the status of a registered clinical study using SHED-conditioned medium for hair loss has shifted from “Complete: Follow-up continuing” to “Complete: Follow-up complete”. Given the rapid updates in registered information and trial statuses of DSC-related studies, regularly reviewing and discussing these developments will benefit the fundamental research, clinical practice, and translational application of DSCs.

Clinical trials based on other NC-MSCs (except for DSCs)

In 2018, a study by Redondo et al[142] (registration ID: NCT03070275) demonstrated that cross-linked serum scaffold combined with autologous alveolar bone MSCs significantly enhanced bone density in maxillary cystic bone defects, suggesting its potential as an alternative therapy for maxillary defects and other bone loss conditions (Table 3). Two additional clinical studies utilized autologous alveolar bone MSCs in combination with collagen scaffolds enriched with autologous fibrin/platelet lysate to investigate their therapeutic effects on chronic periodontitis[143,144] (Table 3). These two studies, sharing the same registration ID (NCT02449005) and grouping strategy, respectively examined bone defect tissue repair and inflammatory factor changes in gingival crevicular fluid. While the MSCs + scaffold group did not show significant clinical improvement in bone defect healing compared to the control group (minimal access flap surgery), it exhibited a biochemical pattern favoring inflammatory control and tissue regeneration over 12 months.

Table 3 Neural crest-derived mesenchymal stem cells (except for dental stem cells) based clinical trials from published articles.

Ref.	Registration ID	Conditions/diseases	Study design	Cell source	Administration route	Interventions		Follow-up period	Outcomes
						Test group	Control group
Redondo et al[142]	EudraCT 2010-024246-30; NCT01389661	Maxillary bone cysts	Nonrandomized	Autologous alveolar bone	Surgical implantation	BioMax scaffold + MSCs (n = 9)	Contralateral control area of spongy alveolar bone without treatment	3-4, 6-8 months	Bone density (↑)
Apatzidou et al[143]	NCT02449005	Intrabony defects and periodontitis	Randomized; single-blind	Autologous alveolar bone	Transplanted into the osseous defect	MAF + Biocomplex (containing BMSCs, n = 9)	MAF + aFPL (n = 10); MAF (n = 8)	Baseline; 6 weeks; 6, 9, 12 months	TNF-α, RANKL levels in GCF gradually decreased, MMP levels were higher than baseline (6-9 months), and RANKL/OPG ratio was lower than baseline (9-12 months)
Apatzidou et al[144]	NCT02449005	Intrabony defects and periodontitis	Randomized; single-blind	Autologous alveolar bone	Transplanted into the osseous defect	MAF + Biocomplex (containing BMSCs, n = 9)	MAF + aFPL (n = 10); MAF (n = 8)	Baseline; 6 weeks; 6, 9, 12 months	Significant clinical improvements (attachment gain and PPD) but no between-group differences

MSCs: Mesenchymal stem cells; MAF: Minimal access flap; BMSCs: Bone marrow-derived mesenchymal stem cells; aFPL: Autologous fibrin/platelet lysate; TNF: Tumor necrosis factor; RANKL: Receptor activator for nuclear factor kappa B ligand; GCF: Gingival crevicular fluid; MMP: Matrix metalloproteinase; OPG: Osteoprotegerin; PPD: Periodontal probing depth.

Beyond the two registered studies mentioned above (NCT03070275, NCT02449005), two additional phase 1/2 registered trials (NCT03070275, NCT06700655) are investigating the potential of autologous alveolar bone MSCs in implant therapy and autologous CSSCs in the treatment of limbal stem cell deficiency (Table 4). Compared to DSCs, the clinical research and applications of other NC-MSCs (excluding DSCs) are still in their early stages, likely due to challenges in cell isolation and a relatively weaker foundation in basic research.

Table 4 Neural crest-derived mesenchymal stem cells (except for dental stem cells) based clinical trials registered at ClinicalTrials.gov.

Registration ID	Status	Diseases	Study design	Cell source	Administration route	Number of patients	Interventions		Follow-up period	Phase	Outcomes	Ref.
							Test group	Control group
NCT03070275	Completed	Implant therapy	Randomized; parallel assignment; single-blind (outcomes assessor)	Alveolar bone	Gently stabilized over the implant platform head partially covering the buccal bone	20	Biocomplex (aBM-MSCs/fibrin glue/collagen fleece)	No use of adjunctive grafting materials	4 months	Phage 1/2
NCT02449005	Completed	Chronic periodontitis	Randomized; parallel assignment; quadruple-blind (participant care, provider, investigator, outcomes assessor)	Autologous alveolar bone	Transplanted into the osseous defect	60	MAF + Biocomplex (containing BMSCs, n = 9)	MAF + aFPL (n = 10); MAF (n = 8)	Baseline; 6 weeks; 6, 9, 12 months	Phage 1/2		Apatzidou et al[143], Apatzidou et al[144]
NCT01389661	Completed	Maxillary cyst bone loss of substance	Single group assignment; open label	Autologous alveolar bone	Surgical implantation	11	BioMax scaffold + MSCs	Contralateral control area of spongy alveolar bone without treatment	0, 2 weeks; 2, 6 months	Phage 1/2		Redondo et al[142]
NCT06700655	Not yet recruiting	Limbal stem cells deficiency	Randomized; parallel assignment; single-blind (outcomes assessor)	Autologous stromal opacities	Cell transplantation	60	Cornea limbal stem cell; cornea limbal stem cell combined corneal stromal stem cell	Corneal transplantation	32, 48, 108 days; 6 months; 1, 2 years	Phage 1/2

aBM-MSCs: Autologous bone marrow mesenchymal stem cells; BMSCs: Bone marrow-derived mesenchymal stem cells; MAF: Minimal access flap; aFPL: Autologous fibrin/platelet lysate; MSCs: Mesenchymal stem cells.

PSCS AS A STABLE SOURCE FOR NC-MSCS

The NC-MSCs have demonstrated promising preclinical and clinical therapeutic potential. However, similar to other adult MSCs, NC-MSCs face several challenges that limit their broader application, including invasive procedures leading to donor site damage, cellular heterogeneity, and restricted cell availability[7,15-18]. In recent years, the rapid advancements in PSC research have provided a potential solution to these limitations.

PSCs refer to a unique class of cells with self-renewal capabilities and the ability to differentiate into nearly all cell types, primarily including ESCs and iPSCs[19-22]. ESCs are derived from early-stage embryos, whereas iPSCs are generated by reprogramming somatic cells (e.g., skin fibroblasts) through the introduction of specific transcription factors such as Oct4, Sox2, Kruppel-like factor 4, and c-Myc[145]. PSCs are characterized by the expression of pluripotency markers (e.g., Oct4, Nanog, and stage-specific embryonic antigen-4), indefinite proliferation in an undifferentiated state, and the capacity to give rise to cells of the three germ layers (ectoderm, mesoderm, and endoderm)[146,147]. These properties make PSCs an attractive source for generating homogeneous MSC populations with therapeutic potential in a controlled in vitro environment.

Several strategies have been developed to induce NCC differentiation from PSCs. Early methods relied on fluorescence-activated cell sorting (FACS) to isolate NCCs from mixed neural precursor cell (NPC) populations[148-150]. In 2005, Pomp et al[151] demonstrated that co-culturing ESCs with the mouse stromal cell line PA6 promoted the differentiation of ESCs into peripheral sensory and sympathetic neurons. Interestingly, prior to neuronal differentiation, PA6 cells first induced the formation of NCC-like cells, which aligns with the developmental origins of peripheral sensory and sympathetic neurons from NCCs in vivo[151]. Using a similar induction approach and FACS, Jiang et al[149] isolated a p75-positive cell population with NCC-like phenotypic and functional characteristics, which exhibited multilineage differentiation potential into peripheral neurons, glial cells, and myofibroblasts in vitro and in vivo. Despite modifications in FACS selection markers (e.g., frizzled-3+cadherin-11 and p75+HNK1), the efficiency and stability of NCC isolation from NPCs remained low[148,150]. Since NCCs typically constitute only a small subset of NPCs, sorting-based strategies suffer from inconsistency. Additionally, variations in NPC induction protocols (e.g., co-culture with stromal cells, neurosphere formation, and small molecule-based induction) introduce differences in cellular composition and proportions, further complicating NCC isolation and downstream applications[152].

To overcome these challenges, researchers have developed adherent culture protocols that simulate in vivo NC development, enabling the direct differentiation of NCCs from PSCs. Canonical Wnt signaling plays a critical role in NCC fate determination during vertebrate embryogenesis[153-155]. By activating Wnt signaling while inhibiting the activin A/Nodal pathway, several studies successfully directed PSCs toward NCC-like cells (p75+HNK1+AP2+), with minimal contamination from Pax6+ neural progenitors[156-158]. However, inhibition of the activin A/Nodal pathway does not appear to be strictly necessary for NCC differentiation. Two studies reported that transient activation of Wnt signaling (for two days) followed by withdrawal for three days was sufficient to induce NCC-like cells expressing SOX10, PAX7, PAX3, MSX1, and TFAP2A, while the addition of the activin A/Nodal inhibitor SB431542 had a negative impact on differentiation[159,160].

Building on these strategies, several studies successfully established protocols for generating MSCs from PSCs via a NC intermediate. In 2014, Fukuta et al[24] reported a two-step differentiation strategy in which PSCs were first induced into NCCs and subsequently differentiated into MSCs. In the first stage, NCC formation was promoted by inhibiting TGF-β signaling (SB431542) and glycogen synthase kinase 3β (CHIR99021), while in the second stage, alpha minimum essential medium supplemented with 10% fetal bovine serum facilitated NCC-to-MSC transition[24]. The derived MSCs expressed typical MSC markers (CD44, CD73, and CD105) but were negative for hematopoietic marker CD45 and exhibited trilineage differentiation potential (osteogenesis, chondrogenesis, and adipogenesis)[24]. Another study incorporated FGF2 into the NCC induction protocol, which further enhanced NCC differentiation efficiency[25]. Additionally, a commercial serum-free MSC medium (CTS StemPro MSC SFM) was successfully employed to induce NCC-to-MSC differentiation in vitro[25]. Compared to MSCs derived from trophoblast co-culture, NCC-derived MSCs exhibited higher purity and superior osteogenic differentiation potential, and their transcriptional profiles more closely resembled adult ADSCs, BMSCs, and umbilical cord MSCs[25]. These findings highlight the importance of lineage-specific differentiation strategies for generating MSCs with enhanced therapeutic potential. In another study, Ouchi et al[26] demonstrated that NC-like cells expressing low affinity nerve growth factor receptor and THY-1 could be induced using a combination of epidermal growth factor, FGF2, Gem21 neuroplex, GlutaMAX, and N2, and these NC-like cells retained robust differentiation potential into mesenchymal and NC lineages[26].

Despite being a major PSC subtype, ESCs pose ethical concerns due to the destruction of blastocysts during derivation, prompting ethical debates among researchers and regulatory bodies[161,162]. iPSCs offer an alternative, but their epigenetic instability may contribute to differentiation bias, raising concerns regarding their clinical applications[163-165]. Recently discovered EPSCs, while also reprogrammed from somatic cells, have demonstrated superior genetic and epigenetic stability compared to ESCs[27-29]. The advantages of EPSCs suggest that they may serve as a more suitable starting point for directed differentiation compared to ESCs and iPSCs. Although there are currently no reports of EPSC-to-MSC differentiation, future studies focusing on MSC generation from EPSCs via a NC intermediate may address current challenges in MSC-based therapies.

On the other hand, while PSC-derived cells exhibit immense therapeutic potential, concerns regarding teratoma formation limit their widespread application[19,166-168]. A major advantage of PSCs is their unlimited proliferative capacity, which enables the large-scale production of human cells for therapeutic purposes. However, this same proliferative ability also increases the risk of tumorigenicity if residual undifferentiated PSCs persist post-transplantation. Even a small number of residual PSCs can potentially form teratomas. For example, a 2022 case report documented the formation of an immature teratoma following the injection of PSC-derived pancreatic β-cells[30]. Therefore, effective detection and removal of residual PSCs from PSC-derived MSC populations are essential for improving the safety of PSC-based therapies. Developing specific clearance strategies for residual PSCs in NC-MSCs could enhance the feasibility of PSC-based MSC therapy and facilitate its clinical translation.

Additionally, NC-MSCs exhibit significant diversity and widespread distribution, with notable differences among various types of NC-MSCs as well as between NC-MSCs and other MSC populations. These differences may be attributed to variations in signaling regulation during MSC development. Although some studies have successfully induced the differentiation of PSCs into MSCs via the NC lineage in vitro[24-26], the discrepancies between PSC-derived NC-MSCs, adult NC-MSCs, and other MSCs remain incompletely understood. Further exploration of the developmental processes of different adult NC-MSCs will facilitate the directed differentiation of distinct NC-MSC subtypes in vitro. In conclusion, PSC-derived NC-MSCs represent a promising supplement and potential alternative to adult NC-MSCs, offering a viable solution to the current limitations in the clinical application of both adult NC-MSCs and MSCs in general.

LIMITATIONS AND PROSPECTS

Despite significant advances in the study of the developmental origin, molecular characteristics, and therapeutic applications of NC-MSCs in recent years, their clinical translation still faces numerous challenges, necessitating further exploration and optimization.

Firstly, heterogeneity and lineage specificity remain central issues in NC-MSC research. Although NC-MSCs originate from a common source (the NC), distinct subpopulations exhibit significant functional differences depending on their tissue of origin[33,169-171]. Therefore, further application of lineage-tracing techniques, single-cell transcriptomic sequencing, and epigenetic analyses is required to delineate the characteristics of different NC-MSC subtypes, thereby refining directed differentiation strategies and enhancing their therapeutic efficacy in specific disease models.

Secondly, expanding clinical applications and optimizing transplantation strategies are critical for the clinical translation of NC-MSCs. Current clinical studies primarily focus on the use of DSCs in periodontal and pulp regeneration, while research on their potential in neurological and systemic diseases remains limited. Moreover, improving the survival rate and integration of transplanted cells remains a major challenge. Future studies should explore scaffold-based delivery, in situ differentiation, and immune-compatible cell sources to optimize the clinical efficacy of NC-MSC transplantation.

In addition to addressing the heterogeneity of NC-MSCs and optimizing transplantation strategies, further investigation into the interactions and potential synergistic effects of key signaling pathways such as Wnt, Notch, TGF-β, FGF, BMP, and Hippo is also of great significance. These signaling pathways play a coordinated role in the development and function of NC-MSCs and exhibit different interaction patterns in various physiological environments. This may provide key insights for discovering new therapeutic targets and enhancing regenerative treatment strategies.

Finally, establishing standardized quality control systems and regulatory frameworks is crucial for the clinical application of NC-MSCs[172,173]. Moving forward, it is essential to develop uniform cell manufacturing standards, activity assessment protocols, and long-term safety evaluation systems to meet regulatory requirements and facilitate the commercialization of NC-MSC-based therapies.

CONCLUSION

NC-MSCs have demonstrated significant potential in regenerative medicine, particularly in craniofacial reconstruction, neural repair, and immunomodulation. However, their clinical application remains limited due to challenges such as restricted cell sources, heterogeneity, and invasive isolation procedures. Recent clinical trials have explored NC-MSCs, particularly DSCs, for treating periodontal diseases, pulp regeneration, and neural disorders, yet large-scale and long-term clinical data are still lacking. PSCs offer an alternative source for generating NC-MSCs, with EPSCs providing greater genetic stability and differentiation capacity. While PSC-derived NC-MSCs hold promise, safety concerns, including teratoma formation, require further investigation. Future research should refine differentiation strategies, optimize transplantation safety, and expand clinical studies to accelerate the translation of NC-MSCs into effective cell-based therapies.