Gingival mesenchymal stem cells: Biological properties and therapeutic applications

Abstract

Our understanding of mesenchymal stem cells (MSCs) and their biological properties is steadily increasing, with more studies focusing on their therapeutic effects in the domains of immunology, tissue engineering and regenerative medicine. MSCs may be derived from tissues such as bone marrow, adipose, the umbilical cord, as well as from dental tissues (e.g., tooth germ, dental follicle, pulp tissue of exfoliated deciduous and permanent teeth, apical papilla, periodontal ligament, gingiva, and alveolar bone). Gingival mesenchymal stem cells (GMSCs) are non-hematopoietic adult stem cells isolated from the gingival lamina propria. When compared to MSCs purified from various dental and non-dental tissues, GMSCs are more abundant in source, relatively non-invasive to obtain, and genetically stable. In recent years, many studies have found that GMSCs possess the ability of self-renewal, multi-directional differentiation, and chemotaxis to inflammatory sites for immunity regulation. Their molecular and stem-cell properties make them highly suitable for both preclinical and clinical research. Extracellular vesicles (EVs) secreted by GMSCs are of key interest due to their ability to emulate the biological and therapeutic activity of GMSCs themselves. This paper will therefore review the current consensus on GMSCs, surveying their sources and isolation methods, their biological properties, and their therapeutic applications on inflammatory and immune-related diseases.

1. Introduction

1.1. Mesenchymal stem cells

In the early 1970's, mesenchymal stem cells (MSCs) were discovered by Friedenstein et al. and in 1991, they were officially named as such by Dr. Arnold Caplan.^{1, 2, 3} MSCs are a type of non-hematopoietic adult stem cells with a high capacity of self-renewal and a considerable potential for multidirectional differentiation. They are derived from the mesoderm and exist in various tissues of the body (e.g., bone marrow, adipose tissue, skin, peripheral blood, thymus, placenta, umbilical cord blood, tendon, and heart).^{4, 5, 6, 7, 8, 9} Current techniques facilitate MSC isolation, culture, and expansion, with various in vitro induction methods leading these cells to differentiate into other mesenchymal cell types, such as osteoblasts, chondrocytes, adipocytes, liver cells, tenocytes, and nerve cells. Low immunogenicity and strong immunomodulatory effects both in vivo and in vitro are few of the incomparable advantages that have led MSCs to be widely used as seed cells in tissue engineering in recent years.

In 2006, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) proposed three minimal criteria to identify MSCs: (1) plastic adherent cells with clonogenic ability under standard cell culture conditions; (2) expression of a series of mesenchymal cell surface markers such as CD73, CD90 and CD105, but negative for hematopoietic and endothelial cells surface markers (e.g. CD34,CD45,CD11b,CD14, CD19,CD79α) and human leukocyte antigen-D related surface molecules (HLA-DR or HMC-II); (3) tri-lineage differentiation potential into osteoblasts, adipose cells and chondrogenic cells in vitro.¹⁰ The currently most well-described MSCs include bone marrow mesenchymal stem cells (BMSC), adipose mesenchymal stem cells (ADSC) and umbilical cord mesenchymal stem cells (UCMSC).

1.2. Dental-derived mesenchymal stem cells

The craniofacial region is home to an abundance of dental-derived MSCs which can be derived from different oral tissues. As of now, eight distinct dental-derived MSCs populations have been isolated. First came the discovery and isolation of dental pulp stem cells (DPSCs), followed by human exfoliated deciduous teeth stem cells (SHED), periodontal ligament stem cells (PDLSCs), dental follicle progenitor cells (DFPCs), alveolar bone marrow stromal cells (ABMSCs), stem cells from the apical papilla (SCAP), tooth germ progenitor cells (TGPCs), and more recently, gingival mesenchymal stem cells (GMSCs).^{9,11, 12, 13, 14} In comparison to MSCs isolated from other tissues, those derived from dental tissues not only have the commonality of MSCs, but also their own unique characteristics. These cells are easier to obtain clinically from their (discarded) donor tissues, and they offer certain advantages in terms of proliferation rate, cell function, homogeneity and tumorigenicity.

1.3. Gingival mesenchymal stem cells

Gingival tissue, one of the four connective tissue pillars within the periodontium, plays a crucial role in supporting teeth. It surrounds the tooth and attaches to the alveolar bone, thereby creating the gingival attachment.¹⁵ The human gingiva is a masticatory keratinized mucosal tissue that is histologically composed of the lamina propria containing spinous and connective tissue, the stratum basale, the stratum spinosum, and superficial layers of keratinized cells. In addition, the gingival tissue appears to be an appealing reservoir of stem cells. For example, if compared to the natural skin healing abilities, gingival tissue stem cells are capable of fast regeneration and rapid wound healing following injury, resulting in minimal to no scar formation.¹⁶

Early studies have pinpointed mesenchymal progenitors capable of multilineage differentiation in the fibroblast-like cell population isolated from skin, lung, umbilical cord, and amnion.¹⁷ Subsequently, Fournier BP et al. analyzed the colony forming units of gingival fibroblasts and found that 92 % of the colonies could differentiate into osteoblasts, 72 % could differentiate into adipocytes, and 50 % could differentiate into chondroblasts. These results suggested the existence of gingival multipotent progenitor cells (GMPCs).¹⁸ At the same time, Zhang et al. identified a population of progenitor cells derived from healthy human gingiva that displayed fibroblast-like characteristics.¹⁹ These progenitor cells were distinguished by their fibroblast-like spindle morphology, their colony forming unit-fibroblasts (CFU–F), their expression of MSC-related cell surface markers (e.g., CD73, CD90, CD105, SSEA-4, STRO-1), the absence of hematopoietic cell markers such as CD34 and CD45, and their multipotent differentiation potential into osteocytes, adipocytes, and neural cells. These properties meet the ISCT's criteria defining MSCs, which allows for these progenitor cells to be designated as gingiva-derived mesenchymal stem/stromal cells (GMSCs).¹⁹ It was further reported by Xu et al. that GMSCs contain two subsets of cells: neural-crest-derived N-GMSCs and mesoderm-derived M-GMSCs, split approximately 90 % and 10 %, respectively. In contrast to M-GMSCs, N-GMSCs show an increased capacity to differentiate to neural cells and chondrocytes, as well as to modulate immune cells.²⁰

Overall, GMSCs are more abundant in source than other MSC-types and can be obtained through relatively minimally invasive methods, exhibiting adequate genetical stability. Moreover, it may be used in autologous transplants, which eliminates the need to search for a matching donor. Since Zhang et al. first named and fully reported the isolation, characterization, and immunomodulatory properties of GMSCs in 2009, these cells with their incomparable advantages have attracted widespread attention of scholars all over the world.

Recent studies aim to determine the optimal source of GMSCs. This kind of mesenchymal cells can be isolated from both inflamed and healthy gingival tissue, each to their own advantages. For example, GMSCs isolated from inflamed gingival tissues retain a high proliferative potential and expression of MSC-associated surface markers.^21,22 A proportion of these ex vivo-expanded clones of GMSC exhibited the potential to differentiate into osteogenic, adipogenic and chondrogenic lineages. They possessed the capacity to generate connective-like structures following ectopic transplantation, which provides evidence for the existence of MSC-like populations within inflamed gingival tissues that are functionally equivalent to those derived from healthy gingival tissues.²¹

A growing body of evidence has demonstrated that, distinct from other types of cell therapy, MSC-based therapy achieves its therapeutic effects, not only through direct cell-cell contacts, but also by releasing secretome-derived bioactive factors.²³ MSC secreted extracellular vesicles (EVs) including exosomes (30-200 nm), micro-vesicles (500-1000 nm), and apoptotic bodies (1-5 μm), have been suggested as a viable cell-free therapeutic alternative for MSCs. Interestingly, The GMSC-derived EVs (GMSC⁃EVs), that GMSCs can produce in a large quantity, have strong potential for immune regulation, tissue regeneration, and disease treatment. This is due to their low immunogenicity, excellent biocompatibility, and lack of cytotoxicity. Additional advantages of MSC-EVs-based therapy are being studied in comparison to cellular therapies, particularly the high drug loading capacity, high specificity, high stability, competitive price, and efficient intercellular communication.²⁴ To understand the broader therapeutic application prospects of GMSCs in tissue engineering, regenerative medicine, and the treatment of inflammatory or immune-related diseases, this review will focus on the current research progress to the biologically characterize GMSCs and their therapeutic application.

2. Source, isolation, culture method, and preliminary identification of GMSCs

2.1. Source, isolation, and culture method of GMSCs

One of the considerable challengers that researchers face is the sourcing and isolation of GMSCs. They can be found in the gingival tissue obtained from the interdental gingival papilla, the buccal marginal region, and the maxillary tuberosity. The theory of sourcing GMSCs from inflamed gingival tissue raises an interesting discussion. It is proposed that hyperplastic gingiva and gingival tissues affected by periodontal diseases possess a regenerative capability more robust than that of GMSCs stemming from healthy gingiva.^21,25 Moreover, these cells also possess the ability to proliferate and express specific markers associated with GMSCs, indicating that inflamed gingival tissue could serve as a valuable reservoir of GMSCs.

To successfully isolate GMSCs, gingival tissue samples are first obtained as remnants or discarded biological waste from standard dental procedures. Specific instruments are occasionally used to collect these samples during gingival punch biopsy procedures. The gingival tissue is subsequently detached from its epithelial layer, and the remaining connective tissue is cut into small pieces, from where it will subject to one of two isolation methods: the enzymatically digested method or the explant culture method.

In the enzymatically digested approach, the gingival tissue samples are cut into small fragments and digested with enzymes such as collagenase and dispase, and then filtered through strainers to achieve a single cell suspension suitable for cultivation. Depending on the given re-search group, different enzymes may be used. While some choose to utilize a specific collagenase (e.g. I,²⁶ II,²⁷ IV,^19,28), others opt for a combination of various types of collagenase and dispase.^26,28 Once the isolation is completed, in vitro culture condition of primary GMSCs is conducted in a humidified tissue culture incubator with 5 % CO₂ and 95 % O₂ at 37 °C. After 24–72 h, the non-adherent cells are removed. The plastic-adherent confluent cells are passaged, continuously subcultured and maintained in the complete growth medium. Cells from the second to the sixth passages are used in the ensuing experiments. However, this method is can frequently damage primary GMSCs, leading to early differentiation of cells and loss of their biological characteristics.^{29, 30, 31} As for the explant culture method, the gingival tissue sample is minced with a sterile scalpel into 1 mm³ pieces and placed directly in tissue culture to adhere. Compared to the alternative, the number of primary cells extracted by this method is small, with low efficiency and an unstable success rate.^{29, 30, 31} Conveniently, the remaining cell culture and passage procedures, as well as the required basal media, is the same for both methods. This involves the Minimum Essential Medium α (α-MEM)^19,26,28,32 or Dulbecco's Modified Eagle's Medium (DMEM).^27,33 Typically, the research groups added the media with antibiotics and fetal bovine serum (FBS) at varying concentrations. This potentially opens the door to supplementing the culture media with different factors to improve efficiency and success rates.

2.2. Preliminary identification of GMSCs

Currently, there exist a number of methods for identifying primary GMSCs. Apart from the evaluation of cell morphology and expression of MSC markers, these methods range from the analysis of proliferation rate and clonogenicity (CFU–F assay), to the assessment of multi-differentiative ability. Assessment of this ability is crucial in determining in vitro differentiation potential towards mesodermal lineages (osteocytes, adipocytes, chondrocytes), or other non-mesodermal lineages. In contrast to BMSCs which need two to three passages to obtain uniformly homogenous populations, GMSCs already exhibit the desired homogeneity at the end of a primary culture.³⁴ These populations are then sorted for MSCs, usually via flow cytometry or magnetic activated cell sorting.

Morphologically, GMSCs have a fibroblast-like appearance, long cytoplasmic processes and many filopodia with distinct extracellular transport apparatus.^26,35 The cell sorting of GMSCs based on specific cell markers is lacking in evidence, in addition to meeting the surface markers of mesenchymal stem cells proposed by SCTI, current literature suggests that GMSCs express some non-specific markers including CD29 (Integrin beta 1),²⁷ CD44,^{26,28,32,33,36} and CD146.^19,27,28 Certain proteins regarded as indicators of pluripotency or markers associated with embryonic stem cells such as Oct-4 (octamer-binding transcription factor 4), STRO-1,^19,26,28 SSEA-4 (stage-specific embryonic antigen 4)^19,28 and Nanog³⁷ are often expressed in the presence of GMSCs. Table 1 provides a detailed summary of human GMSC isolation and identification methods.

Table 1.

| Isolation and characterization of human GMSCs.

Isolation Method	Composition of Culture Medium	Enzymes Used for Digestion	Time of Digestion	Cell surface markers	Multipotency	Ref.
Enzymatic method	α-MEM, 10 % FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM l-glutamine, 100 mM nonessential amino acids	4 mg/mL collagenase IV	2h at 37 °C	CD73, CD90, CD105, CD146, SSEA4, STRO-1	Osteocytes, adipocytes, neurocytes, endothelial cells	19
	α-MEM,15%FBS, 100 U/mL penicillin, 100 lg/mL streptomycin, 200 mM l-glutamine, 10 mM ascorbic acid 2-phosphate	1 mg/mL dispase, 2 mg/mL collagenase IV	2h at 37 °C	CD44, CD73, CD90, CD105, CD146, CD166, SSEA-4, STRO-1	Osteocytes, adipocytes, chondrocytes	28
	α-MEM, 10 % FBS, 2 mM l-glutamine,100 μML-ascorbate-2-phosphate,1 mM sodium pyruvate,50U/mL penicillin, 50 μg/mL streptomycin, 2.5 μg/mL amphotericin B	dispase II, collagenase type I	2h at 37 °C	CD44,CD73,CD90,CD105, CD166, STRO-1	Osteocytes, adipocytes, chondrocytes	26
	DMEM, 10 % FBS, l-glutamine 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 lg/mL fungizone	3 mg/mL collagenase type II	30–45min at 37 °C	CD29, CD146	Osteocytes, adipocytes, chondrocytes	27
Explant method	α-MEM,10%FBS,1 % penicillin, streptomycin, amphotericin	–	–	CD13, CD44,CD73,CD90,CD105	Osteocytes, adipocytes, chondrocytes	32
	DMEM/F12,10%FBS,1 % antibiotic-antimycotic solution	–	–	CD13, CD44, CD73, CD90, CD105	Osteocytes, adipocytes, chondrocytes	33
	IMDM,10%FBS,300 U/mL penicillin, 300 mg/mL streptomycin	–	–	CD44, CD90, CD105, CD166	Osteocytes, adipocytes, myocytes	34

3. Biological properties of GMSCs

3.1. Proliferation

Proliferation is a primary biological property to be taken into account for the clinical utilization of in vitro expanded MSCs. The MTT assay or the Cell Counting Kit-8 (CCK-8) method is preferred by researchers to plot the GMSC growth curve in determining the optimal proliferative rate and capacity of GMSCs. For example, 96-well plates containing GMSCs are treated with MTT or CCK-8 solution. Then, based on the absorbance measured respectively at the 570nm to 630nm or 450 nm to 490 nm wavelengths using a microplate reader, a proliferation curve can be generated.^{38, 39, 40, 41} Cell growth curve typically displays a distinctive ‘S’ shape which it is divided into three stages: Latency, Logarithmic growth period and Plateau.

The logarithmic growth phase was noted to occur on days 3–4 following inoculation, and near days 7 and 8, the cell number would stabilize. Here, Chen's study concluded that second and third generation cells were stronger than fourth and fifth generation cells in their capacity to proliferate.⁴² A remarkable benefit of GMSCs when compared to MSCs isolated from the dental pulp and periodontal ligament is their superior proliferation capability.^{43, 44, 45} Similarly, MSCs derived from gingiva surpass those from the umbilical cord in both proliferation rates and population doubling times.³⁷

Further research on factors that modulate MSC proliferation could improve the yield of current methods. Pham et al. showed that Ascorbic acid (AA) could trigger GMSCs proliferation and upregulate some pluripotent stem cell markers (e.g., SSEA-3, Oct-3/4, Nanog, Sox-2, TRA-1-60). These AA-treated GMSCs maintained MSC phenotype markers and mesodermal cell differentiation markers. More importantly, these cells did not cause any tumors in the athymic mice.³³ Cannabidiol (CBD) has also been reported to activate genes associated with cell proliferation, multipotency and self-renewal in human GMSCs (hGMSCs). Rajan et al. have found that CBD induces hGMSCs towards neuronal progenitor cells differentiation and neurogenesis.⁴⁶ It is just as crucial to identify factors that are detrimental GMSC proliferation. The study by Junaid et al. has revealed that high glucose in GMSCs can obliterate their proliferative capacity. This hyperglycemia was found to increases the expression of TNF family with Fas L by activating apoptosis through the extrinsic apoptotic pathway.⁴⁷ Other studies have also reported Lipopolysaccharide (LPS),⁴⁰ Valproic acid,⁴⁸ cigarette smoke or aerosol⁴⁹ and even some resin composite-based materials as factors inhibiting the proliferation rate and vitality of GMSCs in vitro.⁵⁰

3.2. Clonogenicity

Clonogenicity is the ability of MSCs can self-renew and initiate colony-forming units of fibroblasts (CFU–F) from a single cell.¹⁹ This ability is best assessed by a CFU-F efficiency assay which assesses the frequency of undifferentiated progenitor cells after their isolation or at a given passage during culture.⁵¹ Briefly, the assay consists of seeding 500 to 1000 cells per well in plates, and culturing the experimental generation of GMSCs in basic medium within a humidified atmosphere. The culture medium is changed three times per week. 14 days later, the cultures are washed twice with phosphate-buffered saline, stained overnight, washed with distilled water, and counted using a microscope. Colonies with aggregates of 50 or more cells were scored as one CFU-F.²⁶ The number of CFU-F present in the dish divided by the number of cells initially seeded calculates the colony formation rate (in percentage). In comparison to dental pulp-derived mesenchymal stem cells (PDMSCs), GMSCs are observed to be superior in formation of colony units.⁴⁵ This colony forming ability is typically associated with the prevalence of stromal clonogenic precursors in the gingival tissue. However, GMSCs isolated from healthy gingival tissues exhibited fewer and smaller colonies than those isolated from inflamed gingival tissues showed multiple and larger colonies.⁵² Diseased gingival tissue showed decreased CFU efficiency and alkaline phosphatase (AKP) activity; Furthermore, it displayed diminished osteoblast mineralization capability and a heightened inclination to differentiate into adipocytes when compared to healthy gingival tissue.⁵³

In depth studies comparing various MSCs have marked GMSCs to be superior to in certain aspects. A comparison of the biological characteristics between four types of MSCs showed that UCMSCs were the lowest in CFU, while GMSCs displayed the strongest levels of self-renewal. However, the colony-forming efficiencies of BMSCs and SHED were similar.⁵¹ Another interesting example reports that human GMSCs have a mean population doubling time of 39.6 ± 3.2h, significantly less than that of BMSCs (80.4 ± 1.2h). GMSCs were also more homogenous and nontumorigenic, displaying stable phenotypes, and maintained normal telomerase activity in long-term cultures.³⁴ It is proposed that cultures may be further sustained via supplementary AEDG and KED peptides, prolonging hGMSCs and human periodontal ligament stem cells division in vitro. These peptides may delay senescence and apoptosis by preventing p16 and p21 gene expression and subsequent protein synthesis. More data is required to confirm the use of these peptides to delay the expression of senescence markers in long term stem cell cultures.⁵⁴ One study reports a decrease in the expression levels of oncogenes in GMSCs at later passages. At passage number 41, levels were lower than those at passage number 10, which may indicate a greater safety for GMSCs at high number of passages.⁵⁵ Thus, refining techniques to sustain long-term cultures will reduce the risks and instability of GMSCs, thereby improving their safety and reliability in therapeutic applications.

3.3. Migration capacity

Another significant property of MSCs is their capacity to migrate, as it determines the ability of the cell to reach a target site. Overall, GMSCs show a higher migration capacity than other MSCs such as PDMSCs⁴⁵ and skin-derived MSCs.⁵⁶ Additionally, GMSCs lead to superior oral wound healing which may be partly explained by the decreased matrix contraction in full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix).⁵⁶ A major example is in gingival tissue regeneration, where in order to achieve gingival healing and augmentation, GMSCs need to migrate to the areas of tissue loss. A wide range of parameters can affect cell migration, and the comprehensive analysis of these factors is an emerging field in MSC research. Tests can be carried out by applying membranes with different pore sizes and different treatment modifications. Recent articles suggest the role of bacteria on chemokine/cytokine secretion and cell migration. Notably, Kang's research team had demonstrated that persistent exposure to Fusobacterium nucleatum promoted cell migration, chemokine, and cytokine release, while inhibiting the proliferation and osteogenic differentiation of GMSCs.⁵⁷

In general, the cell migration capacity is evaluated by the cell migration assay (Transwell). In this experiment, a Transwell chamber is placed in a culture plate. The inner side of the chamber, the upper chamber, and the space outside of Transwell chamber, the lower chamber, are separated by a polycarbonate membrane. The cells of interest are planted in the upper chamber and are prone to the effects of the components in the lower culture medium, due to the permeability of the membrane. The migration ability of the detected cells can be obtained by counting the number of cells entering the lower chamber.²⁷

3.4. Multilineage differentiation ability

Multilineage differentiation potential is considered to be the most important biological characteristic of GMSCs. In an earlier study of Marynka-Kalmani et al., single colony-derived GMSCs treated with dexamethasone were seeded on fibrin membranes and then transplanted subcutaneously in severe combined immune deficiency mice. The implant could generate connective tissue-like structures, bone matrix and even form tumors consisting of two germ layer-derived (teratoma-like) tissues consisting of fetal fat, striated, muscle, cartilage, bone, epithelial, and neural tissues.⁵⁸ A large number of in vitro experiments have proved that under appropriate in-duction culture conditions, GMSCs can differentiate into a variety of mesoderm-derived cells (e.g., osteoblasts, chondrocytes, adipocytes, endothelial cells, myocytes) and can also differentiate into ectoderm-derived cells (e.g., neuron-like cells, odontogenic cells and keratinocytes) and endoderm-derived cells such as insulin producing cell clusters.⁵⁹ Fig. 1 depicts the multilineage differentiation potential reported for GMSCs. Table 2 summarizes comprehensive information about this ability of GMSCs.

Fig. 1.

| Differentiation potential of GMSCs. GMSCs were reported capable to differentiate into cell lineages originating from each of the three primary germ layers. Under defined induction conditions, GMSCs could differentiate into cells derived from the mesoderm (e.g., osteoblasts, adipocytes, chondroblasts, endothelial cells and myocytes), the endoderm (insulin producing cells), and ectoderm (e.g., neurocytes, auditory progenitor cells, odontogenetic cells and keratinocytes). The respective time for this cell differentiation is indicated for each cell type. These findings were based on the presence of cell-specific markers (indicated in the box next to each cell type).

Table 2.

Multilineage differentiation ability of GMSCs, induction media, and modulating factors.

Derived Cell		Supplements to Basal Medium	Differentiated Cell-Specific Markers	Inhibitory Factors	Enhancing Factors	Ref
Mesoderm Lineage Cell Differentiation	Osteoblast	Dexamethasone, Ascorbic acid, β-glycerophosphate	RUNX2, COL1A1, ALP, Osteocalcin, Bone sialoprotein, BMP2/4, Osteopontin, Osteonectin	Exposure to F. nucleatum, Extraction of GMSCs from inflamed tissues,Addition of Muscone to Medium	CGF, Trichostatin A, Noni Extract, Resveratrol, Biosynthesized Gold Nanoparticles, Sema3A, Blue-Light Irradiation	32,60,61
	Adipocyte	1-methyl-3-isobutylxanthine, Idomethacin, Dexamethasone/Hydrocortisone, Insulin, l-glutamine	Oil Globules, PPARγ2, Lipoprotein Lipase	N/A	Extraction of GMSCs from inflamed tissues, Addition of Muscone to Medium	18,19,32,38,68
	Chondroblast	TGF-β, Dexamethasone, Ascorbic Acid, Insulin, BMP-6, Selenium Acid, Insulin transferrin	Collagen II/V, SOX9, Glycosaminoglycans, Acid Proteoglycan, ACAN, Cartilage‐specific Proteoglycan Core Protein	N/A	Controlled inflammation, AA/retinol	18,32,61,69, 70, 71
	Endothelial Cell	Hydrocortisone, FGF-b, R3-IGF-1, Ascorbic Acid, EGF, GA-1000, Heparin, VEGF-165, FBS	Capillary-like Tube Formation Test, CD31 (PECAM-1), vWF	N/A	N/A	19,72,73
	Myocyte	Dexamethasone, Ascorbic Acid, Sodium Pyruvate, Forskolin, Recombinant Human bFGF	Myosin II (MF20), MyoD, Myf5	N/A	N/A	74
Ectoderm Lineage Cell Differentiation	Neurocyte	FBS, penicillin, streptomycin, fibroblast growth factor 2, and epidermal growth factor	Immature markers: Nestin, Musashi 1, β-Tubulin III, MASH1, NGN2, SOX1, Nissl bodies; Mature markers: MAP2, NSE, NeuN, NFM, CNPase, GFAP, pan-Nav, GAD67, Nav1.6, NF1, PSD95, synapsin	N/A	Hypoxia Preconditioning, Cannabidiol, Metformin, AEDG peptide	19,20,37,46,58,75, 76, 77, 78, 79, 80, 81, 82
	Auditory Progenitor Cell	IGF, FGF-b EGF in RGD-Coupled Alginate-Matrigel Nanohybrid Hydrogels	GATA3, SOX2, PAX2, PAX8, Myo7a	N/A	N/A	83
	Odontogenic cell	Embryonic Tooth Germ Cell-conditioned Medium (ETGC-CM)	ALP, OPN, BSP, Dentin Matrix Protein 1	N/A	MiR‐3940‐5p	59,84
	Keratinocyte	Plant Extracts of Acalypha indica	Cytokeratin 5/10/14, Involucrin	N/A	N/A	85
Endoderm Lineage Cell Differentiation	Insulin producing cell clusters (IPPCs)	N/A (Fibroblast-like GMSCs are converted into IPPCs)	Insulin, Pdx1, Glucagon, GLUT2, GLUT4, Dithizone staining, GSIS assay	N/A	N/A	86

3.4.1. Mesoderm lineage cell differentiation (osteoblast, adipocyte, chondroblast, endothelial cell, myocyte)

• •Osteoblast

Osteogenic differentiation can be achieved by culturing GMSCs in an osteo-inductive medium supplemented with ascorbic acid, dexamethasone, and β-glycerophosphate.^32,60,61 The culture is then stained using Alizarin Red S to identify calcified deposits. Additionally, an alkaline phosphatase (ALP) activity assay is performed, and gene expression analysis is conducted to assess specific bone markers. In principle, the detection of Runt-related transcription factor 2 (RUNX2), Collagen 1A1 (COL1A1), ALP, bone sialoprotein (BSP), osteocalcin (OCN), bone morphogenetic protein 2/4 (BMP2/4), osteopontin (OPN) and osteonectin (SPARC), may indicate successful osteogenic differentiation.^28,59,61,62

When compared to BMSCs, GMSCs were more positive for ALP and had a stronger osteogenic potential. They formed a greater number of mineralized nodules following osteogenic induction, and expressed ALP, OCN, OSX and Runx2 at significantly higher levels. It was similarly noted that GMSCs presented with a higher number of CD90‐positive cells during osteogenic induction.⁶¹ In terms of osteogenic potential, PDLSCs expressed upregulated bone-related markers ALP and COL1A1 significantly more than GMSCs, and they yielded a greater number of mineralized nodules. However, the adipogenic potential of both MSC types was similar.⁴³ A considerable number of studies have found that many factors or culture environments can promote the osteogenic differentiation of GMSCs, such as concentrated growth factors (CGF),³⁹ Trichostatin A (TSA),⁶³ noni extract,⁶⁴ resveratrol,⁶² gold nanoparticles biosynthesized by an actinomycete,⁶⁵ osteoprotective factor Semaphorin3A (Sema3A),⁴⁰ as well as irradiation by blue light-emitting diode.⁶⁶ Conversely, persistent exposure to F. nucleatum could inhibit the proliferation and osteogenic differentiation of GMSCs.⁶⁷

• •Adipocyte

For adipogenic differentiation, the basal culture medium was enhanced with 1-methyl-3-isobutylxanthine, dexamethasone or hydrocortisone, indomethacin, insulin, and l-glutamine. The differentiation could be confirmed by presence of oil globules, was evaluated with oil red O staining and the increased expression of specific adipogenic markers determined by RT-PCR [e.g., peroxisome proliferator-activated receptor γ(PPARγ2), lipoprotein lipase (LPL) ].^18,19,32 Interestingly, GMSCs extracted from inflamed tissues had a higher adipogenic potential than those derived from periodontally healthy tissues, while their osteogenic potential was inversely correlated.³⁸ One study reported that introducing muscone into the culture medium, a primary constituent of natural musk, promoted the adipogenic differentiation of GMSCs. In turn, this inhibited the osteogenic differentiation of GMSCs by inhibiting the Wnt/β-catenin signaling pathway.⁶⁸

• •Chondroblast

Different mixtures of transforming growth factor-β(TGF-β), ascorbic acid, bone morphogenetic protein-6, dexamethasone, insulin, and insulin-transferrin selenium acid can be added into a culture medium to induce chondrogenic differentiation.^18,32 Following the staining of glycosaminoglycans with safranin O or the highlighting of the acid proteoglycans with toluidine blue, chondrogenic differentiation may be evaluated.^18,32 The expression level of certain chondrogenic markers, such as collagen II (COL2), collagen V (COL5) and sex-determining region Y box protein 9 (SOX9), can further confirm the differentiation status.^32,61 Recent studies have made use of a chondrogenic differentiation kit, which complements researchers' ability to optimize their success rates. Alcian-Blue/nuclear-fast-red staining quantification can detect the presence of cartilage‐specific proteoglycan core protein and Aggrecan (ACAN) mRNA expression, acting as conclusive evidence for chondrogenic differentiation.^69,70 Similar to adipogenic differentiation, the use of GMSCs derived from tissue with controlled inflammation may be associated with enhanced chondrogenic differentiation potential as well. It was noted that GMSCs’ characteristics had changed in the presence of controlled inflammation or AA/retinol, wherein controlled inflammation could restore the AA/retinol-mediated reduction in intracellular phosphorylated β-catenin.⁷¹

• •Endothelial cells and Myocytes

Limited evidence suggests the capability of GMSCs to transform into other mesoderm-derived cells, such as endothelial cells and myogenic cells. These reports seek to challenge our understanding of trilineage differentiation potential. Endothelial differentiation could be achieved in a medium composed of hydrocortisone, ascorbic acid, fibroblast growth factor (FGF-b), R3-Insulin-like growth factor-1 (R3-IGF-1), epithelial growth factor (EGF), GA-1000, heparin, vascular endothelial growth factor-165 (VEGF-165) and FBS.⁷² Successful differentiation is noted by the formation of capillary-like tubes and the expression of specific endothelial surface markers such as CD31 (PECAM-1), vWF.^19,73 Alternatively, Ansari et al. reported myogenic differentiation through the encapsulation of GMSCs in alginate microspheres containing growth factors cocktail. They are cultured in a basal medium containing forskolin (FSK), sodium pyruvate, dexamethasone, ascorbic acid, and recombinant human bFGF. When induced in vitro for two weeks, GMSCs started to exhibit a morphological resemblance to myogenic tissue; after four weeks, immunofluorescence staining revealed the presence of myogenic markers (e.g., Myosin II (MF20), Myf5, MyoD), subsequently validated through gene expression analysis. Overall, encapsulated GMSCs showed greater capacity for myogenic regeneration in vivo after subcutaneous transplantation into immunocompromised mice, in comparison to human BMSCs.⁷⁴

To optimize the success rates of GMSC differentiation, the scientist must properly corelate the amount of culture time required with the nutrient composition of the medium and concentration of the differentiation-inducing components. Notably, previous studies have found that the time for differentiation of chondroblast, adipocyte, osteoblast is about 14, 21 and 28–35 days, respectively. Endothelial differentiation takes approximately 1 week.^19,38 and myogenic differentiation needs 4 weeks.⁷⁴

3.4.2. Ectoderm lineage cell differentiation (neurocyte, odontogenic cell, keratinocyte)

• •Neurocyte

In one of the first studies reporting on the neural differentiation potential of GMSCs, Zhang et al. plated GMSCs in slides coated with poly-d-lysine/laminin, and cultured them in a neuro-inductive medium supplemented with FBS, penicillin, streptomycin, fibroblast growth factor 2, and epidermal growth factor.¹⁹ Following 14–21 days under these neural differentiation conditions, GMSCs displayed positive markers for neural differentiation, including β-tubulin III(TUJ-1), glial fibrillary acidic protein (GFAP), and neurofilament 160/200 (NF-M).^19,75

In contrast, both Heng and Li carried out neural induction in a two-step culture processes.^76,77 In the former, upon GMSCs reaching 40%–60 % confluence, the basal culture milieu was changed to the neuronal induction medium. Subsequently, after 8 days of culture, the media was switched to the neuronal maturation medium. At the 8- and 14-day timepoints, cells were analyzed for expression of neural markers with immunocytochemistry. Immature neural markers contain Nestin, Musashi 1, β-Tubulin III, MASH1, NGN2, and mature neural markers are composed of microtubule associated protein 2 (MAP2), NSE, NeuN, NFM.⁷⁶ In the latter, GMSCs expressed increased neural stem cell markers, nestin and SOX1, in the first phase of differentiation. Neuronal-like cells expressed β-tubulin III, CNPase, GFAP, MAP2, NFM, pan-Nav, GAD67, Nav1.6, NF1, NSE, PSD95, and synapsin after the second phase of differentiation to maturity.⁷⁷ Throughout the transition from immature to mature neural cell differentiation, a combination of markers may be seen. For example, Rao et al. confirmed the existence of Nissl bodies, the increased expression of Nestin, β-tubulin III, as well as MAP2 in GMSCs encapsulated by a 3D bioconjugated protein hydrogel undergoing neuronal differentiation.⁷⁸ Conversely, Marynka-Kalmani et al. demonstrated a reduction in the expression of β-tubulin III and glial fibrillary acidic protein, along with induction of NeuN and MAP2 expression.⁵⁸

Overall, induced GMSCs appear to be more neurogenic compared to other oral stem cells such as UCMSCs, either morphologically or on levels of neurogenic gene expression.^37,77 Among GMSCs, it seems that neural-crest-derived GMSCs(N-GMSCs) are more capable of differentiating into neural cells than mesoderm-derived GMSCs(M-GMSCs).²⁰ Interesting, hGMSCs still retained the capability of spontaneously differentiating into neural precursor cells over prolonged passages, as reported by Rajan et al.⁷⁹ In addition, it has been noted that neurogenic differentiation of GMSCs can be boosted through hypoxia preconditioning,⁸⁰ treatment with Cannabidiol (CBD),⁴⁶ Metformin,⁸¹ or AEDG peptide.⁸²

• •Auditory progenitor cell

Regarding the potential of GMSCs to differentiate into auditory progenitor cells, Pouraghaei et al. proposed encapsulating them inside RGD-Coupled Alginate-Matrigel Nanohybrid Hydrogels, an optimized three-dimensional scaffold, and coculturing them with certain growth factors such as insulin-like growth factor (IGF), FGF-b and EGF. The expression of a set of preplacodal ectoderm (PPE) markers (GA-TA3, SOX2), early otic markers (PAX2, PAX8), and the auditory specific marker Myo7a during differentiation of the encapsulated cells was assessed to evaluate the ability of the hydrogel to induce auditory progenitor cell differentiation of GMSCs, both in vitro and in vivo. Results obtained from the in vitro studies were confirmed in vivo with positive Immuno-fluorescent staining against GATA3, SOX2, PAX2, and PAX8. While this suggests that GMSCs show favorable characteristics for auditory progenitor cell production in the presence of the necessary signaling factors, in vitro results differ in that Myo7a was not detected in the subcutaneous nude mice model.⁸³

• •Odontogenic cell

Odontogenic differentiation can be achieved by inducing GMSCs with an embryonic tooth germ cell-conditioned medium (ETGC-CM).⁵⁹ The ETGC-CM induction promotes the proliferation of GMSCs, resulting in significantly elevated levels of gene expression associated with odontogenesis, including ALP, BSP, OPN, and dentin matrix protein 1 (DMP-1).⁵⁹ MiR‐3940‐5p could encourage osteo/dentinogenic differentiation but may simultaneously hinder the cell proliferation of GMSCs, as evidenced by Han et al.⁸⁴ The increase in differentiation is noted by the expression of the vital transcription factor DLX5 and markers DMP-1, DSPP. Overall, alkaline phosphatase activity and mineralization in vitro was enhanced by the over‐expression of miR‐3940‐5p.

• •Keratinocyte

Differentiating GMSCs into keratinocytes is a considerable challenge, but Girija et al. hinted at this possibility by inducing the morphology of GMSCs with plant extracts of Acalypha indica. This prompts the cells to adopt a characteristic feature of keratinocytes: the polygonal cobblestone shape. Following this morphological change, a significant increase in the expression of basal keratinocytes markers in the cultures was observed either in two‐dimensional or three-dimension forms, notably cytokeratin 10, cytokeratin14 (KRT14), and cytokeratin 5. Moreover, the gene expression of progenitor's markers for maintaining epidermal homeostasis, filaggrin and stratifin, was also up-regulated. Some terminal differentiation markers were also upregulated relative to un-differentiated cells, such as involucrin.⁸⁵

3.4.3. Endoderm lineage cell differentiation (insulin producing cell clusters)

More recently, Kharat et al. converted adherent fibroblast-like GMSCs into floating insulin producing cell clusters (IPCCs) in a ten-day regimen involving a three-step sequence. They combined numerous differentiators, and identified insulin in the IPCCs by dithizone staining. Moreover, it was confirmed that these IP-CCs secrete insulin in response to glucose by glucose-stimulated insulin secretion (GSIS) assay. RT-PCR analysis of these IPCCs confirmed the expression of certain pancreatic markers (e.g., insulin, pdx1, glucagon, GLUT4, GLUT2). While there is insufficient reporting, IPCCs might be used to screen antidiabetic drugs in diabetes research, and emerge as a novel source of autologous tissue for islet transplantation in the treatment of diabetes.⁸⁶

3.5. GMSCs-mediated modulatory effects on immunity

Adaptive immunity and innate immunity are two divisions of the human immune system. The innate immune system is comprises of numerous cells such as monocytes/macrophages, dendritic cells (DCs), neutrophils, and natural killer (NK) cells, this acts as the first line of host defense.⁸⁷ The adaptive immune system is categorized into two lymphocyte-driven subdivisions: a T-cell-mediated and a B-cell antibody-mediated immune responses.^87,88

The immunomodulatory impacts of oral MSCs are thought to be mediated via both direct cell-to-cell interaction and the secretion of soluble cytokines, such as IL-1, IL-6, IL-10, transforming growth factor-β1 (TGF-β1), nitric oxide (NO), indoleamine 2,3-dioxygenase (IDO), and prostaglandin E2 (PGE2).⁸⁹ Notably, many studies have demonstrated that GMSCs exert powerful immune regulation and anti-inflammatory functions. These effects are achieved by regulating cell phenotypes and by activating various subsets of innate and adaptive immune cells, notably mast cells, macro-phages, dendritic cells, various subsets of T helper cells both in vitro and in vivo.^90,91 Fig. 2 presents the immunomodulatory network of GMSCs.

Fig. 2.

| Immunomodulatory effects of GMSCs. Schematic immunomodulatory effects of GMSCs on both innate and adaptive immune cells. In terms of natural immune cells, GMSCs cause M2 polarization of macrophages by secreting IL-6 and GM-CSF. It leads to heightened levels of CD206, IL-10, and TGFβ, as well as a decreased level of TNFα. At the same time, the number of foam cells and M1 macrophages decreased, resulting in a decreased secretion of TNFα, IL-6, IL-1β by M1 macrophages. The activation and differentiation of dendritic cells are inhibited by GMSCs through a mechanism reliant on PGE2, as well as the activation and degranulation of mast cells, resulting in a downregulated expression of TNFα and upregulated expression of IL-6, IL-4. Exogenous TNFα could stimulate the secretion of PGE2 in GMSCs. Contrarily, naive T cells can be stimulated by mitogens to turn into activated T cells, and secrete IFNγ. In response to IFNγ produced by activated T cells, GMSCs release IDO and IL-10, which suppress T cell proliferation and IFNγ secretion. GMSCs exert apoptotic effect on T cells through the FasL/Fas pathway (Fas lig-and/Fas receptor), and hypoxia enhances FasL expression and IL-10 secretion of GMSCs. T cells are classified into two categories: helper T cells (CD4⁺ T cells) and cytotoxic T cells (CD8⁺ T cells). CD4⁺ T cells are able to differentiate into Th17 (helper T cells) and Treg (regulatory T cells) under the stimulation of inflammatory factors. The secretion of PGE2 by GMSCs is responsible for the inhibition of CD8⁺ T cells and Th17 cells, while for the stimulation of Tregs. Additionally, GMSCs could attenuate the activation and proliferation of B cells and PBMCs.

Abbreviations: IL-6 (interleukin 6), GM-CSF (granulocyte macrophage colony-stimulating factor), IL-10 (interleukin10), TGFβ (transforming growth factor β), TNFα (tumor necrosis factor α), IL-4 (interleukin 4), COX2 (cyclooxygenase 2), PGE2 (prostaglandin E2), IDO (indoleamine 2,3-dioxygenase), IFNγ (interferon γ), FoxP3 (forkhead box P3), IgG (immunoglobulin G), IgM (immunoglobulin M), PBMCs (peripheral blood mono-nuclear cells).

3.5.1. Immunomodulatory effects on innate immune cells

• •Regulation of Macrophages

Macrophages are known to be the pillars of the innate immune response system, but they also hold supporting roles in the broader processes of development, tissue repair, and homeostasis. These have been categorized into two major subpopulations: the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype.^92,93 Based off the current understanding, MSCs from different tissue sources can promote M2 macrophage polarization, while effectively inhibiting M1 macrophage polarization. This leads to a decreased expression of genes related to M1 (e.g., CD86, iNOS), and proinflammatory cytokines such as TNF-α, IL -6, IL-1β, but an increased M2-associated expression of CD206, CD163, arginase-1, and secretion of anti-inflammatory cytokines (e.g., IL-10 and TGF-β).⁹⁴

Zhang et al.’s study had demonstrated the capability of GMSCs to polarize macro-phages into the M2 phenotype, characterized by the upregulated expression of CD206, IL-10, and increased phagocytotic activity. A GMSCs co-culture has shown to concurrently suppress M1 macrophage polarization while reducing the expression of TNF-α and CD86. Notably, in a murine skin wound healing model, protein expressions of M2 macrophage-related genes RELMα and arginase-1 were promoted by GMSCs transplanted to the injury site. It may be concluded that the soluble factors secreted by GMSCs, IL-6 and granulocyte macrophage colony-stimulating factor (GM-CSF), enhance cutaneous wound healing.⁹⁵ In an in vivo trial, apolipoprotein E knock out (ApoE−/−) mice with a high fat diet-induced atherosclerosis experienced a significant and similar decrease in the total macrophages frequency following systemic infusion of human GMSCs, specifically F4/80+CD16/32+M1 macrophages in the spleen, in peripheral blood, and in lymph nodes. The decreased expression of MHCII (antigen presentation marker) on macrophages and the expression of CD68⁺ (phagocytic marker) macrophages were observed in aortic tissues of ApoE−/− mice.⁹⁶ Meanwhile, the results indicated that GMSCs promoted the polarization of CD206+ M2 macrophages and inhibited M1 macrophage marker expression such as HLA-DR and CD86. GMSCs also reduced the frequencies of CD11b + monocytes, particularly, the pro-inflammatory subset of CD11b + Ly-6Chi, and inhibited ox-LDL-induced foam cell formation in vitro. These results further corroborate the possible modulatory effect of GMSCs on macrophages, partly through CD73 and IDO signaling pathways.⁹⁶

Additionally, the expressions of inflammatory cytokines TNF-α, IL-1β, and IL-12 were significantly reduced when GMSC-derived exosomes were co-cultured with activated M1 macrophages induced by LPS and IFN-γ. Anti-inflammatory cytokine IL-10 expression levels were moderately increased, while those of the M1 macrophage marker CD86 were significantly lower than that of the control group (only M1 macrophage culture). Due to an insignificant difference in the expression of CD163, an M2 macrophage marker, it may be assumed that GMSC-derived exosomes inhibit M1 macrophage activation, but they are limited in their capacity to stimulate M2 macrophage polarization.⁹⁷ These findings indicate how GMSCs carry a remarkable immunomodulatory effects on macrophages activation and phenotype.

• •Regulation of Mast cells

Mast cells (MCs) are a key component of innate immune cells. Under the activation of various environmental stimuli, mast cells release biologically active mediators through degranulation, and participate in immune regulation. MCs recruit inflammatory cells which amplifies the inflammatory response and promotes an adaptive immune response. These responses regulate angiogenesis, vascular permeability, and fibrosis.⁹⁸ A dysregulation of MC functions could initiate and exacerbate numerous pathological diseases or conditions (e.g., malignancies, allergic and anaphylactic reactions, fibrosis, abnormal wound healing). Equally, co-culture of GMSCs and HMC-1 (human mast cell line) reduced overall CD117 MC marker expression, and inhibit the secretion of TNF-α, IL-6, and IL-4 by HMC-1 through a prostaglandin E2 (PGE2)-dependent mechanism.⁹⁹ GMSCs could reduce the percentage of HMC-1 that produces TNF-α, IL-6, and IL-4, but seemingly has no effect on the proliferation of HMC-1. Meanwhile, activated HMC-1 can upregulate the expression of cyclooxygenase-2 and prostaglandin E2 (COX2/PGE2) in GMSCs through a TNF-α-dependent mechanism. When performed in vivo, the administration of GMSCs significantly suppressed MC degranulation and attenuated chronic hypersensitivity (CHS) of mice skin.⁹⁹ Thus, the TNF-α/PGE2 feedback loop played a key role between GMSCs and MCs.

• •Regulation of Dendritic cells

Messengers of the immune systems, dendritic cells (DCs) are the most functional antigen-presenting cells known; they can efficiently uptake, process, and present antigens, and are important cells bridging innate immunity and adaptive immunity.¹⁰⁰ Deregulated functions of DCs, either hyperactivated or tolerogenic, could set off a cascade of various diseases, such as autoimmune diseases, rejection of organ transplantation, and malignancies.^100,101 There is now limited evidence of the role GMSCs may play as an immunomodulator in the differentiation, maturation, and antigen presentation of DCs. A coculture with human GMSCs has demonstrated a significant inhibition of CD11c and CD80 expression, two mature DC markers, while reducing pro-inflammatory cytokine IL-12 secretion by DCs in response to LPS stimulation. This capability to directly suppress the maturation and differentiation of DCs through a PGE2-dependent mechanism supports the notion that PGE2, but not IL-10, TGF-b1 and IDO, plays a dominant role in the GMSC-mediated immunosuppressive effects on DCs.⁹⁸

3.5.2. Immunomodulatory effects on adaptive immune cells

• •Regulation of peripheral blood mononuclear cells

It has been reported that GMSCs can suppress peripheral blood mononuclear cells (PBMCs) activation and proliferation, especially those activated by allogenic lymphocytes, phytohemagglutinin (PHA),¹⁹ and ConA.¹⁰² The inhibitory effect is positively correlated with the proportion of GMSCs in the coculture.^32,52 GMSCs inhibited PBMCs through either cell-cell contact or through the secretion of soluble factors, among which soluble factors IDO and IL-10 played a significant role. The expression of IDO and IL-10 increased significantly when GMSCs and PBMCs were co-cultured, although adding anti–IFN–γ antibody to the co-culture system decreased the expression of these two soluble factors. It seems as though IFN-γ promotes the release of active IDO and IL-10 from GMSCs in a dose-dependent manner, which suggests that secretion of IFN-γ by PBMC could correspondingly promote the release of IDO and IL-10 from GMSCs.¹⁹ Another factor improving the immune regulation function of GMSCs is hypoxia. A hypoxic environment can improve the inhibitory effect of GMSCs on PBMCs, inhibit the proliferation of PBMCs, and increase their apoptosis, which is related to the expression of Fas ligand (FasL) on the stem cells.^80,102

• •Regulation of B cells

B cells hold pivotal roles in autoimmunity, whether it be antigen presentation, antibody secretion, or complement activation. These immune effector cells of the adaptive immune system can be modulated by the effects of MSCs. DCs mediated antigens indirectly regulated by MSCs can direct B cell proliferation and differentiation, inhibit B cell apoptosis, and suppress the adaptive immune response. For example, MSCs can induce the generation of regulatory B cells (Bregs) characterized by the secretion of IL-10 and potent immunosuppressive functions.^103,104 Dang et al. demonstrated that hGMSCs have immunosuppressive effects on B cells in a spontaneous lupus nephritis murine model.¹⁰⁵ In their in vitro studies, it was proved that GMSCs can suppress the expression of CD25, CD69, CD80 and CD86 on B cells, suggesting that they directly suppress the antigen presenting ability (CD80 and CD86) and B cell activation. GMSCs did display a better capacity to suppress B cells in late activation (CD25) compare to those in early activation (CD69), but affected both activation stages nonetheless.¹⁰⁵ The dose-dependent inhibitory effect on IgG and IgM secretion, and on B-cell proliferation was observed mechanistically through the CD39⁻CD73 signaling pathway. These in vitro results were successfully corroborated by in vivo experiments.¹⁰⁵

• •Regulation of T cells

T cells are the pillars of the adaptive immune response. These lymphocytes can be classified into three broad categories according to their functions: T helper cells, T cyto-toxic cells, and regulatory T cells. T helper cells are a type of CD4⁺ T cells, including Th0, Th1, Th2, Th9, Th17, Th22, Tfh, etc.⁸⁸ Among them, the unactivated naïve CD4⁺ T cells are Th0, which differentiate into different types of T helper cells under the stimulation and regulation of various antigens and cytokines. Whereas, to counter-balance any immune responses generated by T helper cells, CD4⁺CD25+FoxP3+T regulatory cells (Treg) can contribute to immune homeostasis by producing anti-inflammatory cytokines such as IL-10 and TGF-β.

In a mechanism similar to that of PBMC inhibition, GMSC inhibits T cell proliferation, promotes T cell apoptosis, and enhances Treg function through cell-to-cell contact, secretion of enzymes and soluble cytokines. Several research projects have revealed that GMSC can in vitro impede the proliferation and activation of T cells by elevating IL-10 levels and reducing tryptophan through IDO generated by GMSCs-produced IDO under the stimulation of inflammatory cytokines IFN-γ and TGF-α.^19,90,106 Equally, IFN-γ which is produced by activated T cells functions as a regulator in the feedback signaling between T cells and GMSCs.¹⁹ It has additionally been noted that GMSCs express CD39 and CD73 at levels high enough to trigger the CD39/CD73/adenosine signaling pathway which inhibits the proliferation of effector T cells, suppresses the differentiation of Th17 and Th1 cells, reduces the expression of IL-17 and IFN-γ, and up-regulates CD4+Treg cells.^107,108

When assessing the counterbalancing effect that they have on T helper cells, it was noted that GMSCs stimulate the secretion of Foxp3, IL⁃10 and TGF⁃β, and reduce the ratio of IFN⁃γ/IL⁃4. Thus, restoring the balance of Th1/Th2 by releasing PGE2.¹⁰⁹ Proliferation rates of Th1 and NK cells, specifically, were reduced via the FasL/Fas-mediated pathway.¹⁰² The reduced production of IFN-γ and IL-17 by Th1 cells, was contrasted by an enhanced secretion of IL-4 by Th2 cells. In the even that FasL expressed by GMSCs combines with Fas expressed by activated T cells, this Fas/FasL signaling pathway will promote T cell apoptosis. The apoptotic T cells, in turn, can promote M2 macrophages to produce TGF-β, thereby upregulating Tregs, while Hydrogen sulfide significantly contributes to this process.¹¹⁰

By promoting the transformation of B cells into regulatory B cells (Breg), GMSCs can indirectly inhibit the expression of CD4+T cells, the secretion of pro-inflammatory cytokines and the differentiation of Th1 and Th17 cells. Although IDO plays an important role in the immunoregulation of human MSCs, mouse MSCs do not produce IDO,¹¹¹ suggesting that the immune regulation of MSCs may be species specific. In conclusion, GMSCs may form complex regulatory networks to regulate both T cell and B cell subsets through multiple cytokine-dependent pathways.

4. Therapeutic applications of GMSCs

Unraveling the biological properties of GMSCs will improve our understanding of their prospective therapeutic applications. Their potential for self-renewal and multi-lineage differentiation could set a new standard in regenerative therapy and tissue engineering, while their powerful immunomodulatory and anti-inflammatory capacities may challenge the current approaches to treating inflammatory, autoimmune, and other diseases. GMSCs and GMSC-derived cell-free products (i.e. EVs and exosomes) secreted into the conditioned medium (CM) are being evaluated in many preclinical studies and initial clinical trials, as prospective therapeutics for various pathological conditions and diseases.^91,112,113 Fig. 3 details the sequence of adapting GMSCs and their cell-free products for use in medical therapy.

Fig. 3.

| Therapeutic applications of GMSCs and their cell-free products. GMSCs can be turned into optimized primed GMSCs by genetic modification and nongenetic priming. Products of GMSCs such as the cell content, conditioned media (CM), and extracellular vesicles (EVs) may be delivered with or without scaffolds and growth factors. Preclinical models have suggested an unparalleled regenerative and therapeutic potentials of GMSCs in human disorders.

4.1. Applications in tissue engineering

To benefit from the enticing properties of GMSCs in Tissue engineering (TE), it is useful to consider the accumulating evidence suggesting their benefit in the treatment of various diseases. Unlocking this potential will require the support from scaffolds, cell seeding, and growth factors.¹¹⁴ Previous studies have showed that GMSCs under the conditions of a standard two-dimensional (2D) cell culture can adhere quickly to the wall, and take on the shape of a long spindle, as they grow. There are, however, distinct differences between GMSC under 2D and three-dimensional culture conditions, notably in proliferation, morphology, cell differentiation ability, and biological function. Under conditions of low cell adhesion, GMSCs can spontaneously aggregate into compact 3D spheres (3D-GMSCs), having a diameter of 400∼500 μm. Compared to 2D-GMSCs, 3D-GMSCs have an early stem cell phenotype, an enhanced ability to survive, a resistance to oxidative stress, and a capability of multi-lineage differentiation, whereas their proliferation rates are significantly reduced.^115,116 A key concept stem cell tissue engineering is the synthesizing scaffolds inoculated with GMSCs in suspension, and implanting them into tissue defects. Scaffold carriers can provide mechanical support to cells and protect them from harmful microenvironments in vivo, while promoting cell survival, proliferation, and differentiation. At this stage, 3D cell culture methods in vitro based on scaffolds such as polylactide,¹¹⁷ alginic acid/hyaluronic acid¹¹⁸ and bovine pericardium membrane¹¹⁹ can better simulate the mechanical properties and physiological characteristics of a microenvironment similar to one in vivo. The promising results of these early studies have popularized these 3D cell culture methods, thus breaking into the mainstream of GMSC applications in tissue engineering.

4.1.1. Applications in the cranio-maxillofacial region

• • Cranio- Maxillofacial Bone Defects

Conceptually, repairing cranio-maxillofacial bone defects generally requires differentiating locally transplanted hGMSCs mixed with type I collagen gel bound to osteocytes. Past trials have performed such test in vivo on mandibular and critical-sized calvarial bone defects in rats, to promote new bone formation.⁶⁹ Systemically transplanted GMSCs can not only target to the mandibular defect site in a C57BL/6 J mice model but also promote bone regeneration, as confirmed by Xu et al.¹²⁰ The more recent trend is to use carriers, scaffolds, and growth factors to support osteogenic repair. Table 3 summarizes the applications of GMSCs in the tissue engineering of cranio-maxillofacial bone defects.

Table 3.

GMSCs and their cell-free products application in tissue engineering (Cranio-maxillofacial Bone Defects).

Application	Cell Type/Products	Scaffold/Factor	Route	Model	Ref
Calvarial Bone Defect and Mandibular Defect	hGMSCs	Type I collagen gel	Local implantation	Rat	69
Calvaria Bone Defect	Autologous rat GMSCs	RGD-coupled alginate (Alg−RGD) with whitlockite and hydroxyapatite microparticles	Local implantation	Rat	120
Calvarial Defects	hGMSCs	RGD- (arginine-glycine-aspartic acid tripeptide) coupled alginate microen-capsulation system	Local implantation	Nude mice	121
Calvarial Defect	hGMSCs and hGMSCs EVs	A poly-(lactide) (3D-PLA) scaffold	Local implantation	Rat	122
Calvarial Defect	hGMSCs CM and/or hGMSCs	A poly-(lactide) (3D-PLA) scaffold	Local implantation	Rat	123
Calvarial Defect	hGMSCs EVs and/or hGMSCs	Poly (lactide) (3D-PLA) 3D printing	Local implantation	Rat	117
Calvaria Inflammatory Osteolysis	hGMSCs metabolite	–	Local subcutaneous injection	Rat	124
Mandibular Bone Defect	hGMSCs	–	IV injection	Mice	125
Maxillary Alveolar Defect	hGMSCs	Hydrogel scaffold PuraMatrix™ (PM)/BMP2	Local implantation	Nude rat	126
Maxillary Bone Defects	Autologous pig GMSCs	Bio-Oss®/SB431542	Local implantation	Mini pig	127

Alginate-based scaffolds and hydrogels may act as carriers of GMSCs for bone defect repair. In the study by Moshaverinia, GMSCs were encapsulated in an RGD-coupled alginate microspheres and transplanted into immunocompromised mice with critical-size calvarial defects. Bone regeneration was achieved with the use of GMSCs, however, the bone regeneration efficiency in the treatment group using PDLSCs was higher.¹²¹ Another team led by Sevari made similar use of RGD-coupled alginate (Alg−RGD), this time adding in a commercially modified alginate hydrogel, whitlockite microparticles (WHMPs), and hydroxyapatite microparticles (HapMPs). They assessed this compounded alginate's effect on the osteo-differentiation of encapsulated autologous rat GMSCs in the calvaria defect rat models. Substantial amounts of bone repair/fill was achieved in the WHMP-loaded hydrogel group, and there was a higher expression of osteogenic markers OCN and RUNX2 compared to other control groups.¹²²

A notable alternative to alginate is commercial poly (lactide), a three-dimensional scaffold (3D-PLA). Diomede et al. aimed to treat a critical-sized rat calvarial defect using a modified 3D-PLA, combining different concentrations of hGMSCs and their derivatives (e.g., hGMSC-EVs, and hGMSCs derived conditioned medium [CM]). Implanting the modified 3D-PLA into the site of cortical calvaria bone tissue damage lead to an improvement in bone healing, demonstrating better osteogenic properties.^123,124 Moreover, transcriptomic analysis in vitro showed the upregulation of genes in-volved in ossification and regulation of ossification in this experimental group.¹²⁴ In a subsequent study published by the same researcher, they proved that 3D-PLA, in combination with hGMSCs and hGMSCs-EVs, promoted both bone regeneration along and enhanced vascularization of induced calvarial defects in Wistar male rats, in comparison to control group. In vitro results showed an upregulation of miR-2861, miR-210, osteogenic and angiogenic markers (e.g., RUNX2, VEGFA, OPN, COL1A1), as observed from the cell culture in contact with the biomaterial and EVs.¹¹⁷

In addition to usage of scaffold or carriers, the incorporation of growth factors can remarkably improve the efficacy of bone regeneration in pre-differentiated GMSCs (dGMSCs) undergoing osteogenic differentiation. The addition of bone morphogenetic protein2 (BMP2) to a self-assembling hydrogel scaffold PuraMatrixTM (PM) showed a significant enhancement of bone regeneration in athymic nude rats with a critical-size maxillary alveolar defect by surgically created, compared to the group without the growth factor or the scaffold.¹²⁵ Recently, it was reported that metabolites from GMSCs have the ability to decrease the expression of transcription factors related to bone resorption such as tartrate-resistant acid phosphatase (TRAP), nuclear factor-activated T cells-1(NFATc1), and sclerostin in LPS-associated calvaria osteolysis of Wistar rats through immunohistochemically analysis.¹²⁶ Moreover, a mixture of a TGF-β signaling inhibitor (SB431542)-treated autologous pig GMSCs (pGMSCs) composited with Bio-Oss® were transplanted into circular confined defects in minipigs maxillary, and it could be remarkably observed that SB431542 treatment advanced new bone formation of pGMSCs in minipig maxillary bone defect model. Meanwhile, it was found that hGMSCs treated by SB431542 markedly increased bone-related proteins expression, and BMP2 as well as BMP4 gene expression. Conversely, SMAD3 protein-dependent TGF-β signal pathway phosphorylation was decreased.¹²⁷

• •Periodontal Diseases

Periodontitis is a chronic infectious disease caused by dental plaque biofilm, which manifests as an inflammatory response in the tooth-supporting structures, leading to periodontal attachment loss and alveolar bone resorption. Clinically speaking, conventional treatment strategies are limited in their efficacy to promote the regeneration of damaged periodontal tissues. GMSCs systemically administered in vivo could pinpoint to the periodontal injury area and differentiate into osteoblasts, cementoblasts, or periodontal ligament (PDL) fibroblasts. They have been proven to significantly enhance the injured periodontal tissue regeneration, specifically cementum, functional PDL, and alveolar bone in beagle dogs models subjected to class III furcation.¹²⁸

The implantation or systemic transplantation of GMSCs is yielding new insights on potential treatment of periodontal defects. Tested on miniature pigs model, the implantation of autologous GMSCs combined with IL-1ra-releasing hyaluronic acid synthetic extracellular matrix (HA-sECM) promoted periodontal regeneration in defects pertaining to molar and premolar sites.¹²⁹ GMSCs may specifically target to injured areas and enhance periodontal tissue regeneration when systemically transplanted. Following systemic transplantation via the tail vein, C57BL/6 J mice healed with significantly greater alveolar bone heights, in comparison to control groups. In fact, GMSCs were present in the regenerated alveolar bone and surrounding periodontal ligament.¹³⁰ Another report suggests that systematic transplantation of hGMSCs equally attenuates hyperlipidemia and inflammatory responses. An extensive periodontal tissue regeneration of hyperlipidemic mice with periodontitis was observed by comparing the increase in alveolar bone height and new bone formation relative to the control groups.¹³¹

There is a growing body of evidence suggesting that incorporating a scaffold with GMSCs may induce periodontal regeneration. In a recently published study, Sanchez et al. established a dog model with gingival recession type 1, a dehiscence-type gingival defects, and investigated the therapeutic impact of a xenogeneic collagen matrix (CMX) seeded with autologous GMSCs and combined with a coronally advanced flap (CAF). Incorporating autologous GMSCs into the CMX led to decreased inflammation and varying levels of newly formed cementum or bone.¹³² With regard to intrabony periodontal defects, a recent randomized clinical trial explored the potential clinical applications of gingival fibroblasts (GF) and GF-derived MSCs. The transplantation of a β‐calcium triphosphate (β‐TCP) scaffold loaded with autologous GMSCs into the defects under a collagen membrane sharply reduced the vertical pocket depth (VPD), clinical attachment loss (CAL), and raised the radiographic bone gain as compared with other control groups at 6 months after surgery.¹³³

GMSC derivatives such as EVs and conditioned mediums similarly play an important role in periodontal regeneration. Small extracellular vesicles (sEVs) derived from GMSC are capable of decreasing the secretion of pro-inflammatory cytokines from monocytes/macrophages and T cells. By suppressing T-cell activation and inducing the formation of Tregs in vitro and in a rat model with periodontal disease, Zarubova's group achieved similar therapeutic effects in both models relative to the baseline cells. They successfully developed a delivery platform that engineered metalloproteinases at the affected site to release EVs sensitive to their microenvironment, complemented by antibiotics to inhibit local bacterial biofilm development.¹³⁴ GMSC-conditioned mediums (GMSC-CM) have been likewise proven to enhance periodontal tissue regeneration by regulating the expression of inflammatory factors TNF⁃α, IL⁃1β, IL⁃10. This regulation promoted osteoblast differentiation in a rat model with periodontal defects, where PDLSC-CM and GMSC-CM groups exhibited a significantly greater quantity of regenerated periodontal tissue in comparison to the control.¹³⁵ Exosomes consist of another derivative of GMSCs possibly capable of periodontal regeneration. Nakao et al. found that local injection of TNF- α-preconditioned exosomes significantly reduced periodontal bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in ligature-induced periodontitis mice model.¹³⁶ Therefore, it appears that the soluble factors secreted by GMSCs are adequate to induce periodontal regeneration. The therapeutic benefits of GMSC-CM, PDLSC-CM, and other GMSC derivatives such as exosomes on periodontal regeneration are promising, but the current evidence is limited.

• •Peri-Implantitis

In a well-established model of a rat with peri-implantitis, the application of a adhesive hydrogel biomaterial (AdhHG) encapsulating GMSCs resulted in complete bone regeneration around ailing dental implants with peri-implant bone loss, followed by a site-specific attenuation of the inflammatory response.¹³⁷ Further experiments have concluded that GMSC proliferation and viability might be inhibited by a high a concentration of titanium, to the extent of triggering apoptosis. While zirconia nanoparticles did not inhibit GMSC in these aspects, both types of implant surface materials affected the production of inflammatory mediators by hGMSCs.¹³⁸ Moreover, a Poly D, l-lactide (PDLLA)- Laminin 332 composite coating can regulate the behavior of GMSCs and keratinocytes, fundamentally enhancing the implant soft tissue-sealing delivery vehicle carrying antimicrobial properties.¹³⁹ The alternative use of a RGD-modified alginate hydrogel containing a silver lactate (SL) to encapsulate GMSCs did strengthen the antimicrobial properties on the surface of titanium discs and improved the GMSCs’ capability of osteogenic differentiation, however these results were limited to A. actinomycetemcomitans bacteria.¹⁴⁰ Overall, these studies demonstrate the promising potential of GMSC-based regenerative therapy of peri-implantitis.

• •Regeneration of Submandibular Salivary Glands

A study by El-Latif et al. demonstrated the possibility of regenerating salivary glands cells at the cut borders of a partially dissected submandibular salivary gland. Injecting GMSCs directly at the cut area of a rat model and gluing with autologous fibrin glue significantly ameliorated ductal, acinar, and myoepithelial cell regeneration, compared to the use of fibrin glue alone.¹⁴¹

• •Palatal/Gingival Defects

Testing on female mice with full-thickness circular gingival wounds in the palate showed that local injection of GMSCs-EVs significantly facilitated the palatal defect healing, and unveiled the biochemical mechanism behind such a process. It is Fas binds with Fas-associated phosphatase–1 (Fap-1) and caveolin-1 (Cav-1) axis that regulates a common soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE)-mediated small extracellular vesicles (sEVs) and interleukin-1 receptor antagonist (IL-1RA) secretion in stem cells, which contributes to accelerated wound healing.¹⁴² Similarly, human fetal GMSCs significantly promoted wound closure and re-epithelialization of rat gingiva following local transplantation of the defect. The defect had decreased one week following transplantation, and at 3 weeks, the gingival tissue at the defect site regained color, morphology, and height comparable to healthy gingiva of the control group.¹⁴³

• •Tongue Defect and Muscle Regeneration

Preliminary attempts have been made to regenerate tongue and muscle defects with a combinatory transplantation of small intestinal submucosa–extracellular matrix (SIS-ECM) and GMSCs or their derivative exosomes. The stem cell construct successfully facilitated the restoration of lingual papillae and the regeneration of taste buds in a rat model with a critical-sized tongue defect, as evidenced by increased expression of CK14, CK8, BDNF, shh and markers for type I, II, and III taste bud cells. Additionally, GMSCs or their derivative exosomes promoted innervation of the regenerated taste buds, evidenced by elevated expressions of neurofilament and P2X3 at the injury areas.¹⁴⁴ In another study, the same research group found that if the GMSC/SIS-ECM construct was engrafted at the host recipient site, it would effectively reduce fibrosis, promote soft tissue healing, and regenerate the muscular layer at the wound healing site. Compared to SIS-ECM alone or the nontreated defect controls, the GMSC/SIS-ECM group exhibited a significantly increased expression of several myogenic transcriptional factors such as MyoD, PAX7, Myf5, while suppressing the expression of type I collagen at the wounded area of the tongue.¹⁴⁵ In sum, these findings suggest that GMSC/SIS-ECM and exosome/SIS-ECM constructs could define novel approaches in regenerative tongue reconstruction and post-surgical rehabilitation.

• •Chemotherapy-induced oral mucositis

Oral mucositis is generally characterized by an impaired regenerative capacity of the oral and alimentary epithelium. The vicious circle of atrophy, erythema, and ulcer-ation eventually leads to the loss of mucosal barrier functions. A study by Zhang's re-search group showed how systemic administration of hGMSCs or 3D-GMSC spheroids can remarkably mitigate oral mucositis in mice. Balb/c mice were given an intraperitoneal (i.p.) injection of 5-fluorouracil to induce oral mucositis. Compared to their adherent cells, the spheroid-derived GMSCs demonstrated a greater therapeutic efficacy in reversing body weight loss and promoting the regeneration of disrupted epithelial lining of the mucositic tongues.¹¹⁶ These promising results underscore the regenerative potential of GMSCs in the treatment of chemotherapy-related mucositis. A summary of studies on the applications of GMSCs for periodontal, oral, and tongue defects is presented in Table 4.

Table 4.

| GMSCs and their cell-free products application in tissue engineering (Periodontal diseases, Tongue defects, Oral mucositis).

Application	Cell Type/Products	Scaffold/Factor	Route	Model	Ref.
Class III furcation defects	hGMSCs	–	IV injection	Dog	128
Periodontal bone loss	hGMSCs-Exosomes	TNF-α precondition	Local injection	Mice	136
Periodontitis	hGMSCs	–	IV injection	Mice	130
Periodontitis bone loss	hGMSCs	–	IV injection	ApoE−/− mice	131
Dehiscence-type defect	Autologous canine GMSCs	Collagen matrix and a coronally advanced flap	Local implantation	Dog	132
Intrabony periodontal defect	hGF and hGMSCs	β-calcium triphosphate (β-TCP) followed by collagen membrane defect coverage	Local implantation	Clinical trail	133
Periodontitis	hGMSC sEVs	“Dual delivery” microparticles and antibiotics	Local injection	Rat	134
Periodontal defects	hGMSCs CM	Collagen scaffolds (Bio-Gide)	Local implantation	Rat	135
Periodontal defects	Autologous pig GMSCs	IL-1Ra-hyaluronic acid synthetic extracellular matrix (HA-sECM), collagen membrane (Bio-Gide) defect coverage	Local implantation	Mini pig	129
Peri-implantitis	Rat GMSCs	Alginate-based adhesive, photocrosslinkable hydrogel (AdhHG)	Local implantation	Rat	137
Partially dissected submandibular salivary glands	Rat GMSCs	Fibrin glue	Local injection	Rat	141
Palatal wound	Mice GMSCs-EVs	–	Local submucosal injection	Mice	142
Gingival defects	Human fetal GMSCs	–	Local injection	Rat	143
Tongue defects	hGMSCs	small intestinal submucosa–extracellular matrix (SIS-ECM)	Local implantation	Rat	145
Tongue defects	hGMSCs-EVs	SIS-ECM	Local implantation	Rat	144
Chemotherapy-induced oral mucositis	hGMSCs	3D GMSCs spheroids, or 2D GMSCs	IV injection	Mice	116

4.1.2. Application of non-craniomaxillofacial region

• •Nerve Regeneration

4.1.2.1. Facial nerve injury

Under different experimental conditions, numerous studies have explored the nerve regeneration potential of GMSCs and their EV-derived byproducts. One report demonstrated that certain culture conditions may consistently reprogram or induce human GMSCs into neural crest stem-like cells (NCSC) without the addition of genetic transcriptional factors. Compared to parental GMSCs, induced NCSC population had an increased expression of NCSC-related genes and displayed a robust differentiation into neuronal and Schwann-like cells.¹⁴⁶ In a rat model with a facial nerve defect, NCSC-like cells induced from human GMSCs in combination with a nerve conduit, an extracellular matrix coaption aid, were trans-planted into the site. The transplanted cells exhibited a significantly enhanced ability to facilitate the regeneration and the functional recovery of the subject's facial nerve, relative to their parental GMSC counterparts.¹⁴⁶ Furthermore, a scaffold-free 3D bio-printer system may use GMSC spheroids to print nerve constructs without the use of exogenous scaffolds, which is subsequently matured in a bioreactor. The 3D-bioprinted GMSC-laden scaffolds was capable in promoting regeneration, axonal regeneration, and functional recovery of injured facial nerves following in vivo transplantation tion.¹⁴⁷ These findings lay down the foundation for employing GMSCs for repair and regeneration of peripheral nerve defects.

4.1.2.2. Sciatic nerve injury

In a sciatic crush injury model in female Sprague-Dawley rats, GMSCs transplanted to the injured area could differentiate into neuronal cells, whereas GMSC-derived neural progenitor cells (NPC) were able to differentiate into both neuronal and Schwann cells in vivo. Following transplantation, GMSC-derived NPCs displayed superior therapeutic effects on axonal regeneration at both the injury site and the distal segment of the injured sciatic nerve. The complete mechanism behind this regenerative ability is yet to be deciphered, but it is hypothesized that transplantation of GMSCs and NPCs promotes peripheral nerve repair/regeneration by promoting remyelination of Schwann cells mediated via the regulation of the antagonistic myelination regulators, c-Jun, and Krox-20/EGR2.¹⁴⁸ Additionally, locally wrapping gel-foam embedded with GMSC-derived EVs can significantly encourage functional recuperation and axonal regeneration of crush-injured sciatic nerves in mice models.¹⁴⁹ In sum, GMSC-EVs or GMSC-derived NPCs repair peripheral nerve injury presumably by encouraging Schwann cells adaptation towards a repair phenotype, distinguished by the increase of key transcriptional factor c-Jun expression and proliferation.^148,149

Similarly, Rao et al. reported that exosomes derived from GMSCs could markedly enhance the proliferation of Schwann cells and the growth of DRG axons in vitro. Moreover, it was observed that the use of a chitin conduit along with GMSCs exosomes substantially augmented both the quantity and diameter of nerve fibers in vivo studies. This promoted a myelin formation to repair the sciatic nerve segmental defect, achieving the therapeutic aims of recovering motor and muscle function, and nerve conduction.¹⁵⁰ Conversely, GMSCs can differentiate into Schwann-like cells (GiSCs) once mixed with methacrylated collagen and cultured for 48 h. When a functionalized nerve protector is repopulated with GiSCs and is implanted in rats with crush-injured sciatic nerves, it accelerates axonal regeneration and functional recovery. The accompanying increase in pro-regenerative macrophages (M2) infiltration is contrasted by a decrease in pro-inflammatory macrophages (M1) infiltration. This alteration was evidenced by a significant increase in IL-10 secretion, and remarkable reduction in TNF-1α and IL-1β by LPS-stimulated secretion.¹⁵¹

4.1.2.3. Spinal cord injury

The versatility of GMSCs equally encompasses the ability to repair or regenerate spinal cord injuries (SCI). Mammana et al. investigated the anti‐inflammatory, anti‐apoptotic, and regenerative effects of GMSCs pretreated with nanostructured liposomes enriched with MOR in ICR (CD‐1) mice model of SCI. The results showed that intravenous injection of MOR‐treated GMSCs could promote SCI repair and restore normal spinal cord morphology. This is marked by a decrease in inflammatory cytokines IL‐1β and IL‐6, and by a reduction of spinal cord COX‐2 and GFAP levels. MOR‐treated GMSCs influenced the apoptotic pathway by suppressing Bax, caspase 3, and caspase 9 expressions.¹⁵² It is important to note that other factors regulating the inflammatory response in spinal cord injury and repair have been identified. A hemi-transection spinal cord injury study in rats reported that neuronal stem cells derived from human gingiva locally alleviate the inflammatory response, promote the repopulation and engraftment of transplants, and stimulate the new formation of synaptic vesicles at the injury site, when directly transplanted in an injectable caffeic acid bio-conjugated hydrogel (CBGH).¹⁵³

4.1.2.4. Cavernous nerve injury

Cavernous nerve injury (CNI) is the main cause of erectile dysfunction (ED) following pelvic surgery. Injecting hGMSCs around the bilateral major pelvic ganglia (MPG) in a male rat model of CNI showed that the transplantation significantly improved CNI-related ED. It appears to provide protection against CNI-induced penile atrophy, apoptosis, as well as fibrosis. In this potential mechanism, hGMSCs exerts these functions by shifting macrophage polarity from the M1 to the M2 anti-inflammatory phenotype.¹⁵⁴

4.1.2.5. Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer disease, and it is characterized by tremor, rigidity, postural instability, and bradykinesia.¹⁵⁵ In 6-hydroxydopamine-induced PD rat model, forelimb misalignment behavior and rotation were improved due to intravenous injection of GMSCs. The injected GMSCs promoted the anti-apoptotic B-cell lymphoma 2/B-cell lymphoma 2-associated X axis (BCL-2/BAX), which was complemented by an increased protection of tyrosine hydroxylase neurons and a decrease in astrocytes activation. Since Parkinson's disease is a degenerative disorder of the substantia nigra, a reduction of certain markers may be noted in the substantia nigra and striatum of PD rats. For example, the astrocyte marker, glial fibrillary acidic protein (GFAP), and the microglia marker, ionized calcium-binding adaptor molecule 1(IBA-1), are remarkably reduced. These findings will set up the foundation for future GMSC-based therapeutic applications of Parkinson's disease. This is in part due to GMSCs' potent capacity to protect neurons in vivo by reducing mitochondrial membrane potential damage and impeding reactive oxygen species accumulation.¹⁵⁵

4.1.2.6. Retinal ischemia-reperfusion injury

Retinal ischemia-reperfusion injury (IRI) is one of the main pathogenic mechanisms of glaucoma, and its pathological manifestations include neuroinflammation and neuronal death, however pathological process is unclear. Recently, a study on IRI mice models reported a reduction of inflammation and cell loss in the vitreous body following the local injection of exosomes obtained from the CM of TNF-α-stimulated GMSCs-exosomes (TG-exos), compared to the GMSC-exosomes injection group. This study shows a new therapeutic pathway for neuroprotection against IRI by delivering miR-21-5p-enriched exosomes through MEG3/miR-21-5p/PDCD4 axis.¹⁵⁶ Similar results were obtained in in vitro experiments by Rajan et al.¹⁵⁷ They employed a conditioned medium derived from GMSCs to address scratch-induced damage in murine motor neuron-like NSC-34 cells. This led to a reduction in the levels of caspase 3, Bax, SOD-1 (superoxide dismutase 1), iNOS, and TNFα, while increasing the levels of Bcl-2, IL-10, and neurotrophic factors like BDNF (brain-derived neurotrophic factor) and NT3 (neurotrophin 3). In short, GMSCs trigger specific mechanisms to treat nerve injury diseases. This involves replacing damaged neurons, secreting nerve growth factors, and rebuilding nerve axons. Table 5 gives the details of the papers on neural regeneration.

• •Wound healing

Table 5.

| GMSCs and their cell-free products application in tissue engineering (Nerve).

Application	Cell Type/Products	Scaffold/Factor	Route	Model	Ref.
Facial nerve defect	NCSCs induced by hGMSCs	AxoGuard nerve conduits	Local implantation	Rat	146
Facial nerve defect	2D hGMSCs or 3D hGMSCs spheroid	3D bio-printed nerve grafts	Local implantation	Rat	147
Sciatic nerve crush-injury	hGMSC-NPCs or hGMSCs	GelFoam	Local implantation	Rat	148
Sciatic nerve crush-injury	hGMSCs-EVs or hGMSCs	GelFoam	Local implantation	Mice	149
Sciatic nerve segmental defect	GMSCs-EVs	Biodegradable chitin conduits	Local implantation	Rat	150
Sciatic nerve crush-injury	GiSCs induced by hGMSCs	Methacrylated 3D-collagen hydrogel, the SIS-ECM nerve protector	Local implantation	Rat	151
Spinal cord injury	hGMSCs	Moringin (MOR)-enriched liposomes treatment	IV injection	Mice	152
Hemitransection spinal cord injury	hGMSCs	Caffeic acid‐based bioconjugated hydrogel (CBGH)	Local implantation	Rat	153
Cavernous nerve injury	hGMSCs	–	Local injection	Rat	154
Parkinson Disease	hGMSCs	–	IV injection	Rat	155
Retinal Ischemia-reperfusion Injury	hGMSCs-Exo	TNF-α stimulation	Local injection	Mice	156
Chemotherapy-induced oral mucositis	hGMSCs	3D GMSCs spheroids, or 2D GMSCs	IV injection	Mice	116

Wound healing is a complex process involving immune cells, host cells and numerous growth factors. Preliminary reports revealed that systemically infused hGMSCs could migrate to the wound site and develop a tight spatial interaction with host macrophages. The GMSCs prompted the macrophages to shift toward the anti-inflammatory M2 phenotype, significantly enhancing re-epithelialization, collagen deposition and angiogenesis. The accelerated wound repair mediated by the GMSCs also mitigated local inflammatory cell responses in the excisional full-thickness skin wound splinting of mice models.⁹⁵ In terms of extending full-thickness skin allograft survival and delaying rejection, a similar systemic injection of hGMSCs achieved a stronger beneficial impact and immunosuppressive function than that of BMSCs. This may be related to the enhanced immunomodulatory effect of Tregs in vivo.⁵² Certain modifying factors have been identified that enhance these newfound properties of GMSCs. For example, the use of hypoxia-preconditioned hGMSCs on skin wounds exhibited an enhanced full-thickness repair in mice.¹⁰² Similarly in NOD/SCID mice, an excisional skin wound was treated with IL-1β-primed GMSCs. This human epidermal substitute engraftment model promoted cell migration, dermal-epidermal junction formation, and inflammation reduction in vitro. Thus, GMSC modulation could improve the in vivo engraftment of epidermal substitutes and promote skin wound healing.¹⁵⁸

Incorporating GMSCs into hydrogels and similar structural scaffolds has allowed for an increasingly stable wound healing process with added benefits. In a rat model, Shafiee et al. demonstrated that applying a 3D-printed medical-grade polycaprolactone (mPCL) dressing to a splinted full-thickness excisional wound would significantly improve skin granulation, re-epithelialization, and regeneration, while decreasing wound contracture. By 3D-printing a biomimetic wound dressing and delivery it with reinforced GMSCs will reduce the formation of scar tissue and enhance physiological wound closure.¹⁵⁹ A study led by Kalachaveedu combined GMSCs with an electrospun nanofibrous guar gum/PVA based scaffold matrix alongside the extracts of four herb medicinal plants, and its transplantation of the nanofibrous scaffold seeded with human GMSCs yielded similar results.¹⁶⁰

Conversely, the encapsulation of GMSCs in an engineered hydrogel sheet can accelerate wound closure and healing. As reported by Ansari et al. implanting a GMSC−hydrogel construct based on alginate and gelatin methacrylate (GelMA) onto skin defects of wild-type (WT) mice contributed to angiogenesis and the suppression local proinflammatory cytokines.¹⁶¹ Recently, mice with 137Cs γ-radiation-induced with skin wounds experienced improved wound healing of irradiated skin tissues following treatment with a different hydrogel: one with Nap-GDFDFpDY (pY-Gel) self-assembled peptides encapsulating GMSCs. The study effectively assessed the skin damage score, hind limb extension measurement, in addition to histological and immunohistochemical analysis. In vitro studies of the human keratinocyte cell line (Ha-CaT) and normal human dermal fibroblasts (HFF) showed that GMSCs-CM could upregulate the expression of epidermal growth factor receptor (EGFR), signal transducers and activators of transcription 3 (STAT3), as well as matrix metalloproteinases (MMPs). Here, the results suggest that, through the EGFR/STAT3 signaling pathway, the activation GMSCs-CM promoted the proliferation, migration, and DNA damage repair ability of the skin cells following irradiation.¹⁶² Considering the evidence discussed in these studies, topical application of GMSC and derived EVs has potential benefits¹⁴² in promoting full-thickness skin wound healing, including diabetic skin defects in rats.¹⁶³

• •Bone, Cartilage, Tendon, Muscle Regeneration

Few studies have elaborated on the potential use of GMSCs in the tissue engineering of bone, cartilage, tendon, and muscle. The myogenic differentiation potential of hGMSCs was investigated by Ansari et al. with the use of an injectable 3D RGD-coupled alginate scaffold. The subcutaneous transplantation of hGMSCs encapsulated by the scaffold and multiple growth factors led to a higher expression of gene markers related to muscle regeneration and to an overall muscle cell-like morphology. Compared to hBMSCs, GMSCs displayed a stronger ability for myogenic regeneration in the immunocompromised mice.⁷⁴ Notable growth factors significantly affecting this process include TGF-1 and TGF-β3, TGF-1 loaded RGD-modified alginate microspheres encapsulating PDLSCs or GMSCs could significantly promote chondrogenic differentiation in vitro and enhance the regeneration of ectopic cartilage tissue in vivo.¹⁶⁴ Similarly, TGF-β3 loaded RGD-coupled alginate micro-spheres encapsulating PDLSCs or GMSCs could differentiate into tendon tissue in vitro, as confirmed by increased mRNA expression for gene markers related to tendon regeneration (e.g., Scx, DCn, Tnmd, and Bgy). In a comparable in vivo animal model, ectopic neo-tendon regeneration was observed in subcutaneous transplanted MSC-alginate constructs.¹⁶⁵

Alternatively, GMSCs or BMSCs may be loaded onto scaffolds. The NanoBone scaffolds displayed its regenerative capacity on surgically created critical-sized tibial bone defects in rabbit models, significantly enhancing the new bone formation. Moreover, there was no difference in the new bone area percentages between the bone defects treated with GMSCs and BMSCs, suggesting that locally transplantation of NanoBone scaffolds stacked with either GMSCs or BMSCs held a similar degree of bone regenerative potential. This demonstrates how that GMSCs may represent a reliable substitute to BMSCs in regenerative therapies.¹⁶⁶ On a broader scale, these findings indicate that GMSCs may be a suitable source of stem cells for bone, cartilage, tendon, and muscle tissue engineering. A detailed summary of the studies in this section can be found in Table 6.

Table 6.

| GMSCs and their cell-free products application in tissue engineering (Wound healing, Tibial bone defect).

Application	Cell Type/Products	Scaffold/Factor	Route	Model	Ref.
Full-thickness excisional skin wound	hGMSCs	–	IV injection	Mice	95
Full-thickness skin allografts	hGMSCs	–	IV injection	Mice	52
Excisional skin wound	hGMSCs	Hypoxia-primed	IV injection	Mice	102
Full-thickness excisional skin wound	hGMSCs	IL-1β primed, human plasma-based epidermal substitute (hPBES)	Local implantation	NOD/SCID mice	158
Excisional skin wound	hGMSCs	RGD-Modified alginate−gelatin methacrylate (GelMA) hydrogel	Local implantation	Mice	161
Splinted full-thickness excisional wound	hGMSCs	Electrospun nanofibrous Guar gum/PVA based scaffold matrix, extracts of four traditional medicinal plants	Local implantation	Rat	160
Splinted full-thickness excisional wound	hGMSCs	3D printing polycaprolactone (mPCL) dressings	Local implantation	Rat	159
Radiation-induced skin injury	hGMSCs	Nap-GDFDFpDY (pY-Gel) Self-Assembling Peptide Hydrogels	Local injection	Mice	162
Full-thickness excisional skin wound	Mice GMSCs -sEVs	–	Local injection	Mice	142
Diabetic skin defects	hGMSCs EVs	Chitosan/silk-based hydrogel sponge	Local implantation	Rat	163
Tibiae bone defect	Rabbit GMSCs	NanoBone scaffold	Local implantation	Rabbit	166

4.2. Therapeutic effects of inflammatory diseases

4.2.1. Atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the arterial intima. Its pathogenesis is mainly based on the accumulation of lipoproteins under the endothelium, the activation or dysfunction of endothelial cells, and the infiltration of foam cells, inflammatory cytokines, and chemokines.⁹⁶ According to a report by Zhang, the systemic infusion of hGMSC significantly diminishes and lipid deposition plaque size in blood vessel wall. Inflammatory monocytes/macrophages experienced a decrease in frequency following the infusion, and macrophage foam cell formation met a noticeable decline in an apolipoprotein E knockout (ApoE−/−) mouse model with atherosclerosis.⁹⁶ These findings provide further insight on how GMSCs could promote inflammatory monocyte/macrophage activity in potential atherosclerosis therapies.

4.2.2. Experimental colitis

Inflammatory bowel disease (IBD) includes two major chronic inflammatory intestinal conditions: Crohn's disease and ulcerative colitis.^19,167 Current evidence indicates that IBD is caused primarily by a wide spectrum of pathologies, ranging from mucosal immunity deregulation to bacterial factors, host genetics, and exaggerated inflammatory responses, resulting in an imbalance between epithelial barrier function and tissue inflammation.

The systemic infusion of hGMSCs or mice GMSCs appears to significantly attenuate the severity of colon inflammation, both histopathologically and clinically.^20,110,168 It demonstrated the ability to reverse diarrhea and weight loss, to mend gastrointestinal mucosal wounds, and to suppress disease pathophysiology in mice with colitis induced from dextran sulfate sodium (DSS). The therapeutic outcome was affected by T-regulatory cell infiltration increase and the inhibition of inflammatory mediators, cytokines, and infiltrates (e.g., interleukin [IL]-10+ regulatory T cells). In many cases, anti-inflammatory cytokine expression, specifically IL-10, was more prominent at colonic sites.^19,167 The infusion of human GMSCs similarly reduced the secretion of pro-inflammatory cytokines, however it also increased the production of anti-inflammatory cytokines and prolonged survival. The symptom relief in this murine colitis model may be attributed to GMSCs’ key role in alleviating inflammatory bowels through the immunoregulation of the IL-10/IL-10R signaling pathway.¹⁶⁷

The data analysis of GMSC-based immunomodulation and its therapeutic effects on colitis supports a connection to FAS ligand (FASL), a transmembrane protein. One study attributes the superior effects of neural crest-derived GMSCs(N-GMSCs) in strengthening inflammatory-related disease phenotype to an increased expression of FasL in N-GMSCs.²⁰ In comparison, the mesoderm GMSCs (M-GMSCs) treatment group did not display such an expression of the protein, resulting in a lessened therapeutic effect. Conceptually, the regulation of FasL in GMSCs should directly corelate with GMSCs' efficacy in treating colitis. Yu et al. demonstrated that Acetylsalicylic acid (ASA) could upregulate the expression of FasL in inflamed gingival tissues (iGMSCs), leading to an improvement in iGMSC-mediated T cell apoptosis and therapeutic efficacy in the treatment in colitis mice. IGMSCs, nonetheless, exhibited a decreased immunomodulatory capacity compared to GMSCs.¹⁶⁸ Further investigation of FASL's role led Yang R et al. to compare the immunomodulatory effects between control and hydrogen sulfide (H₂S)-deficient GMSCs in an in-vitro coculture system and in a mouse colitis model. They found that H₂S-deficient GMSCs exhibited an attenuated therapeutic effect on colitis in vivo. This revealed that H₂S was required for the Fas/FasL coupling-induced T-cell apoptosis, a fundamental mechanism behind GMSCs' ability to regulate chronic intestinal inflammation.¹¹⁰

4.3. Therapeutic effects of autoimmune diseases

4.3.1. Diabetes

Type 1 diabetes (T1DM) is a chronic autoimmune disease in which insulin-secreting pancreatic beta cells are attacked and destroyed by autoreactive T cells. In a study inducing T1DM in mice through multiple low-doses streptozotocin (STZ) injections, Zhang showed how performing an adoptive transfer of hGMSCs significantly controlled blood glucose levels, delayed diabetes onset, and improved pathologic scores in the pancreas. A down-regulation of IL-17 and IFN-g production in CD4⁺ and CD8⁺ T cells was appreciated in the spleen, pancreatic lymph nodes (pLN), and other lymph nodes. Overall, the level of CD4⁺ Treg induced in the periphery was also up-regulated by GMSCs,¹⁰⁸ thus demonstrating the potential clinical applicability of GMSCs in treating T1DM.

4.3.2. Contact hypersensitivity

Allergic contact dermatitis (ACD) is the most common diseases caused by repeated skin exposure to contact allergens. ACD is classified as a type IV or a delayed-type hypersensitivity reaction: an immune response mainly mediated by T cells encountering exogenous haptens or antigens. Murine contact hypersensitivity (CHS) is widely used as a model for ACD. In a murine CHS model of ear skin induced by oxazolone solution, systemic GMSC infusion prior to the phases of sensitization and challenge has drastically suppressed CHS via PGE2-dependent mechanisms, manifested as a decreased infiltration of DCs, CD81 T cells, TH17 and MCs, a suppression of several inflammatory cytokines, and a reciprocal increased infiltration of regulatory T cells and expression of IL-10 at the regional lymph nodes and the allergic contact areas.⁹⁹

Relative to BMSCs and adipose-derived stem cells, GMSCs showed the greatest efficacy in inhibiting CHS. Moreover, a local injection rather than an intravenous injection of GMSCs led to a more marked attenuation of CHS, especially in the late phase of CHS. This manifested a decrease in inflammatory cells infiltration, a suppression of various proinflammatory cytokines, the reestablishment of the disrupted Th1/Th2 balance, and an upregulation of regulatory T cells in the allergen contact areas.¹⁰⁹ These studies additionally revealed that COX2/PGE2 signaling has a crucial effect in decreasing skin CHS mediated by GMSC in mice.

4.3.3. Rheumatoid arthritis

Rheumatoid arthritis (RA) is a common chronic autoimmune disorder which is clinically observed as an irreversible symmetric polyarticular arthritis, progressively exacerbated by inflammatory synovitis, eventually leading to the destruction of articular cartilage and marginal bone. An attempt to treat collagen‐induced arthritis (CIA) in mice with or without GMSCs treatment, compared the effect on bone morphology, mineral density, and stiffness of the tibiae. Microstructure and mechanical behavior analysis revealed attenuated CIA-related bone erosion in the epiphyseal and metaphyseal trabecular bone of the tibiae. The reduction in the severity of the disease was accompanied by a significantly improved load-bearing function of the proximal tibia.¹⁶⁹ Infusion of human GMSCs into DBA/1 J mice with CIA markedly decreased inflammatory cytokine production and lowered histopathological scores.¹⁷⁰ Systemically infusing GMSCs into mice remarkably reduced the intensity of experimental CIA in DBA/1 J mice. Failing to treat colitis, FasL−/− GMSCs induce apoptosis of activated T cells provide no therapeutic benefit, implying that the lack of a FasL signal critically alters the GMSC-mediated therapeutic effect.¹⁷¹

A deeper investigation of the mechanism behind GMSCs' effect on RA explored several cell surface receptors that could potentially affect therapeutic potential. As expected, an infusion of hGMSC in mice with CIA significantly reduced the severity of arthritis, the pathology scores, the frequency of osteoclasts, and the bone erosion related to a decreased expression of RANKL in synovial tissues. However, an in vivo blockade of the receptors for adenosine and CD39/CD73 hindered any of GMSC's possible benefits on bone erosion.¹⁷² Furthermore, a study by Wu demonstrated that B7–H1 (PD-L1), is a costimulatory molecule widely ex-pressed on activated T cells, B cells, monocytes, and many types of cancer cells and displays a negative effect on immune actions, plays a significant role in the immuno-suppressive function of hGMSCs in the CIA murine model that is dependent on STAT3 signaling. In depth evaluation of B7–H1 expression in GMSCs enables the identification of a new subpopulation of MSCs with greater immunosuppressive properties.¹⁷³ Interestingly, systemic administration of GMSCs-derived exosomes (GMSCs- Exo) equally reduced the inflammation and bone erosion of joints in a CIA mice model. The team behind this study compared the immunomodulatory functions of GMSCs-Exo with those of GMSCs. They confirmed that GMSCs-Exo had a similar if not stronger potential in IL-17A inhibition and in amplifying IL-10. The coinciding reduction in incidence and in bone erosion is attributed to an inhibition of the IL-17RA-Act1-TRAF6-NF-κB signal pathway.¹⁷⁴ Ultimately, the treatment of RA with GMSCs and GMSCs-Exo is strongly supported by the literature, but attaining dependable therapeutic application requires further clinical validation.

4.3.4. Graft-versus-host disease

Allogenic graft-versus-host disease (allo-GVHD) is a severe complication of organ or bone marrow transplantation. In a xenogenic GVHD model established by intravenously injecting human CD25-depleted PBMC into NOD/SCID mice, the co-transfer of hGMSCs with human PBMC (hPBMC) markedly prolonged mice survival. This similarly reversed pathological changes, improved inflammation, and reduced human T cell expansion, thus suppressing the immune responses in vivo. Mechanistically, it was demonstrated that GMSCs inhibited hPBMC-initiated xenogenic responses via CD39/CD73/adenosine and IDO signals.¹⁷⁵ According to a recent report on two GVHD murine models, the systemic implantation of hGMSCs on C57BL/6-to-BALB/c and C57BL/6-to-B6D2F mice prevented and treated acute GVHD (aGVHD). GMSCs prevented the effects of aGVHD by regulating the conversion of Tregs to Th1 and/or Th17-like cells. Intriguingly, Foxp3 expression was stabilized by CD39 signaling, which plays an important role in the function and stability of Tregs. Furthermore, these findings suggest that GMSCs carry a heavier weight in therapeutic potential for aGVHD, compared to BMSCs and ADSCs.¹⁷⁶ These generally positive findings emphasize the possible benefits of GMSCs in the treatment of GVHD.

4.3.5. Psoriasis

Psoriasis is an autoimmune skin disease characterized by T cell-mediated inflammation, marked by an alteration in the balance of Th2 and Th1/Th17 cytokines.¹⁷⁷ The engineering of MSCs is emerging as a modern approach to treatment of psoriasis. When administered to the diseased site, MSCs have the potential to alleviate psoriasis skin lesions by reducing the local levels of angiogenic and proinflammatory substances, possibly by inhibition of the activation and differentiation of CD4⁺ T cells mediated by DCs.¹⁷⁸ Ye et al. found that infusion of GMSCs in 2D and 3D culture conditions can significantly improve erythema, skin thickness and scaling, provide protective benefits against skin inflammation resembling psoriasis in a murine model induced by imiquimod (IMQ). Both 2D and 3D GMSCs cultures affected the site similarly; the treatment of GMSCs reduced the Th1- and Th17-related cytokine levels (e.g., TNF-α, IFN-γ, IL-6, IL-17A, IL-17F, IL-21, and IL-22). A particular finding was the upregulation of the spleen's CD25+CD3⁺ T cells percentage and downregulation of its IL-17+CD3⁺ T cells percentage.¹⁷⁹

Interestingly, in 2020, Wang SG et al. reported on the safety and effects of one of the first psoriasis cases treated with repeated GMSCs administration. Considering the patient's severe plaque psoriasis which was refractory throughout 5 years despite various systemic and topical treatment methods, the 19-year-old male received two consecutive weekly administrations of allogeneic human GMSCs (3 × 10⁶/Kg/infusion). Clinically, complete regression was achieved after 5 infusions with no adverse reactions.¹⁸⁰ The patient was followed for three years, with no recurrent sign of disease. In sum, these findings reveal that therapeutic application of GMSCs could outperform traditional clinical approaches for treating psoriasis.

4.3.6. Osteoporosis

Osteoporosis is a multifactorial chronic disease where bone quality and density are affected. A fracture can occur from a mild fall or stressor since the bone has become weak and brittle. The incidence of this disease is greater in women than in men, of which postmenopausal women and the elderly carry a higher risk. Systemic administration of hGMSCs has been reported to regulate the osteoclast/osteoblast balance promoting dynamic bone formation. In an ovariectomized mouse model, a significant attenuation of ovariectomy (OVX)-induced osteoporosis was observed between a higher osteoblast frequency, a respective decrease in osteoclast frequency, and an increasingly dense trabecular bone. Moreover, the results further demonstrated how CD39 produced by GMSC exerted its osteogenic capacity through the Wnt/b-catenin pathway.¹⁸¹ Based off this study, GMSCs carry potential for the management of patients with osteoporosis and similar skeletal disorders.

4.3.7. Bone marrow failure

Human aplastic anemia (AA) is a rare autoimmune disease characterized by severe pancytopenia and bone marrow failure (BMF). The destruction of hematopoietic stem and progenitor cells is mediated by activated T lymphocytes containing Th1, Th17 cells. This pathological response causes pancytopenia, marrow hypoplasia, and an obliteration of the hematopoietic microenvironment. A mouse model investigation of the effects of GMSCs on preventing T cell-mediated BMF showed how systemically infused hGMSCs could migrate to the inflamed site within bone marrow (BM) and impede disease progression. This improved survival by attenuating BMF and modulating the equilibrium of Tregs, Th1 and Th17. The histological findings displayed evidence of decreased CD8⁺ cells, Th1 and Th17 cells levels, with increased CD4+Foxp3+ regulatory T cells in lymph nodes.¹⁰⁷ It appears that GMSCs therapy is a promising approach to the prevention and treatment of patients with conditions related to bone marrow failure, such as aplastic anemia.

4.3.8. Lupus nephritis

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with a predilection for women of childbearing age. The involvement of other organs is most common in kidneys, with SLE manifesting as lupus nephritis. The administration of GMSCs was proven to carry therapeutic effects in a lupus nephritis mouse model by decreasing proteinuria, frequency of plasma cells, levels of autoantibodies and lupus nephritis immunopathology. As demonstrated by Dang J et al., GMSCs could directly suppress B cells activation, proliferation, differentiation, and autoantibody production in an inflammatory milieu through targeting of CD39-CD73 signaling pathway.¹⁰⁵ These results suggest that GMSCs can fundamentally change the baseline of therapy for patients with SLE and other autoimmune diseases.

4.4. Therapeutic effects of other diseases

4.4.1. Cancer

As of yet, only a limited number of studies focus on the application of GMSCs in anticancer treatments, most of them are related to oral carcinomas, particularly tongue squamous cell carcinoma (TSCC). GMSCs were found to impede the growth of oral cancer cells both in vitro and in vivo. In vitro direct co-culture of GMSCs with two human oral cancer cell lines, CAL27 and WSU-HN6, displayed an inhibition oral cancer cell growth. Additionally, the GMSCs conditioned medium exerted a comparable anticancer effect, indicating that soluble factors within the GMSCs-CM, such as IL-6, IL-8, and GM-CSF, may play a significant role in antiproliferation. Mechanistically, GMSCs upregulate the expression of pro-apoptotic genes in cancer cells (e.g., p-JNK, cleaved PARP, cleaved caspase-3, and Bax) and downregulate proliferation and anti-apoptosis related gene expression (e.g., p-ERK1/2, Bcl-2, CDK4, cyclin D1, PCNA and survivin). The volume of the tumor in the CAL27+GMSCs nude mice group was significantly smaller than that of the CAL27 group, suggesting that GMSCs inhibited the growth of CAL27 in vivo.¹⁸² Further in vitro experiments discovered that GMSCs could migrate towards TSCC cell lines. The GMSCs were transduced with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and carried out anti-tumor functions on TSCC cell lines; this resulted in an elevated count of necrotic and apoptotic tumor cells. Analogous in vivo experiments determined that transduced GMSCs injected into nude mice systemically suppress the growth of TSCC cells via apoptosis, following the selective engraftment onto tumor tissues.¹⁸³

The recent understanding of the function and mechanism of action of GMSCs on oral SCC has laid the groundwork for loading GMSCs with anti-neoplastic drugs, or genetically modifying them to inhibit cancer cell proliferation. For example, GMSCs have the ability to up-take and releasing anti-neoplastic drugs, such as doxorubicin, paclitaxel, and gemcitabine. It was discovered by Coccè that GMSCs efficiently incorporated the drugs and then released them in active form and in sufficient amount to produce a dramatic inhibition of squamous cell carcinoma growth in vitro.¹⁸⁴ While this study was limited to the human squamous cell carcinoma line (SCC154)14 and the human pancreatic adenocarcinoma cell line CFPAC-1, a broader report evaluated the therapeutic value of the secretome of GMSCs. The secretome alone did not affect the growth of cancer cell lines tested in vitro, but those primed with paclitaxel exerted a significant anticancer effect.¹⁸⁵ In order to evaluate the benefit of genetically modifying GMSCs for cancer treatment, Du et al. evaluated the therapeutic potential of Interferon-β gene-modified GMSCs (GMSCs/IFN-β) on TSCC. The modified GMSCs notably restrained the proliferation of TSCC cells and induced apoptosis in cancer cells in vitro. In a TSCC xenograft model developed by subcutaneous injection of CAL27 cells into BALB/c nude mouse, intravenous injections of GMSCs/IFN-β, engrafted selectively in the TSCC xenograft, expressed a remarkably high level of IFN-β. GMSCs and GMSCs/vectors presented with the potential to inhibit CAL27 cell growth, both in vitro and in vivo, although the perceived effect was weaker than that of GMSCs/IFN-β.¹⁸⁶ There was a smaller tumor volume and a lower number of Ki67-positive cells in the GMSCs/IFN-β group relative to the other control groups. Effectively, modifying GMSCs to produce particular antitumor substances appears to hold great potential as a strategy in anticancer treatment. It could guarantee a sustained and sufficient release of specific factors at the tumor site, in amounts unachievable through systemic administration.

4.4.2. Tracheal defect/stenosis

Repairing tracheal defects and stenosis is a challenging undertaking. In a recent study, Zeng et al. conceptualized a cartilaginous extracellular matrix (cECM), which was formed by coculturing human GMSCs with chondrocytes isolated from auricular cartilage of rabbits. This combination produced abundant “matrix bound human gingi-val mesenchymal stem cells derived EVs (gMVs)”. Following a mild, short-period de-cellularization, the gMVs were preserving in acellular cECM (cACM). The newly formed gMVs-cACMs demonstrated the ability to anchor themselves onto the polyglycerol sebacate microporous patch in vitro, enhancing surgical suturability and mechanical strength accordingly. A rabbit tracheal defect model utilizing this concept showed the gMVs-cACM patch inducing a rapid regeneration of vascularized ciliated columnar epithelium and proliferation of tracheal progenitors-basal epithelial cells. The evidence indicated that this is in part due to the activation of the JAK2/STAT1 pathway in reparative cells.¹⁸⁷

More recently, Fayzullin et al. developed alternatives to human trachea donation and lays the groundwork for treating tracheal stenosis with these novel biosynthetic prostheses. Donor trachea samples were isolated and washed to discard unwanted human cadaver tissue, then separated from the remaining adipose. The resulting tissue was decellularized and cross-linked with ethylene glycol diglycidyl ether (EGDE) prior to being treated with lasers to create wells on the surface. Afterwards, human GMSCs were planted into the structured matrices and grown in the culture medium. Once matured, they implanted the synthesized cartilage structure orthotopically into rabbits as well as heterotopically into immunosuppressed, nude and healthy mice. In vivo studies demonstrated how the tracheal equivalent samples differed from the matrix ones by a thinned capsule, an increased submucosal resorption, and ingrowth of vessels into the neighboring tissue. This analysis subsequently proved that the developed cartilage constructs had low-immunogenicity, high biocompatibility, and good efficiency in treating tracheal defects.¹⁸⁸ Collectively, the data from these studies show how tissue-engineered products of GMSCs and their derived EVs or ECM may be applied in tracheal regeneration.

5. Limitations on GMSC practical use

Despite the meaningful progress of GMSC research, there are still many challenges to overcome. In vitro culture conditions and identification methods need to be improved due to GMSCs lacking specific surface markers. This could lead to changes in gene levels throughout the process of isolation and culture. The discrepancy in results between in vitro and in vivo differentiation presents another technically difficulty. In vitro differentiation conditions could not completely simulate the microenvironment in vivo; complicated multiple factors in vivo may misrelate the types and numbers of differentiated cells, as well as the direction of cell differentiation. Moreover, the immunogenicity of GMSCs is still somewhat ambiguous. While current literature describes a low immunogenicity, increasing evidence suggests they could still induce immune rejection and tumors.

It remains unclear whether current preclinical treatment methods modeled on GMSCs may be applied in vivo and expand on current disease-specific therapies. A uniform and authoritative standard for the selection and monitoring of medical administration of GMSCs would provide researchers with a baseline to further unlock the potential of GMSC therapy. The ideal standardization should establish criteria for clinical trials, including selection of suitable donors, identification of target patient populations, and a complete methodology for appropriate operating procedures (e.g., cell isolation, expansion, storage, transportation, priming prior to administration). The lack of a definitive guideline in GMSC treatments may also be a major obstacle to large scale clinical trials. The few registered GMSC clinical trials have taken the first steps in increasing our understanding, but further clinical validation is required. Box 1 provides more detail on the clinical application of GMSC therapies.¹⁸⁹

Box 1. Clinical Application of GMSC Therapies.

Applications.

Initial isolation and characterization of GMSCs have demonstrated their strong potential for modulating the immune system, reducing inflammation, and offering regenerative therapeutic benefits in a various preclinical models of human diseases.

Benefits.

GMSCs are shown to maintain their typical karyotype and continue to exhibit telomerase activity throughout long-term cultures, while showing no signs of tumorigenicity in nude mice succeeding long-term inoculation. Similarly, when human GMSCs were transplanted into various animal models and even some autoimmune disease models, GMSCs showed good tolerance from all recipient hosts without any obvious systemic adverse effects. These findings provide an insight on how GMSCs and their derivatives are attractive prospects for clinical application.

Current trials.

Clinical trials employing stem cells originating from gingival tissue were identified on the clinicaltrials.gov website, with key words relating to “Gingival Mesenchymal Stem Cells”, “Gingival Stem Cells,” or “GMSC”. The search yielded three registered clinical trials focusing on GMSCs applications in periodontal diseases: one completed trial (NCT03638154), one active but not recruiting (NCT03570333), and one of unknown status (NCT03137979). Current translational applications of GMSCs seem to face considerable challenges due to their intrinsic and extrinsic heterogeneity, which remains a significant technical barrier.

Alt-text: Box 1

6. Conclusion

Over the last two decades, interest in MSC therapies has been gaining rapid momentum within the field of stem cell research. These scientific advancements are allowing for a gradual transition of these therapies into clinical practice. From an ethical standpoint, GMSCs derived from discarded gingival tissue are easier to obtain compared to traditional cell seeding, their applications however, are currently limited to in vitro cell culture and animal studies. Various disciplines of medicine will benefit from the remarkable biological properties of GMSCs, such as self-renewal, multi-directional differentiation, immune regulation, and promotion of tissue regeneration. Although these therapies require fine tuning for comprehensive clinical application, future innovation, and continued progress of GMSC research will create new opportunities for clinical treatment of various diseases.

Declaration of competing interest

All authors declare no competing interests.