Introduction

Mesenchymal stromal cells (MSCs) are a population of stromal cells capable of self-renewal and differentiation into various cell lineages given specific environmental cues.1 MSCs can be extracted from various human tissues which is a pluripotent stem cell that are widely present in adult bone marrow and can differentiate into other cell types and related lineages of mesenchymal tissue.2 MSCs differentiate into various cells at the single cell level in vitro including osteocytes, cartilage tissue cells,3 cardiomyocytes4 and epithelial cells.5 The umbilical cord (including the Wharton’s jelly, a gelatinous substance found inside the umbilical cord),6,7 fetal tissue,8 dental pulp9 and placenta10 have been investigated as sources of various MSCs types. Furthermore, the induced pluripotent stem cells (iPSCs)11 have attracted much attention as a new source of MSCs, which are becoming an emerging cell therapy product.

Due to their ability to self-renew, be pluripotent, and have immunomodulatory properties, MSCs are frequently utilized in adoptive cell therapy, tissue engineering, and regenerative medicine.12,13 Of particular note, MSCs as an adoptive therapy exhibit vast potential in alleviating inflammation-related diseases including inflammatory bowel disease (IBD). Many clinical trials demonstrate both the safety and efficacy of MSCs therapies on IBD. MSCs and their secreted bioactive factors (eg, extracellular vesicles (EVs), proteins, RNAs) could regulate immunity, repair inflamed tissues as well as modulate gut microbiota, thereby showing a protective effect on IBD. Although there have been great progresses on promoting MSCs for IBD therapy, several challenges emerge.

Therefore, this review aims to comprehensively discuss the current understanding of applying MSCs on IBD treatment based on their actions and mechanisms. Importantly, through a review of clinical trials of MSCs therapy, insights on current challenges and future directions are provided. This review will advocate a safe and effective treatment of MSCs in IBD in future.

An Overview of IBD

IBD is a chronic inflammatory disease that can be divided into ulcerative colitis (UC) and Crohn’s disease (CD) by its etiology and pathogenesis, which has a high recurrence rate and is not easily curable, seriously affecting patients’ quality of life. The clinical symptoms of IBD include gastrointestinal symptoms such as diarrhea, abdominal pain, blood in the stool and systemic symptoms such as weight loss, fatigue, anemia, etc.14 IBD is currently prevalent in Western countries, particularly northern Europe and north America, with approximately 1.6 million Americans affected by IBD, including about 700,000 for CD patients and 900,000 for UC patients.15 Up to 2 million people in Europe suffer from the disease, with the frequency being higher in wealthy Western countries.16 In contrast, the prevalence of IBD is lower in newly industrialized countries in Asia, and in epidemiological studies of IBD in Asia-Pacific countries, the highest IBD prevalence was in India at 9.3/100,000 person per year, and in China at 3.3/100,000 person per year.17 Overall, IBD has created tremendous financial pressure on the families of patients. The etiology of IBD has not been well studied, and genes, environment, gut microbiome and immune system all contribute to the development of IBD.18–21 Among them, environmental factors are key factors in the pathogenesis of IBD, as most environmental triggers can mediate the pathogenesis of IBD through their effects on the gut microbiome.22

Pathogenesis of IBD

Pathogenesis of IBD is related to genetic inheritance, intestinal mucosal barrier, environment, gut microbes, immune system, and potentially other factors.23 The main causes and symptoms are shown in Figure 1. Further exploration of the pathogenesis of IBD can contribute to a better comprehension of the function of current treatments and provide new ideas for the future development of new therapeutic drugs.

Figure 1 Pathogenesis and main symptoms of IBD. The interaction among environment, genetic inheritance, intestinal mucosal barrier, immune system and underlying factors leads to the development of IBD. The main clinical symptoms are diarrhea, abdominal pain, blood in the stool, and weight loss due to loss of appetite.

Abbreviations: DC cell, dendritic cell; IBD, inflammatory bowel disease; NK cell, natural killer cell.

Genetic Factor

It is reported that first-degree relatives of IBD patients have a 3–20 times higher risk of developing the condition than first-degree relatives without the condition in the general population.24 Studies have confirmed that IBD has some family heritability and its pathogenesis is closely related to genetic inheritance.25 Identical twin concordance in UC is 10–15%, but concordance in CD is 30–35%, indicating that CD is more likely to be genetically influenced than UC, and that monozygotic twins are more likely to have IBD than dizygotic twins.26 It is identified that NOD2 is a susceptibility gene for CD through positional cloning and candidate gene approaches.27,28 The identification of NOD2 opens up research direction for the study of the pathogenesis of IBD at the genetic level. New genotyping and sequencing technologies have aided to identify 242 common susceptibility loci for IBD, with 45 of them being identified as statistically conclusive causative variations, while 50 genes being linked to inflammatory disorders.29 Notably, most of the genes in the loci associated with IBD, although shared in different ancestral groups, are only partially present in people with IBD. For instance, most European patients have variations in the NOD2 and IL23R genes, but southeast Asian patients rarely do so.30 Similarly, there were genetic IBD risk scores disparities between African Americans and Europeans, identifying genetic heterogeneity in populations of different ethnic origins.31

Despite substantial progress in determining the genetic inheritance of the IBD gene, there is still a long way to go before the cause of IBD is fully understood.

Gut Microbiota

The balance of the gut microbiome plays an important role in maintaining the health of the organism and mediating disease development, which is influenced by both environmental and host factors.32–34 Over-immunization to bacteria in genetically susceptible hosts disrupts the gut homeostatic environment, which is one of the reasons for the development of IBD. When gut microbiota diversity is reduced or altered, in which case the host becomes more susceptible to pathogens or pathogenic microorganisms, as well as the IBD.23,35 Reflecting on the fact that the incidence of IBD has been rising in industrialized nations over the past decades, it is more likely that lifestyle, diet and environmental changes other than the genetic inheritance or natural selection are to blame for this sudden rise.36 These dietary and environmental changes can exacerbate immunological imbalances in the body and promote the development of IBD in genetically predisposed people, because the composition and function of the human gut microbiome are particularly sensitive to these changes.37,38

Several studies comparing the intestinal microbial diversity and the abundance of specific bacteria in IBD patients found differences in the composition of the intestinal microbiota between IBD patients and healthy individuals. IBD patients had reduced biodiversity, reduced abundance of some bacteria belonging to the thick-walled phylum (eg, Enterococcus faecalis) and increased bacteria belonging to the Aspergillus phylum (eg, Escherichia coli) compared to healthy individuals.39–41 Schaubeck et al colonized already ecologically dysregulated microbiota in inflamed mice into healthy mice and found inflammation in healthy mice.42 Meanwhile, in another animal experiment investigating the effect of sugar on the pathogenesis of colitis, major alterations in the intestinal microbiota were discovered in mice fed sugar by short-term gavage.43,44 When mice were treated with antibiotics or kept in a germ-free environment, there was no sugar-induced exacerbation of colitis, but transfer of the intestinal microbiota from sugar-treated mice to germ-free mice resulted in an exacerbation of colitis, indicating that changes in the microbes play an essential part in the development of colitis.43 All these studies in animal models provide strong arguments for the role of microbiome in inducing intestinal inflammation.38,45

While most of the current studies have focused on bacteria and less on the role of fungi and viruses, some studies have shown that the role of viral and fungal communities in the pathogenesis of IBD cannot be underestimated as well.39,46 Notably, intestinal inflammation promotes fungal proliferation, and conversely, some fungi can modulate host susceptibility to inflammation.47 Although the exact role and mechanism of the virus in IBD has not been fully explored, some evidence suggests that it may be indirectly involved in the development of intestinal inflammation. For future directions of IBD research, modulation of the intestinal microbiota and local immune response can be indirectly achieved by altering viral diversity.

Environmental Factor

Recent findings in the epidemiology of IBD suggest that the environment is one of the key pathogenic mechanisms of IBD.48 Among the environmental factors, diet is the main contributor to IBD.49 Genetically susceptible individuals have intestinal dysregulation and abnormal immune responses, a process that may be caused by changes in environmental factors, including diet.23 Excessive intake of simple sugars is the main culprit of IBD because ingestion of large amounts of simple sugars can cause changes in gut microbes.44 Interestingly, rectal insulin drip reduces colonic inflammation in mice,50 suggesting that increased intake of simple sugars is an environmental risk factor for colitis.43,44

In addition, vegetables and fruits can help decrease the occurrence of CD.51 Increased dietary intake of animal protein was considered a factor in the development of CD decades ago.52,53 The harmful effects of animal proteins are also evident in chronic colitis and are associated with significant changes in intestinal bacteria and fungi.54 Other environmental factors influence the development of IBD, such as sterility,55 antibiotics, and other appendectomies and smoking.56–58 In general, environmental factors trigger IBD mainly by disrupting the balance of the intestinal microbiome, and another possible cause is the destruction of intestinal epithelial cells.

Barrier Factors

The disturbance of the epithelial barrier is the root cause of IBD. The colonic epithelium facilitates host-microbe interactions, regulates mucosal immunity, coordinates nutrient circulation, and generates a mucus barrier to maintain the intestinal environment in equilibrium.21,59 Existing studies have reported increased intestinal epithelial cell permeability and impaired intestinal mucosal barrier function in patients with IBD compared to normal subjects.60 Furthermore, mucin and antimicrobial proteins secreted by mucus create a physical barrier to microbial contact, and the formation of internal and external mucus layers is crucial for maintaining homeostasis in the body. Of particular note is that goblet cells produce mucus to form the intestinal mucus layer, which is a crucial part of intestinal epithelial cell protection.61,62 The intestinal flora and immune system’s dysbiosis may be impacted by impaired intestinal mucosal barrier function, which could lead to an ongoing immunological response and chronic intestinal inflammation. Therefore, a key factor in the etiology of IBD is the breakdown of the intestinal mucosal barrier.15

Innate and Adaptive Immunity

The innate immune system clears foreign pathogens and activates the body’s immune response, which consists of natural barriers such as the mucosal epithelium, immune cells such as neutrophils and natural killer cells (NKs), pattern recognition receptors, complement proteins and cytokines that allow for a rapid and effective response to foreign pathogens.63 In rats with trinitro-benzene-sulfonic acid-induced colitis, dendritic cells (DCs) in the medullary lineage of mesenteric lymph nodes increased inflammatory responses by using IL-12 to polarize T helper cells into pro-inflammatory Th1 subpopulations.64 In addition, immature DCs caused local intestinal inflammation, a process that is mediated by the production of IL-23.64 Macrophages can target the antigenic specificity of pathogens and cause inflammation by activating the adaptive immune response of the body.65 Based on their function and amount of inflammatory factor release, macrophages are classified into two subpopulations: M1-type and M2-type macrophages, M1 being associated with the induction of inflammation, while M2 being associated with inhibition of inflammation and stimulation of tissue healing.66 The role of NKs is to induce and maintain inflammation by producing IFN-γ and IL-15, which stimulates the recruitment of additional NKs and thus enhances the immune response. The IL-12 and IL-18 from macrophages can amplify the NKs-mediated immune response.67

Adaptive immunity is also crucial in the etiology of IBD, mainly involving T cells and their subpopulations. Abnormal activation of naive T cells lead to differentiation of different cell subtypes and the production of inflammatory cytokines, thereby mediating inflammation.67,68 DCs stimulate T cells after they have taken up antigen, causing them to transform into Th1, Th2, or Th17 cells, which participate in the inflammatory response.69 In these subtypes, cytotoxic T cells are stimulated by Th1 cells to become active, and assault infected intestinal epithelial cells, resulting in impaired intestinal epithelial barrier function. IL-18 is a key inflammatory factor involved in the activation of Th1 cells and NKs.70,71 Th2 cells activate B cells by transmitting activation signals, proliferate and differentiate into plasma cells, and secrete antibodies against pathogen invasion, but on the other hand, when IL-13 is present in the organism, Th2 cells are induced by it and secrete IL-4, IL-5 and IL-13 that are involved in the pathogenesis of UC.70 Th17 cells can be induced by TGF-β, IL-6, and IL-23 and produce a range of cytokines such as IL-17A and IL-21/22 to mediate inflammation.72

Current Treatments for IBD Including MSCs

Since the current research on the process of IBD pathogenesis is not well studied, and there is a lack of effective drugs. In CD, medical treatment is aimed at reducing abdominal pain, normalizing bowel movements, and promoting ulcer recovery, whereas in UC, the purpose of treatment is to stop the symptoms of rectal bleeding and obtain endoscopic remission.73 Traditionally, conventional treatments include 5-amino salicylic acid, corticosteroids, immunomodulators, etc. It is worth noting that with the gradual maturation of drug development technology, the development of TNF-specific inhibitors as biologics is a milestone achievement that enables IBD patients to achieve long-term remission.74 An increasing number of drugs, including biological agents and small chemical molecules, have been developed to treat IBD, which has also led to an increasing variety of drug options for the treatment of IBD.20,75

However, these treatments do not respond well to a large proportion of patients or are too difficult to tolerate due to adverse reactions, and clinical studies face significant challenges due to the efficacy and safety of existing medicinal medicines. For refractory disease, patients urgently need effective alternatives, and the heterogeneity of UC and CD clinical manifestations also make it difficult to find the best treatment for all patients.76,77 Therefore, it is crucial to use the right and effective therapy, and further development and research of IBD treatment drugs are needed to obtain the best treatment options.

Recently, the MSCs provide more therapy ideas for IBD, which will help in the long-term management of IBD in future. The newer treatment for IBD, MSCs differentiate into different cells depending on their extensive differentiation and self-renewal capacity, and are infused after ex vivo expansion to reduce the production of intestinal inflammation by balancing the immune system and repairing the intestinal mucosa, as shown in Figure 2. In addition, it is worth noting that MSCs have low immunogenicity and immunomodulatory properties and have active therapeutic effects in a variety of inflammatory diseases.78 Numerous animal experiments have shown the alleviating effect of MSCs and its exosomes on intestinal inflammation in mice.79,80 Various clinical trials are also underway.81,82 The safety and short-term efficacy of MSCs administration has been demonstrated, and the potential challenges associated with the treatment of MSCs are slowly being addressed.83,84

Figure 2 Different origins of MSCs possess self-renewal effect enabling allogeneic and autologous cell therapy. MSCs of different origins treat diseases through their broad differentiation potential. MSCs are widespread in the umbilical cord, tonsils, bone marrow, placenta, and dental pulp, and MSCs are able to self-renew and differentiate into various cell lineages in specific environments, and allogeneic or autologous mesenchymal stem cells cultured in vitro are infused into the body for rapid remission of disease symptoms and therapeutic effects.

Application of MSCs exhibits distinct mechanisms of action compared to conventional drug interventions, as shown in Table 1. Firstly, MSCs can selectively migrate to damaged intestinal tissues through homing effects and replace damaged tissues. Secondly, MSCs secrete growth factors via paracrine action to facilitate intestinal epithelium regeneration and angiogenesis and reduce intestinal inflammation. Finally, MSCs can also encourage T cells and macrophages to develop anti-inflammatory characteristics through regulation of the immune system, regulating the inflammatory response.

Table 1 Characteristics of IBD Treatment: Conventional Therapeutic Drugs and MSCs Therapy

Actions of MSCs for IBD Treatment and the Underlying Mechanisms

Traditional therapies for IBD have many side effects and poor efficacy, while MSCs, as an emerging cell therapy, have a wide range of prospects in the treatment of IBD. Because of their strong proliferation and differentiation, immunomodulation and ability to repair damaged tissues, MSCs show strong IBD alleviating effects in preclinical and clinical studies.85 MSCs are considered as the most promising cellular drug for IBD.86

MSCs have low immunogenicity because MSCs induce an immune tolerance phenotype through cell-to-cell interactions, as evidenced by low to moderate levels of MHC class I, lack of expression of MHC class II antigens and no expression of co-stimulatory molecules (CD40, CD40L, CD80 and CD86).87 Regarding this, MSCs avoid clearance by the immune system and allows for better migration, differentiation and regulation in the body. Through its paracrine action, MSCs can release EVs, anti-inflammatory factors, growth factors, anti-apoptotic factors and soluble enzymes to suppress inflammation, promote healing of damaged intestinal tissues and regulate host immune response.88 Herein, this review will summarize the modes of action of MSCs as well as the underlying mechanisms.

Modes of Action of MSCs Homing Effect

The arrival of a certain number of MSCs in the damaged tissue is a prerequisite for their active function, The homing of MSCs targeting damaged tissue is key to MSC therapy for various inflammatory diseases, including IBD.89 So far, only a few studies have studied the aggregation of MSCs in intestinal epithelial tissue, and some of these studies have elucidated homing patterns of intravenous MSC in animal models. MSC homing refers to the directional movement of MSC to the damaged tissue site under the influence of various factors and the replacement of damaged cells in the tissue site.90 The homing effect workflow is shown in Figure 3. In brief, the homing effect of MSCs is divided into five steps:89

Rolling: MSCs express CD44, which captures the selection and drives the cells to start rolling along the blood vessel wall.91 Activation: This step is typically advanced by G protein-coupled chemokine receptors in response to inflammatory signals, and the chemokine receptor CXCR4 ligand stromal cell-derived factor-1 is critical in this process as they allows MSCs to homing more smoothly to target tissues through binding.92 Firm adhesion: Integrins determine the adhesion process. MSCs express very late appearing antigen-4, which is then activated by chemokines such as stromal cell-derived factor (SDF)-1. After activation, in endothelial cells, vascular cell adhesion molecule-1 then produces a firm bond with very late appearing antigen-4 integration.93 Crawling: After the adhesion process is finished, MSCs scurry along the blood vessel’s interior wall in search of an appropriate spot for focused migration.94 Transendothelial migration: In this process, any migrating cells must cross the endothelial cell layer and the basement membrane, and MSCs break down the endothelial basement membrane by secreting matrix metalloproteinases;93 Finally, MSCs migrate through the mesenchyme to the damaged tissue. This step is guided by chemotactic signals released in response to tissue injury.95

Figure 3 Non-immunomodulatory mechanisms of MSCs in the treatment of IBD. The non-immunomodulatory mechanisms of MSCs in IBD mainly include homing effect, mitochondrial transfer and improving gut microbiome balance. Through homing effect, MSCs acts directly on intestinal epithelial cells to replace injured cells to play a therapeutic role. In addition, healthy mitochondria of MSCs transfer to replace damaged mitochondria in the intestine, produce ATP to treat IBD, and finally change the intestinal microbiome and improve the intestinal microenvironment to promote the regression of inflammation.

Abbreviations: CXCR4, C-X-C chemokine receptor type 4; VCAM-1, vascular cell adhesion molecule 1; MMPs, matrix metalloproteinases.

The completion of the homing process requires the involvement of molecules such as adhesion molecules, chemokines and metalloproteinases.96 Chemokine CXCR4 is an important molecule involved in MSCs homing.97 CXCR4 stimulates the transfer of MSCs to damaged tissues, and MSCs homing and survival are reduced when CXCR4 is knocked out.98 Notably, the expression of CXCR4 can be increased by upregulation of pro-inflammatory factors stimulation, such as IL-3, TNF-α, IL-1β, etc. so that MSCs show better homing and migration properties in vivo.99–101 The homing rate of MSCs is related to various aspects, such as the degree of aging of MSCs, intercellular oxidative damage, and the pathway of transplantation.102 In addition, the MSCs amplification process in vitro affects the expression of homing molecules and also affects the homing rate.103 This leads to the inspiration that MSCs could be pretreated in vitro by gene therapy for MSCs to improve their homing efficiency.

Paracrine Effect

MSCs were capable of secreting many bioactive factors which facilitate MSCs to exert beneficial effect. These bioactive factors include the EVs and EVs-contained components, apoptotic bodies, chemokines, cytokines, soluble enzymes and membrane-bound active proteins.

MSCs-Derived EVs (MSC-EVs)

MSC-EVs have strong biological activity similar to that of MSCs, which are cell-secreting nanoscale vesicles with a phospholipid bilayer structure that secrete proteins, microRNAs, mRNAs, and other substances involving various bioactive components under certain conditions.104 Studies have shown that MSC-EVs are highly effective in treating a variety of inflammatory disorders, suggesting that MSC-EVs as a cell-free therapy may have high research value in the treatment of IBD.105 This is because EVs-based therapy has some advantages over cell-based therapies. For example, it is previously reported that there is an increased risk of cancer associated with MSC therapy, which has not been reported for EVs. EVs have a more stable nature compared to MSCs.106

Adipose-derived MSC-EVs reduce the secretion of the pro-inflammatory cytokines IL-1β and TNF-α, increase the proportion of Treg cells and reduce the production of helper T cells, maintaining homeostasis in the body.107 It was shown that EVs derived from umbilical cord MSCs (UC-MSCs) were injected intraperitoneally into mice with enterocolitis, and by increasing TNF-α-stimulated gene 6 protein (TSG-6) expression, MSC-EVs dramatically decreased mortality and relieved symptoms of IBD in colon tissue.108 Additionally, olfactory ecto-derived MSC-EVs prevented T cells from differentiating into Th1 and Th17 cells, and also exerted its immunosuppressive function by aggregating Tregs cells to alleviate enteritis.109 In addition to the effect on adaptive immunity, MSC-EVs can also act on innate immunity by polarizing macrophages and producing M2-type macrophage changes to improve intestinal inflammation, and by increasing the level of the cellular immunosuppressive factor IL-10 through macrophages.110

It is reported that microRNAs in MSC-EVs are associated with inhibition of inflammatory development.105 In addition, MSC-EVs contain several important enzymes involved in glycolysis: glyceraldehyde-3P dehydrogenase, phosphoglycerate kinase, phosphoglucomutase, enolase, and pyruvate kinase M2 isoform, which are speculated to be involved in glycolysis to produce ATP.111 Surprisingly, MSC-EVs have been reported to increase the level of ATP in myocardial tissue,112 which results in a decrease in oxidative stress and a decrease in local and systemic inflammation.113 CD73 may be present in MSCs, which can dephosphorylate adenosine monophosphate to adenosine, and the same process has been observed in MSC-EVs.114 Notably, transmembrane proteins are also found in MSC-EVs, which mainly includes tetraspanins and integrins (CD9, CD63, CD81 and CD82). These transmembrane proteins are essential for cell targeting and adhesion,111,115 showing the importance of transmembrane proteins in mediating the biological activity of MSC-EVs.

Recently, attention has been paid to apoptotic bodies that are the main products of MSCs apoptosis. Apoptotic bodies are EVs rich in deoxyribonucleic acid, ribonucleic acid, proteins, and organelles.116,117 Interestingly, it has been found that a large number of MSCs exhibit apoptosis after transplantation into a skin wound healing model, and it is speculated that apoptosis plays an important role in activating the inflammatory regulation ability of MSCs.118 Moreover, a research team successfully isolated apoptotic bodies derived from MSCs. In their study, these MSC-derived apoptotic bodies demonstrated significant efficacy in reducing bone loss in an animal model of periodontitis.119 MSC-derived apoptotic bodies have been found to show similar biological activity to MSCs, which can promote muscle growth, skin healing, angiogenesis, and exhibit powerful anti-inflammatory and tissue regeneration effects.120,121 Preliminary evidence suggests that apoptotic bodies may serve as a potential therapeutic strategy to help improve the symptoms and disease progression of IBD.

In conclusion, the study of MSC-EVs can help us to further investigate the mechanism of action of MSCs-based cell therapy. The new cell-free EVs-based therapy is emerging that can overcome some limitations of cellular therapies, and IBD treatment will take on new meaning with the help of cell-free therapy and may have the same therapeutic effect in future.

Secretion of Chemokines, Cytokines & Growth Factors

Chemokines are chemical inducers of the body’s immune cells.122 In the presence of certain inflammatory factors such as TNF-α and IFN-γ, MSCs are stimulated to produce chemokines. The C-X-C chemokine receptor 3 and C-C chemokine 5 ligands are the most common chemokines generated by MSCs triggered by cellular inflammatory stimuli.123 These chemokines produce chemotaxis to recruit T cells in the vicinity of MSCs, which subsequently exert immunomodulatory effects on T cells by expressing inducible nitric oxide synthase or indoleamine 2,3-dioxygenase (IDO).124 The mechanism of inhibition is that inducible nitric oxide synthase acts on the JNK signaling transducer and the activator of transcription signaling pathway through catalytic production of NO, which causes cell cycle arrest in T cells.125 In addition to its effect on T cells, NO production acts on macrophages to reduce the production of pro-inflammatory cytokines, and the regulation of NF-κB and mitogen-activated protein kinase activity may be the underlying mechanisms.126

In addition to the chemokines, MSCs can also secrete a variety of cytokines. MSCs are capable of reacting to inflammatory or harmful signals from the body and are stimulated by the release of soluble bioactive substances, which can serve as feedback signals to encourage MSCs’ immunomodulation.127 In a study, IL-10 released by MSCs prevented immature CD4+ T cells from differentiating into Th17 cells in vitro by downregulating RORγ+ T and the related signaling pathways.128 Interestingly, the recently reported soluble mediator IL-10 is not directly produced by MSCs, but indirectly regulates the release of IL-10 through cell-to-cell communication mechanisms that affect the function of other cells.129 One study suggests that MSCs may promote IL-10 secretion by inducing monocytes and macrophages in the presence of high levels of TNF-α and IFN-γ. MSCs may indirectly produce anti-inflammatory effects by acting on neutrophils during inflammatory episodes.130 In addition, inflammation-activated MSCs also act on macrophages, causing them to polarize to anti-inflammatory macrophages and secrete the anti-inflammatory factor IL-10.131,132 Research investigating MSC interaction with T cells revealed that while MSCs cultured in isolation did not secrete IL-10, co-culture with T cells induced IL-10 production by MSCs. Additionally, IL-10 levels were notably elevated in T cells co-cultured with MSCs.133 This change in perspective provides us with a deeper understanding of the role of MSCs in immunomodulation and therapy that does not simply directly produce and release mediators, but influences the release of mediators by regulating the functions of other cells. This contributes to a more comprehensive understanding of the mechanism of action and clinical application of MSCs.

In the presence of inflammatory cytokines, MSCs produce a range of growth factors including vascular endothelial growth factor, basic fibroblast growth factor, keratinocyte growth factor, insulin-like growth factor and hepatocyte growth factor, which activates the regenerative potential of resident stem cells, promotes angiogenesis, inhibits apoptosis and remodels the stroma.134,135 The embryonic-derived MSCs were found to alleviate Dextran Sulfate Sodium-induced enteritis in mice, improve colonic epithelial proliferation and barrier integrity, and increase the level of insulin-like growth factor-1 in the body circulation, which was found with no improvement in enteritis by MSCs with insulin-like growth factor-1 receptor inhibitors.136

Secretion of Soluble Enzymes and Other Proteins

MSCs express heme oxygenase-1, which is an important soluble enzyme in heme metabolism, with antioxidant and anti-inflammatory effects. Its potential regulatory mechanisms include regulation of toll-like receptors (TLRs)-dependent cytokine gene expression and regulation of inflammatory vesicle-dependent cytokine maturation, and macrophage polarization.137–139 IDO as a soluble enzyme is one of the key factors of immune regulation in MSCs. MSCs induce differentiation and maturation of anti-inflammatory Th2 cells by increasing the expression of IDO, resulting in tryptophan depletion, tryptophan metabolite synthesis, and Th1 cell apoptosis.140,141 Species-specific differences exist in the immunosuppressive mechanisms mediated by MSCs. In humans, MSC-mediated immunosuppression in response to inflammatory cytokines is primarily mediated by IDO, whereas in mice, it is mediated by NO. Similarly, MSCs, regardless of their origin, secrete several factors in the presence of inflammatory factors, with NO and IDO being the most active.142 The research team found that MSC-EVs overexpressed by IDO had better cell proliferation than MSC-EVs and inhibited renal tubular cell apoptosis and fibrosis.143 It has been shown that IDO-depleted MSCs do not have the ability to modulate immune responses.144

MSCs can also help to relieve inflammation by promoting the secretion of anti-inflammatory factors such as prostaglandin E2 (PGE2), TSG-6, and IL-10.145 PGE2 promotes the production of Treg cells, enhances their activity and inhibits the activity of NKs and DC cells.146,147 Besides, TSG-6, a hyaluronan-binding protein produced by MSCs in response to TNF-αstimulation, plays a key role in various immune-mediated inflammatory diseases.40 TSG-6 has multiple anti-inflammatory functions, regulating lymphocyte migration and adhesion through binding to the cell surface receptor CD44, and inhibiting the migration of neutrophils, monocytes and macrophages to inflammatory tissues.134,148 Recently, it was discovered that TSG-6 might speed up mucosal regeneration and encourage epithelial cell proliferation in iPSC-derived MSCs, reducing the signs of enteritis in mice.149

Cell-to-Cell Contact

In addition to paracrine effects, MSCs control immune response mechanisms by interacting with other cells. MSCs engage with cell surface molecules and receptors, and they directly control a number of immune cell downstream pathways that have an impact on immune cell survival, proliferation, and production of effectors.134 The PD-1/ PD-L1 axis is crucial for intercellular interactions. PD-1 is triggered to be expressed on the surface of some activated immune cells, and PD-L1 is the ligand of PD-1, which is expressed in T cells, B cells, DCs, macrophages and some non-hematopoietic cells.150 However, it has been shown that the expression of PD-L1 and PD-L2 in MSCs,151 therefore, MSCs can inhibit T cell activity by binding to PD-1 and its MSC-expressed ligands on the surface of immune cells to achieve immunosuppression and control the development of inflammation.152

In addition, MSCs also have beneficial effects on cell-to-cell contact with non-immune cells. One study found that direct cell-to-cell contact between MSCs and endothelial progenitor cells induces MSCs differentiation into a pericycle-like phenotype, promoting angiogenesis.153

Cell Fusion

Cell fusion mechanism refers to the replacement of damaged cells by cell fusion for tissue repair when cells are damaged in tissues, which occurs widely in prokaryotes and eukaryotes under both natural and pathological conditions, such as in tissue and organ repair, immune response, and tumorigenesis.154 In a prior study, bone marrow derived MSCs (BM-MSCs) from a healthy donor group were transplanted into an injury model group, and long-term proliferation of donor-derived cells was seen in major intestinal epithelial lineages, including cupped cells and enterocytes, suggesting that BM-MSCs are engaged in the repair of damaged intestinal epithelial tissues and also demonstrating that this process occurs through cell fusion.155

Mechanisms of Action Immune Regulation

Numerous studies have shown that MSCs have a wide range of immunomodulatory capabilities. Its immunomodulatory function interacts with immune cells mainly through cell-to-cell contact and paracrine activity. The detailed immunoregulatory mechanisms are shown in Figure 4. For example, MSCs are involved in immune regulation, acting on immune cells, recruiting Treg lymphocytes and reducing Th1, Th17 and B cell differentiation to treat IBD.156 Similarly, it has also been extensively studied in refractory systemic lupus erythematosus,157 graft-versus-host disease158 and rheumatoid arthritis.159

Figure 4 Immunomodulatory mechanisms of MSCs in the treatment of IBD. MSCs produce immunomodulatory effects by secreting chemokines, extracellular vesicles, and a series of cytokines interacting with immune cells such as T cells, B cells, natural killer (NK) cells, macrophages, and dendritic cells (DCs). In addition, MSCs can mediate the immune process through mutual contact with immune cells.

MSCs show a dual role in immunoregulation. MSCs recognize different danger signals through TLRs.160 On the one hand, through their specific recognition pattern, MSCs can cause inflammation by activating the immune system when the host’s immune system is underactive, and on the other hand, MSCs mediate immune regulation to avoid excessive self-attack when the immune system is overactive.161 The initial line of defense is TLR recognition from harmed cells or pathogens, and TLR activation can increase immune system stimulation and activated MSCs respond to TLR ligands and release anti-inflammatory substances. Therefore, in MSCs, TLR is essential for controlling immune responses and signal reception.162 Interestingly, differences in the type of TLR activation also differentially affect the generation of anti-inflammatory or pro-inflammatory phenotypes in MSCs.163–165 TLR3 induces an anti-inflammatory phenotype in MSCs, namely MSC2, and secretes inflammatory mediators such as IL-6 and IL-8. Conversely, TLR4 activation induces a pro-inflammatory phenotype, namely MSC1, and secretes anti-inflammatory mediators like IFN-γ inducible protein-10 and IL-1 receptor antagonist, which inhibits T lymphocyte proliferation through expression of PGE2 and IDO.163,166 TLR also has an important role in that when exogenous MSCs are transplanted into the host, which are slowly removed by NKs, while TLR can activate MSCs and regulate their susceptibility to NKs, thus avoiding the killing of NKs.167 This is why MSCs can be present in the body and produce immunomodulatory effects.167

Several factors can affect the immunomodulatory process of MSCs. For example, there is a difference in immunosuppressive effect between tissue-resident and newly infused MSCs, and the speculation is that its potential could be due to a change in the number of MSCs. Studies have shown that the immunosuppressive ability of MSCs is not inherently expressed, but requires stimulation by inflammatory factors. In certain infections, injuries, or immune-related conditions, MSCs can dampen the immune response when exposed to environments abundant in inflammatory cytokines. Interestingly, this specific performance of MSCs in the presence and absence of inflammatory mediators is called MSC polarization.168 The levels of pro-inflammatory factors such as IFN-γ, TNF-α and IL-1β affect their immune processes and inflammation tend to change in the active state of the disease, which may alter the immune properties of MSCs.166,169–171 When levels of inflammatory factors such as IFN-γ and TNF-α are low, MSC demonstrate a pro-inflammatory phenotype, and MSCs produce chemokines such as MIP-1α/β, CCL5, CXCL9 and CXCL10 to activate T cells to regulate immunity.124 In addition, in the presence of IFN-γ and IL-1, the production of pro-inflammatory M1 macrophages is induced, and M1 further responds to T cell activation.172 Conversely, in the case of high levels of inflammation, MSCs can inhibit the activation and proliferation of T lymphocytes.173 In addition to the above-mentioned inflammatory factors, alarmins like IL-1α,174 IL-33,175 and heat shock proteins176 all have an impact on MSC biology and show a potent ability in tissue repair and showed positive promoting effects on MSCs. MSCs also produce a large number of cytokines such as IDO, PGE2, and TGF-β, which are directly involved in the activation of Treg cells.177 Pro-inflammatory MSCs and anti-inflammatory MSCs have opposite biological functions, and they play different roles depending on the inflammation status in the body, helping to maintain immune balance and tissue health. Therefore, inflammatory status and tissue location are the main factors determining immune regulation of MSCs.

Intestinal Epithelial Repair

MSCs produce the epithelial repair effect through cell fusion mechanism and paracrine action. By intravenously injecting human embryonic stem cell-derived MSCs into Dextran Sulfate Sodium-induced colitis mice, elevated insulin-like growth factor levels were detected. Through the elevated insulin-like growth factor levels, the intestinal epithelial cells were repaired and the epithelial cell integrity was maintained.136 In an in vitro experiment, by co-culturing colon tissue with iPSCs, it was found that iPSCs could also promote the proliferation of colon in vitro.149 In addition, MSCs also improve colitis by promoting intestinal epithelial repair and reducing epithelial cell apoptosis through the derived EVs.178

MSC-Mediated Mitochondrial Transfer

MSCs may also be used in the treatment of IBD through mitochondrial transfer. It has been shown that MSCs can replace damaged mitochondria and leave them intact through replacement, making mitochondrial transfer a potential strategy to promote tissue repair and regeneration.179 Mitochondrial transport in MSCs requires a key Rho-GTPase, and MSCs are capable of expressing high levels of mitochondrial transport protein. It is with the help of this enzyme that the smooth transfer of mitochondria is possible.180 Healthy mitochondria from MSCs can be transferred to target cells in a variety of ways, replacing damaged mitochondria, restoring energy supply and ensuring cell survival.181 The mitochondria transferred by MSCs can act on intestinal epithelial cells, increase ATP levels, provide epithelial cells with the necessary bioenergy for growth and differentiation, reduce oxidative stress, and alleviate the intestinal symptoms of IBD in multiple aspects.85,182

MSCs mediate mitochondrial transfer mainly through tunneling nanotubes, gap junctions, microvesicles, and cell fusion.183 Among them, tunneling nanotubes are a new mode of intercellular communication, and tunneling nanotubes are also the most common mode of mitochondrial transfer.184 There are numerous studies on MSCs-mediated tunneling nanotubes transfer of mitochondria for tissue repair. For example, by implanting MSCs into the injured cerebral vascular system, MSCs transferred healthy mitochondria to cells in the injured tissue sites by means of tunneling nanotubes, which significantly improved the mitochondrial activity of blood vessels, enhanced angiogenesis, and promoted tissue repair and functional recovery.185 In addition, BM-MSCs-derived mitochondria were transplanted into the damaged spinal cord of rats, and mitochondrial transfer through gap junctions could reduce neuronal apoptosis and promote neurological recovery.186 As a new organelle-derived therapy, MSCs-mediated mitochondrial transfer has great promise in promoting epithelial cell survival and reducing apoptosis in IBD patients. However, further studies are needed to understand the precise mechanism of MSCs transfer in mitochondria for the treatment of IBD and the influencing factors that affect the transfer.

Regulating Gut Microbiome

MSCs can achieve the anti-IBD goal by altering the gut microbiota. Variation in the composition of the gut microbiota, ie abundance and species, is a key pathogenic factor in the prevalence of IBD, and it is possible that the gut microbiota may change in the early stages of IBD.35 In one study, intraperitoneal injection of UC-MSCs into mice with enteritis effectively alleviated colitis, and analysis of mouse feces by 16S rRNA sequencing revealed that the variety and number of gut microbes were altered by UC-MSCs.187 Another study found that MSCs changed the gut microbiome, reversed the abnormal microbiome to normal, improved overall gut health and healing, and alleviated enteritis by administering adipose-derived MSCs (AD-MSCs) to mice with colitis.188 However, it is not known whether MSCs act directly on the microbiota to improve enteritis or whether MSCs administration promotes epithelial cell repair and thus normalizes the microbiome. The mechanisms responsible for the altered microbiota have not been determined. The potential mechanisms may be the improvement of host metabolic processes by restoring intestinal microbial diversity and abundance, as intestinal bacteria usually target host metabolism, thereby further activating the immune system and promoting inflammation.189

Anti-Fibrosis, Anti-Bacteria and Angiogenesis

In the early stages of intestinal inflammation, fibrosis is the initiating factor. The therapeutic potential of MSCs has been extensively studied in various organs to combat fibrosis. The expression of immune cells and their cytokines plays a key role in the progression of intestinal fibrosis, the TGF-β signaling pathway is a fundamental driver of intestinal fibrosis. Studies have shown that IL-10, as an anti-inflammatory factor, can reduce the expression of collagen 1 and TGF-β, which plays an important role in inhibiting fibrosis.190 However, their specific role in addressing intestinal fibrosis remains relatively underexplored.191 Different types of T cell subsets, including Th1, Th17, Th22, and Treg cells, along with the cytokines they produce, have been implicated in the progression of intestinal fibrosis, among them, MSCs and their EVs can reduce fibrosis by modulating the immune system.190,192 In addition, MSCs and their derived EVs secrete hepatocyte growth factor and TGF-β to reduce fibrosis, and also inhibit dermal fibroblast-myofibroblast transformation by inhibiting the TGF-β1/Smad2/3 signaling pathway.193,194

Antibiotics can target bacterial infections in the gut, reduce inflammation, and may help improve symptoms.195 The cytokines hepatocyte growth factor, IL-6 and IL-8 secreted by dental pulp MSCs have good antibacterial effects.196 In addition, a preclinical study found that BM-MSCs have good antimicrobial effects on infection of Mycobacterium avium in vivo and in vitro. In addition, the study also found that MSCs and their secretions can enhance the efficacy of antibiotics and reduce side effects.197 These results suggest that MSCs combined with antibiotic therapy is expected to reduce patients’ dependence on long-term antibiotics and is expected to be a promising treatment. We speculate that on the one hand, MSCs may reduce the occurrence of intestinal inflammation through antimicrobials, and on the other hand, MSCs may fight against harmful intestinal bacteria and regulate microbiota balance.

MSCs and their derived EVs can promote endothelial cell proliferation and migrate to form new blood vessels, and are widely used in diabetic wound and infectious wound repair research.198,199 MSCs secrete vascular endothelial growth factor, platelet-derived growth factor, TGF-β, and angiopoietin-1 to promote the regeneration of blood vessels.200,201 Intestinal epithelial tissue regeneration requires the formation of new blood vessels to provide oxygen and nutrients,202 and it is hypothesized that the induction of angiogenesis by MSCs is another major mechanism of action of MSCs in promoting tissue regeneration.

Clinical Trial

Due to their varied actions, MSCs have been demonstrated in preclinical research to be effective for the treatment of IBD.203 However, the data from animal studies are not necessarily applicable to human clinical trials, so the safety and efficacy of MSCs in human IBD treatment still need to be further verified in clinical trials. Therefore, clinical studies of stem cell therapy for IBD are underway and have been reported. In 2003, García-Olmo et al pioneered AD-MSCs for IBD in a woman with a rectovaginal fistula,204 and since then, the effectiveness and safety of MSCs in the treatment of IBD have been shown in an increasing number of Phase I, II, and III clinical studies.205–207 In 2012, a Phase III randomized, double-blind, parallel-group, placebo-controlled trial is underway in Europe and Israel using expanded allogeneic adipose-derived stem cell type to assess the efficacy of treatment of perianal fistula CD at 24 weeks and up to 104 weeks of follow-up.207 It was eventually approved for marketing in the Europe for the treatment of perianal fistula CD.208 As of December 2022, a broad count of clinical trials in MSCs found that the NIH Clinical Trials Database (https://ClinicalTrials.gov/) registered more than 1400 clinical trials for MSC-based treatments, with more than 700 trials for immune-related diseases, accounting for about half of the overall trials. A total of 50 trials are for IBD, including 40 trials for CD and 10 for UC. The current findings suggest that no serious complications have been reported in the clinical trials that have been conducted with MSCs for IBD. However, different MSCs sources, administration methods and doses may have different degrees of clinical symptom improvement. The sources of MSCs varied among studies, including both autologous and allogeneic differences, differences in the tissues taken, insufficient number of randomized controlled studies, unclear criteria for the assessment of side effects, and inconsistent healing criteria, all of which require further pilot studies with more rigorous and rational design and clearer criteria to provide convincing findings.209

Representative clinical trials of MSCs for IBD are shown in Table 2. Currently registered MSCs clinical trials for the treatment of IBD are mainly in the following directions: 1) Different tissue sources of MSCs for CD and UC. 2) Validation of the efficacy and safety of autologous and allogeneic sources of MSCs in IBD. 3) MSCs for the treatment of IBD administration modalities, doses administered, interval of injection and number of injections and clinical trials related to cell-free therapies.

Table 2 Clini