Therapeutic potential and mechanistic insights into adipose-derived stem cells and mesenchymal stem cell-derived exosomes in patients with Crohn’s disease-associated fistulas: challenges and future perspectives

Abstract

Exosomes, as noninvasive biological carriers, have emerged as promising therapeutic agents for a wide range of diseases because of their ability to deliver bioactive molecules and modulate intercellular communication. Their interaction with stem cells (SCs), which naturally secrete regenerative factors, further enhances their therapeutic potential. In particular, the use of adipose-derived stem cells (ADSCs) and exosome-enriched mesenchymal stem cell (MSC) preparations has been explored for treating gastrointestinal (GI) fistulas associated with Crohn’s disease (CD), a debilitating form of inflammatory bowel disease (IBD). Preclinical studies, clinical trials, and systematic reviews have evaluated the safety and efficacy of these approaches, with ADSCs—commercially available as Darvadstrocel—emerging as one of the most promising options. While notable progress has been made in both experimental and clinical research, the overall effectiveness of exosomes and stem cell-based therapies is still an area of active investigation. Further studies are needed to develop optimized, innovative treatments with minimal adverse effects. Given the substantial morbidity and healthcare burden associated with IBD-related fistulas, there is a critical need to find cost-effective, accessible, and reliable therapeutic options and strategies. Critically, none of these innovations will reach patients without regulatory convergence or cost-effectiveness data. Exosome therapies still lack a dedicated approval pathway, and GMP-scale purification is still confined to a handful of facilities, driving prices beyond current reimbursement limits. Future phase III trials must therefore embed economic modeling and adopt composite end points that link molecular engagement to MRI-verified fistula healing and patient-reported outcomes.

Graphical abstract

Introduction

Stem cells (SCs) derived from various sources are widely recognized for their remarkable ability to produce and secrete paracrine factors that drive tissue repair and regeneration. The relative ease of isolation, expansion, and application of these materials has positioned them as focal points for extensive research in both in vitro and in vivo models. SCs have been used in many experimental and clinical studies through various delivery methods, including direct injection, incorporation into engineered hydrogels, and transplantation as two-dimensional (2D) sheets or three-dimensional (3D) aggregates into damaged tissues. Importantly, compared with their nonconditioned counterparts, preconditioned SCs have shown superior therapeutic efficacy, with enhanced regenerative capabilities and improved support for cellular stabilization and proliferation at sites of injury [1].

Mesenchymal stem cells, or MSCs, are adult stromal cells that can be isolated from many tissues and expand easily in the laboratory. They have low immunogenicity and release a wide range of factors that help dampen inflammation and repair tissue. Bone marrow-derived MSCs were the first to be described, but similar cells have since been obtained from the umbilical cord, placenta, dental pulp, and other sources [2]. Adipose-derived stem cells (ADSCs) are a tissue-specific subset of MSCs derived from fat via liposuction. Fat yields far more stem cells than bone marrow does, so ADSCs are often chosen for autologous therapies. Like other MSCs, they express CD73, CD90, and CD105, but they secrete larger amounts of angiogenic growth factors such as VEGF and HGF, making them especially useful for conditions where new blood vessel growth is needed. Both MSCs and ADSCs release extracellular vesicles that include nanosized exosomes [3]. These vesicles carry proteins, lipids, and regulatory RNAs that can reproduce many of the effects of the parent cells without the risks associated with transplanting live cells. Because exosomes are cell-free, they are easier to store, are less likely to provoke immune rejection, and cannot form unwanted tissue.

Recently, exosomes have been identified as potent therapeutic carriers in regenerative medicine [4, 5]. These nanoscale extracellular vesicles, which are secreted by various cell types, serve as effective carriers of bioactive molecules that are crucial for cellular signaling, regulation, and tissue repair. Exosomes derived from both preconditioned and unmodified SCs exhibit distinct molecular profiles and functional properties, increasing their potential for a variety of therapeutic applications [4, 5]. Mesenchymal stem cell-derived exosomes (MSC-Exos) have shown considerable therapeutic potential by modulating inflammatory responses, stimulating angiogenesis, enhancing collagen synthesis, promoting cellular proliferation, and improving cell survival—crucial processes that together support tissue repair and regeneration [6]. Given their multifaceted therapeutic properties, MSC-Exos are promising candidates for the treatment of various conditions, including wound healing, cardiovascular diseases, and inflammatory disorders such as Crohn’s disease (CD).

Exosomes carry diverse cargo of miRNAs that modulate immune responses and epithelial integrity in IBD. For example, exosomal miR-223 can both worsen inflammation (by activating the IL-32 cascade) and confer protection (by regulating CX3CR1 + macrophage differentiation). Other miRNAs, such as miR-21, are highly enriched in exosomes during active IBD and take part in modulating the intestinal barrier and immune cell function. These miRNAs facilitate cell-to-cell communication and coordinate the immune response, further supporting the biological plausibility of exosome-based therapies [7].

Gastrointestinal (GI) disorders, particularly inflammatory bowel diseases (IBDs), such as CD, represent a significant global health burden, often leading to complications such as perianal fistula formation. CD pathogenesis involves genetic predisposition, gut dysbiosis, and environmental triggers that collectively drive aberrant immune activation and chronic inflammation [8]. Sustained release of proinflammatory cytokines (TNF-α, IL-6, and IFN-γ) disrupts the epithelial barrier, leading to deep ulceration and transmural inflammation. This progressive tissue damage culminates in the formation of a fistula, a hallmark of severe disease (Fig. 1) [8]. If left untreated, these fistulas can progress to more severe conditions, including colorectal cancer, ultimately increasing the risk of mortality. Current treatment options for fistula management are limited to immunosuppressive therapies, monoclonal antibodies, surgical interventions, and nutritional support, each with varying degrees of success. Given the limitations of existing therapies, exploring innovative and effective approaches such as ADSCs and MSC-Exos presents a promising avenue for advancing fistula treatment. This study focused on evaluating recent clinical trials and systematic reviews investigating the potential of ADSCs and MSC-Exos as viable therapeutic strategies for fistula management in patients with CD [9].

Fig. 1.

Pathogenesis of Crohn’s Disease and Perianal Fistulas. The figure outlines the multifactorial etiologies of Crohn’s disease, highlighting genetic susceptibility, environmental triggers, and immune dysregulation. Key processes include chronic inflammation leading to deep mucosal ulceration, elevated proinflammatory cytokines (TNF-α, IL-6, and IFN-γ), intestinal barrier disruption, and subsequent fistula tract formation

Here, we critically synthesize clinical and preclinical evidence on the use of ADSCs and MSC-Exos for the treatment of Crohn’s disease-related perianal fistulas, identify knowledge gaps, and outline priorities for future trials.

Mesenchymal stem cells and their derivatives in crohn’s disease

MSCs possess a unique combination of characteristics that make them well suited for cell-based therapies, including their ability to adhere to plastic surfaces, robust proliferative capacity, multilineage differentiation potential, and low immunogenicity [10]. Studies have shown that MSCs and their derivatives exhibit strong anti-inflammatory and immunomodulatory effects in CD models by regulating cytokine production and promoting macrophage polarization [11–14]. Specifically, modified MSCs, such as deconditioned umbilical cord MSCs, demonstrate enhanced therapeutic effects through PGE2-mediated T-cell suppression and ERK pathway activation [11]. MSC-Exos also show promise in reducing intestinal inflammation via ubiquitination and inhibiting fibrosis through Smad/TGF-β pathway modulation [14–16].

The TGF-β/Smad pathway is central to both the pathogenesis and potential treatment of CD and its complications, including perianal fistula. TGF-β binds to its type II receptor, recruiting and activating the type I receptor, which then phosphorylates Smad2/3. Phosphorylated Smad2/3 forms a complex with Smad4, translocates to the nucleus, and regulates the transcription of target genes involved in extracellular matrix (ECM) production, fibrosis, and wound healing [17, 18]. In CD, aberrant activation of the TGF-β/Smad pathway contributes to intestinal fibrosis and epithelial–mesenchymal transition (EMT), both of which are implicated in fistula formation and persistence [18, 19]. Therapeutic interventions targeting this pathway, such as MSC-Exos, can modulate TGF-β signaling, reduce fibrosis and promote tissue repair. For example, exosomes from umbilical cord MSCs have been shown to activate the HIF-1α/TGF-β/Smad axis, thereby increasing collagen synthesis, angiogenesis, and overall healing in fistulous tissue [18].

The therapeutic mechanisms of MSCs involve immunomodulation, tissue repair, and anti-inflammatory actions, with distinct surface markers (CD73/CD90/CD105) that help identify them [10, 20, 21]. In CD treatment, local MSC administration is more effective than systemic delivery, achieving 75% fistula closure versus 30% [22–24]. Long-term follow-up confirms durable clinical benefits, with 67% of patients showing improved fistula tracts on MRI after BM-MSC therapy [25]. Both autologous and allogeneic MSCs have demonstrated safety and efficacy in the treatment of fistulizing CD, while umbilical cord-derived MSCs (UC-MSCs) and placenta-derived MSCs have also shown promising therapeutic potential [26–33].

ADSCs are a highly potent population of multipotent mesenchymal stem cells isolated from adipose tissue and are increasingly recognized for their significant regenerative potential [10, 34, 35]. Compared with other MSCs, ADSCs offer several advantages, making them a preferred choice for various therapeutic applications. One of their most notable benefits is the ease of isolation through minimally invasive procedures such as liposuction, which yields a significantly higher concentration of stem cells—up to 500 times greater than that of bone marrow-derived MSCs [35]. This high yield, combined with their self-renewal capacity and multipotency, supports their application in treating numerous pathological conditions, including osteoarthritis, diabetic foot ulcers, cardiovascular diseases, and IBDs [36].

The therapeutic efficacy of ADSCs is largely attributed to their potent immunomodulatory properties, enabling them to regulate immune responses, suppress inflammation, and ease tissue regeneration through the secretion of cytokines, chemokines, and growth factors. A key advantage of ADSCs is their angiogenic potential, which contributes to neovascularization and supports tissue repair in ischemic and damaged environments. Their capacity to promote angiogenesis, combined with their antiapoptotic effects, underscores their effectiveness in treating chronic wounds and other conditions that require enhanced vascularization and tissue regeneration [37–39].

In the context of CD, particularly in cases of perianal fistulizing CD, ADSCs have appeared to be promising therapeutic options. The complex pathology of perianal fistulas, which are frequently resistant to conventional medical and surgical interventions, has spurred increasing interest in the application of ADSCs to promote fistula healing and improve patient outcomes [36]. The immunomodulatory properties of these compounds, which have been proven in both preclinical and clinical studies, contribute to improved healing outcomes. Furthermore, ADSC therapy offers a noninvasive alternative with advantages such as reduced pain, shorter recovery periods, and minimal damage to surrounding healthy tissues, enhancing patient quality of life during treatment [40]. The European Medicines Agency (EMA)-approved harvester (allogeneic adipose-derived MSCs) has demonstrated significant healing in complex perianal fistulas, with a 60% success rate and mild adverse effects [41–43]. Phase III trials highlight improved outcomes with expanded MSCs, although rare complications, such as abscesses or EBV-associated lymphoproliferation, warrant caution.

Exosomes, a subset of small extracellular vesicles (EVs) typically ranging from 30 to 150 nanometers in diameter, have attracted considerable attention in the field of regenerative medicine [4, 5, 44–47]. These vesicles are secreted by a variety of cell types, including ADSCs, through a tightly regulated endosomal sorting pathway [36]. Exosome biogenesis begins with the inward budding of the plasma membrane to form early endosomes, which subsequently mature into multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs) [36]. Upon fusion of MVBs with the plasma membrane, ILVs are released into the extracellular space as exosomes [36].

A critical mechanism by which ADSCs and their exosomes exert therapeutic effects is through the polarization of macrophages from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype. ADSC-derived exosomes (ADSC-Exos) are rich in immunomodulatory cytokines such as prostaglandin E2 (PGE2) and IL-6, which induce M2 polarization. This is achieved via the JAK/STAT6 signaling pathway: IL-4/IL-13 binding leads to JAK1 activation, STAT6 phosphorylation, and the upregulation of M2-associated genes (e.g., Arg-1, IL-10, and CD206). Moreover, exosomal delivery of phosphorylated STAT3 further enhances M2 polarization and anti-inflammatory gene expression. M2 macrophages promote tissue repair, angiogenesis, and the resolution of inflammation, which are all essential for fistula healing [48]. Given the limited efficacy of current medical and surgical interventions for CD-associated perianal fistulas and the significant role of inflammation in gastrointestinal pathology, stem cell-derived exosomes have appeared to be promising therapeutic alternatives. Their potent immunomodulatory effects, ability to promote tissue regeneration, and critical role in mediating intercellular communication make them attractive candidates for clinical application in the management of inflammatory and fistulizing conditions. The lipid-rich composition of exosomes, which includes cholesterol, sphingomyelin, and ceramide, provides structural stability and protects their bioactive contents during transport [49]. Notably, exosomes are enriched in ether lipids, which contribute to membrane stability, cellular differentiation, and protection against oxidative stress (Fig. 2).

Fig. 2.

Cellular interactions and mechanisms of ADSC-exos in fistula repair. ADSC-Exos that modulate key cell populations within the fistula tract. Exosomal IL-10 promotes macrophage polarization from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype via the JAK/STAT6 pathway. miR-21-5p in exosomes aids epithelial healing by converting diseased epithelial cells to a treated state. Exosomes also deliver IL-10 and TGF-β, supporting regulatory T (Treg) cell activation and immunosuppression. Activation of the Wnt/β-catenin pathway enhances fibroblast recruitment and tissue remodeling

In addition to their lipid composition, exosomes contain a diverse array of proteins, including tetraspanins (CD81, CD82, and CD63), heat shock proteins (HSP60 and HSP70), lactadherin, and annexins, all of which contribute to their biological functions [50]. Furthermore, exosomes encapsulate a diverse array of nucleic acids, including DNA, messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA (lncRNA), which are selectively packaged through active sorting mechanisms and contribute to gene regulation and intercellular signaling [51]. These components enable exosomes to modulate recipient cell behavior, mediate anti-inflammatory responses, and promote tissue healing through paracrine signaling mechanisms.

MSC-Exos have shown substantial potential in modulating inflammatory pathways, upregulating vascular endothelial growth factor (VEGF) expression, and promoting tissue repair, making them particularly promising for the treatment of perianal fistulas in CD patients. The ability of these compounds to deliver bioactive molecules directly to target cells enhances their therapeutic efficacy. In addition, exosomes offer several advantages over whole-cell transplantation, including a lower risk of immune rejection and tumorigenicity, thereby representing a safer and more controlled alternative for regenerative medicine applications [52]. Additionally, their natural targeting properties and biocompatibility make them promising candidates for drug delivery, further expanding their potential in clinical therapies (Fig. 3).

Fig. 3.

Overview of exosome biodistribution and ADSC mechanisms of action in fistula repair. Schematic representation of three delivery approaches: (1) hydrogel delivery via a chitosan hydrogel to increase the local retention of exosomes at the treatment site; (2) systemic intravenous delivery involving time-dependent biodistribution (0, 10, or 60 min) of exosomes to major organs; and (3) local injection targeting the fistula area. ADSC-secreted factors, including interleukin-10 (IL-10) and vascular endothelial growth factor (VEGF), mediate key therapeutic effects, such as immunomodulation and angiogenesis

Emerging data show that the clinical benefits of MSCs, ADSCs and their EVs in perianal fistulizing CD are driven by a small set of convergent signaling cascades. ADSC‑Exos transfer miR‑21‑3p, miR‑126‑3p and hepatocyte growth factor (HGF) to endothelial cells, triggering PTEN inhibition and robust PI3K/Akt phosphorylation; this up‑regulates VEGF and accelerates tube formation and granulation tissue perfusion, which are prerequisites for durable fistula closure. Loss‑of‑function of Akt or PI3K abolishes these proangiogenic effects in in vitro and murine wound models [53]. In a rat model of complex perianal fistulas, umbilical‑cord‑MSC‑Exos increased HIF‑1α, TGF‑β1/β3, and phosphorylation of Smad2/3, driving collagen deposition, fibroblast activation, and epithelial sealing; the pathway was dose‑dependent and correlated with faster external‑orifice closure [54]. ADSC‑Exos potently activate Wnt2b/β‑catenin in dermal (and anal) fibroblasts; pharmacological blockade with XAV‑939 or β‑catenin siRNA reverses fibroblast migration, collagen I/III synthesis and wound contraction, confirming the centrality of the pathway to fistula tract re‑epithelialization [55]. Proteomic profiling of bone‑marrow‑MSC‑Exos revealed that metallothionein‑2 (MT2A) is an abundant cargo that enhances IκBα transcription in macrophages, preventing NF‑κB p65 nuclear translocation, curtailing TNF‑α/IL‑1β secretion and skewing macrophages toward an IL‑10‑producing M2 phenotype. Silencing MT2A or depleting macrophages abrogates the anti‑colitic and, by extension, anti‑fistulous effects of MSC‑Exos [56]. Hypoxia‑preconditioning or rapamycin‑induced autophagy in parental MSCs amplifies EV yield and selectively enriches miRNAs that co‑activate PI3K/Akt and dampen NF‑κB while simultaneously promoting the LC3‑II‑mediated survival of transplanted cells; these synergistic effects explain the superior fistula healing observed with “autophagy‑enhanced” MSC‑EVs reported previously. These studies indicate that MSC/ADSC therapies do more than secrete generic anti‑inflammatory cytokines: they re‑program local immunity (NF‑κB/IκBα), restore vascular supply (PI3K/Akt), orchestrate fibro‑epithelial repair (HIF‑1α/TGF‑β/Smad and Wnt/β‑catenin) and are further potentiated by autophagy induction. Mapping these pathways provides rational targets to (i) bioengineer EVs with defined cargo, (ii) select synergistic biomaterial carriers, and (iii) stratify patients who may benefit most from combined pathway activation (e.g., Akt agonists plus EV hydrogel). Future phase III trials should incorporate pathway‑specific biomarkers (p‑Akt, nuclear β‑catenin, p‑Smad2/3 and IκBα levels) to correlate molecular engagement with clinical response.

Accumulating evidence shows that the therapeutic effects of MSCs, ADSCs and their exosomes are mediated by discrete, experimentally validated signaling cascades. Deconditioned umbilical cord MSCs suppress pathogenic CD3⁺ T-cell activity via a prostaglandin E₂–ERK 1/2 axis, an effect that is lost with pharmacologic inhibition of either component [57]. MSC-Exos dampen intestinal inflammation by inducing K48-linked ubiquitination of TRAF6, which blocks NF-κB activation, and by simultaneously reducing Smad2/3 phosphorylation within the canonical TGF-β pathway to limit fibrosis [14–16]. ADSC-Exos transfer miR-21-3p, miR-126-3p and hepatocyte growth factor to endothelial cells, silencing PTEN and triggering robust PI3K/Akt activation; blockade of PI3K or Akt abrogates the resulting VEGF-driven angiogenesis essential for fistula closure [53]. In rat models of complex perianal fistulas, human umbilical-cord MSC-Exos upregulated HIF-1α and TGF-β1/β3, increased Smad2/3 phosphorylation and accelerated collagen deposition and epithelial sealing in a dose-dependent fashion [54]. ADSC-Exos also activate Wnt2b/β-catenin signaling in fibroblasts, and β-catenin inhibition (XAV-939 or siRNA) reverses collagen I/III synthesis and wound contraction, confirming pathway specificity [55]. Proteomic analyses revealed that metallothionein-2, as a dominant MSC-Exo cargo that increases IκBα transcription, prevents p65 nuclear translocation and polarizes macrophages toward an IL-10-producing M2 phenotype, effects that are abolished after metallothionein-2 knockdown [56]. Finally, hypoxia- or rapamycin-induced autophagy in parental MSCs enriches vesicles with miRNAs that concurrently stimulate PI3K/Akt and inhibit NF-κB, yielding superior fistula healing compared with that of naïve vesicles [57, 58]. Collectively, these studies confirm that ADSC/MSC therapies act through a tightly defined network of signaling pathways that can be selectively harnessed or engineered to maximize clinical efficacy (Fig. 4).

Fig. 4.

Key molecular signaling pathways activated by ADSC-Exos. Exosomes from adipose-derived stem cells (ADSCs) regulate multiple pathways that promote tissue repair and immune modulation. TGF-β/Smad signaling: Exosomal TGF-β activates Smad2/3 and Smad4, leading to the nuclear translocation and transcription of anti-inflammatory genes. PI3K/AKT pathway: Exosomal miR-126-3p inhibits PTEN, thereby activating PI3K/AKT signaling and enhancing VEGF-mediated angiogenesis. NF-κB pathway: Exosomes modulate metallothionein-2 and IκBα to suppress p65 activity and reduce TNF-α production, limiting inflammation. Wnt/β-catenin pathway: Exosomal Wnt2b activates β-catenin signaling, increasing collagen expression for tissue remodeling

Given these properties, ADSCs and MSC-Exos hold significant promise for advancing treatment strategies for Crohn’s perianal fistula. Further research and clinical studies are essential to improve their therapeutic applications and establish standardized protocols for their use in clinical practice.

Therapeutic potential of ADSCs in complex perianal fistulas

ADSCs play pivotal roles in tissue engineering and regenerative medicine. This review explores the biological properties and therapeutic applications of ADSCs, with a particular focus on their clinical use in the treatment of fistulizing perianal Crohn’s disease, including insights from recent clinical trials (Tables 1 and 2).

Table 1.

Several published studies and ongoing clinical trials have applied adipose-derived stem cells for the management of crohn’s disease-related fistulas

Trial/Study (Year)	Intervention	Cell Type	Administration Mechanism	Design/Population	Main Outcome(s)	Key Findings	Ref.
Garcia-Olmo et al. (2009)	ASCs + fibrin glue	Allogeneic MSCs	Local injection	RCT, 49 patients	Healing at 8 weeks, 1 year	71% healing vs. 16% control; first RCT to show significant efficacy	[73]
Lee et al. (2013)	Autologous ASCs	Autologous ASCs	Local injection	Phase II, 43 patients	Healing at 8 weeks	82% healed; 88% maintained closure at 1 year	[54]
Panés et al. (2016)	Cx601 (Darvadstrocel)	Allogeneic MSCs	Local injection	Phase III RCT	Combined remission at 24 weeks	50% remission vs. 34% placebo; pivotal registration trial	[41]
Panés et al. (2018)	Cx601 (Darvadstrocel)	Allogeneic MSCs	Local injection	RCT, 212 patients	Combined/clinical remission	56.3% remission vs. 38.6% placebo; confirmed efficacy in larger cohort	[23]
Furukawa et al. (2022)	Darvadstrocel	Allogeneic MSCs	Local injection	Phase III, 22 Japanese adults	Combined remission at 24/52 weeks	68.2% achieved remission at 52 weeks; validated efficacy in Asian cohort	[38]
Bacsur et al. (2024)	Darvadstrocel	Allogeneic MSCs	Local injection	Real-life cohort, 223 patients	Remission at 26/52 weeks	62.3% remission at 52 weeks; confirms effectiveness in routine care	[43]
Brochard et al. (2024)	Darvadstrocel	Autologous ADSCs	Local injection	Multicenter retrospective, 116 patients	Clinical and deep remission	44% clinical, 29% deep remission; better outcomes in young/high fistulas	[78]
Fathallah et al. (2024)	Darvadstrocel	Autologous ADSCs	Local injection	Postmarketing study, 652 patients	Clinical response, remission, safety	76.1% remission; 6.9% anal abscess most common AE; no treatment-related deaths	[79]

Keys: SVF: Stromal Vascular Fraction; PRP: Platelet-Rich Plasma; MSC: Mesenchymal Stem Cell; pCD: Perianal Crohn’s Disease; ASCs: Adipose-Derived Stem Cells; ADSC: Adipose-Derived Stem Cell; QoL: Quality of Life; RCT: Randomized Controlled Trial; AEs: Adverse Events; MRI: Magnetic Resonance Imaging; DVS: Darvadstrocel; ERAF: Endorectal Advancement Flap; CDAI: Crohn’s Disease Activity Index; PDAI: Perianal Disease Activity Index; VAS: Visual Analog Scale; FIA: Fistula-in-Ano; SoC: Standard of Care; RVF: Rectovaginal Fistula; AT: All-treated cohort; TEAEs: Treatment-emergent adverse events; ECM: Extracellular matrix

Table 2.

Summary of clinical trials investigating the use of adipose-derived stem cells (ADSCs) for the treatment of crohn’s disease-associated perianal fistulas

Clinical Trial No. Name Location	Phase Patients No.	Protocol Design	Outcome Measure in Main Endpoints
[NCT04118088] Postauthorization Safety Study of the Long-Term Safety and Efficacy of Repeat Administration of Darvadstrocel in Patients With Crohn’s Disease and Complex Perianal Fistula EU, USA	Phase 4 50	Local injection into the fistula received a single repeat dose of 12 × 107 ADSC (Cx601—Darvadstrocel— ALOFISEL®) (5 million cells/mL).	TESAE, Combined Remission of Perianal Fistula(s), Clinical Response, Time to Relapse and PDAI at weeks 6, 24, 52, 104, and 156.
[NCT04612465] Phase 3 Clinical Study to Evaluate Efficacy and Safety of ADSC (Autologous ADSCs) and Fibringlue or Fibringlue in Patients With Crohn’s Fistula.: A Randomized Study Seoul, Korea	Phase 3 36	The autologous ADSCs injection dose is approximately 1 × 10^7 cells of ADSC per 1 cm^2 of the surface area of the fistula, and the additional injection dose is 1.5 times the initial injection dose. and up to 30% of the ADSC injection dose is administered in combination with Fibrin glue.	Fistula blockage, Visual improvement effect of perianal fistula wound during 12 months after injection.
[NCT05177003] Study of the 12-month Efficacy of Stem Cell Injection in Crohn’s Disease With Complex Ano-perineal Fistula Paris, France	Observational study	Injection of allogeneic mesenchymal stem cells (ALOFISEL®)	12-month clinical remission rate, Primary clinical failure rate at month 3 and Evolution of the severity of the disease at 12 months.

This table provides an overview of selected clinical trials evaluating the safety and efficacy of ADSC-based therapies for the treatment of perianal fistulas in patients with CD. It includes information on trial identifiers, study phases, patient populations, treatment protocols, and primary outcome measures. Trials enrolling fewer than 10 patients were excluded to ensure data robustness. Abbreviations: CD: Crohn’s disease; IL-10: Interleukin-10; TNFα: Tumor necrosis factor α; AE: Adverse event; TRAE: Treatment-related adverse event; PDAI: Perianal Disease Activity Index; Cx601: Darvadstrocel (Alofisel^®); VAS: Visual analog score; CDAI: Crohn’s Disease Activity Index; IBDQ: Inflammatory Bowel Disease Questionnaire; IDO: Indoleamine 2,3-dioxygenase; mPP: Modified per-protocol analysis; mITT: Modified intention-to-treat analysis; MRI: Magnetic resonance imaging; TESAE: Treatment-emergent serious adverse event

Unlike conventional surgical methods such as the endoanal advancement flap, which can result in fecal incontinence rates as high as 30%, ADSCs are minimally invasive and do not impair fecal continence [58]. Compared with standard surgical approaches involving curettage and seton management without introducing additional adverse effects, ADSC injection has been shown to increase healing rates by approximately 17% [38]. While other techniques, such as anal fistula plugs and fibrin glue, have varying success rates, they often have significant drawbacks. Analistula plugs have a high early success rate (70–100%) but a lower late healing rate (under 50%). Similarly, fibrin glue treatment has a success rate ranging from 14% to 63% [73]. Mucosal advancement flaps achieve a cure rate of 66–87%, but they are associated with a high recurrence rate and a 35% incidence of mild to moderate incontinence. Ligation of the intersphincteric fistula tract (LIFT) has inconsistent results (51–94%) and is suboptimal for complex fistulas [74].

ADSC therapy has demonstrated superior short- and long-term outcomes for patients with Crohn’s disease-related fistulas. Over the past decade, advancements in ADSC and stromal vascular fraction (SVF) applications have been well documented. Clinical trials have investigated various formulations, delivery methods, and adjuncts, such as platelet-rich plasma (PRP), to enhance refractory fistula healing. These approaches promote tissue regeneration, inflammation suppression, and durable closure with minimal side effects. Notably, ADSC treatment preserves quality of life and sphincter function, with no reported cases of therapy-induced incontinence. The safety profile remains excellent across studies, as supported by phase I/II trials confirming autologous ADSC feasibility and early efficacy.

Patients with complex, treatment-resistant fistulas often achieve significant healing post-ADSC injection after conventional therapies fail. Adjunct PRP use, as in Arkenbosch et al. (2025), yielded a 68% clinical response at 12 months with SVF + PRP. Comparative studies have shown that ADSCs match or surpass endorectal advancement flap (ERAF) outcomes with fewer complications. The phase III ADMIRE trial demonstrated 50% healing with allogeneic ADSCs versus 34% placebo (sustained at 52 weeks) [23], underscoring their value for conventional therapy-refractory cases. While antibiotics remain first-line agents, trials confirm their inadequacy for durable closure.

Advanced cell-based products such as Darvadstrocel (Cx601), which is composed of expanded allogeneic mesenchymal stem cells (MSCs), have been the subject of multicenter randomized controlled trials. Compared with the placebo, the ADMIRE-CD study and its extensions [23, 41, 59] demonstrated superior combined remission and safety. Additional real-world studies from France, Spain, and Austria [43, 60–62] supported these findings, noting improved quality of life, decreased disease activity indices, and acceptable adverse event profiles.

Longitudinal studies assessing the durability of healing suggest that ASC therapy offers long-term benefits. For example, Cho et al. (2015) [63] and Panés et al. (2022) [64] reported that healing persisted in more than 75% of patients after 2 years. Predictive modeling by Karki et al. (2024) [65] reinforced these clinical outcomes, resulting in higher remission rates and delayed relapse rates when stem cell therapies were compared with standard-of-care treatments. Furthermore, institutional and compassionate use programs [66, 67] in Spain and elsewhere confirmed both the accessibility and practical benefits of these therapies for patients outside traditional trial settings.

Trials targeting specific subtypes, such as rectovaginal fistulas [22], further expand the applicability of these therapies across diverse fistula presentations.

Overall, the use of ADSCs represents a significant advancement in the treatment of perianal fistulas, offering improved healing rates, better preservation of anal function, and a favorable safety profile. Despite these advantages, the regulatory and economic pathways for ADSC-based therapies remain complex. As ADSCs are classified as advanced therapy medicinal products (ATMPs) under the EMA framework, their clinical translation requires compliance with stringent manufacturing, quality, and pharmacovigilance standards, which can delay widespread clinical availability [68]. Moreover, the cost-effectiveness of ADSC therapy remains uncertain, with limited health economic models available to assess its long-term impact compared with conventional surgical and biologic strategies [69]. These regulatory and economic challenges may represent significant barriers to broader adoption. Nonetheless, the regenerative and anti-inflammatory properties of ADSCs and their ability to address complex fistulas make them valuable components of modern fistula management.

Therapeutic potential of MSC-Exos in complex perianal fistulas

MSC-Exos have emerged as a promising therapeutic strategy for the treatment of complex perianal fistulas, particularly in patients unresponsive to conventional medical and surgical interventions.

Unlike ADSCs, MSC-Exos carry bioactive molecules such as proteins, lipids, and nucleic acids and exert immunomodulatory, anti-inflammatory, and pro-regenerative effects without the risks associated with whole-cell transplantation, including uncontrolled differentiation or tumorigenicity [37, 54]. Preclinical studies have demonstrated that exosomes derived from human umbilical cord MSCs (hUC-MSC-Exos) significantly enhance wound healing in rat models of perianal fistulas by promoting collagen synthesis, angiogenesis, and immune regulation via activation of the HIF-1α/TGF-β/Smad signaling pathway (Fig. 5). This mechanism facilitates tissue repair by improving the oxygen supply, reducing inflammation, and stimulating the activity of fibroblasts, which are critical factors for fistula closure (Fig. 5). Additionally, exosome therapy has been shown to restore the gut microbiota balance and modulate metabolic pathways, further supporting its therapeutic potential [54].

Fig. 5.

Therapeutic Mechanisms of ADSC and MSC-Exos in Inflammatory Bowel Disease and Perianal Fistulas. This figure illustrates the therapeutic potential of exosomes derived from ADSCs and MSCs in IBD and fistula treatment. Key effects include the modulation of inflammatory cytokines, promotion of M2 macrophage polarization, mucosal healing, epithelial regeneration, and fistula closure. The diagram highlights how ADSC- and MSC-derived exosomes target inflamed tissues in the IBD colon to restore homeostasis

Clinical trials have further validated the efficacy and safety of MSC-Exos in treating refractory perianal fistulas in CD patients (Tables 3 and 4). In a phase I trial, local exosome injections resulted in complete healing in three out of five patients, with no reported adverse effects [37]. Another study involving 11 patients with complex perianal fistulas reported that exosome therapy led to complete resolution in five patients and reduced discharge in eight patients, reinforcing its potential as a minimally invasive and well-tolerated intervention [52]. A subsequent phase II trial confirmed these findings, with 60% of patients achieving complete fistula closure and 69.7% of treated tracts healing fully, as assessed by clinical examination and MRI [70]. These results suggest that exosomes not only match the therapeutic efficacy of whole MSCs but also offer practical advantages, such as easier storage and reduced immunogenicity.

Table 3.

Summary of clinical trials investigating the use of MSC-Exos for the treatment of perianal fistulas in patients with crohn’s disease

ClinicalTrials.gov Identifier	Phase/Design (masking, centers)	Treated N	Primary outcome definition & time-point	Type of Exosomes	Mechanism of Action	Follow-up	Results	Key limitations	References
NCT05402748	Phase 1 open-label dose-escalation, single-center (Iran)	5	External-orifice closure (clinical) at 12 wk.	Umbilical Cord MSC-Derived Exosomes	Immunomodulation, Anti-inflammatory effects	6 months	4/5 patients responded, 3 had complete healing, and no adverse effects	Uncontrolled; tiny cohort; no MRI confirmation; durability unknown	[37]
IRCT20200413047063N3	Pilot, open-label, single-center	11	Complete fistula closure (clinical) or ↓ discharge at 12 wk.	Placenta-Derived MSC Exosomes	Immunomodulation, Tissue Regeneration	3 months	5/11 patients had complete fistula closure, 8 had reduced discharge, and no adverse effects	No randomization/blinding; heterogeneous endpoints; very short FU	[52]
NCT05402749	Phase II nonrandomized, single center	20	Combined clinical + MRI tract closure at 24 wk.	MSC-Derived Exosomes (Source Unspecified)	Immunomodulation, Anti-inflammatory effects	6 months	12/20 patients had complete closure, 69.7% fistula tract closure, significant reduction in inflammation	No comparator; modest N; manufacturing & dose rationale not reported	[70]

This table provides an overview of selected clinical trials assessing the safety and efficacy of MSC-Exos in the treatment of Crohn’s disease-associated perianal fistulas. Key information includes trial identifiers, exosome sources and types, proposed mechanisms of action, principal findings, and relevant references

Table 4.

Comparative table of key clinical trials that have shaped the MSC/MSC-Exo field in IBD

Trial (lead author & year)	IBD subtype /indication	Cell or EV product (origin)	Administration & schedule	Sample size (treated n)	Main efficacy readout (time-point)	Safety signals	Ref.
ADMIRE-CD (Panés 2016)	Crohn’s – complex perianal fistula	Expanded allogeneic ADSCs (darvadstrocel/Cx601)	Single intralesional injection after curettage & internal-opening closure	103	51.5% combined remission at 24 wk; 56.3% at 52 wk vs. 38.6% placebo	No new safety signals through 104 wk	[59]
Furukawa 2023 (Japan phase III)	Crohn’s – complex perianal fistula	Same darvadstrocel product	Single 120 × 10⁶-cell intralesional dose	22	59.1% combined remission at 24 wk; 68.2% at 52 wk	2 mild treatment-related AEs (4. %)	[38]
Molendijk 2015	Crohn’s – complex perianal fistula	Allogeneic BM-MSCs (30 × 10⁶ optimal)	Local fistula-wall injections (single)	21	80% complete closure with mid-dose at 24 wk; dose-response seen	No severe AEs	[93]
Ciccocioppo 2011	Crohn’s – complex external fistulae	Autologous BM-MSCs	Serial intrafistular injections every 4 wk (median 4)	10	70% sustained complete closure at 12 mo	No adverse effects reported	[26]
Lee 2013 (phase II)	Crohn’s – fistula-in-ano	Autologous ADSCs + fibrin-glue carrier	1–2 intralesional doses proportional to tract size	33	82% complete healing at 8 wk; 88% sustained at 12 mo	No therapy-related AEs	[94]
Jiang 2024	Ulcerative colitis (moderate–severe, steroid/biologic refractory & non-refractory)	Allogeneic umbilical-cord MSCs (UMSCs)	Two monthly IV infusions (1 × 10⁶ cells/kg)	41	41.5% clinical remission & 73.2% response at 2 mo; 34.2% remission at 6 mo	No serious infusion reactions	[95]
Soleimani 2022 (phase I)	Crohn’s – refractory perianal fistula	UC- MSC-Exos	Single peritract injection (50 µg × 5 mL)	5	60% complete healing at 6 mo (3/5); one partial responder	Zero systemic or local AEs	[96]
Hadizadeh 2024 (phase II)	Crohn’s – refractory perianal fistula	UC- MSC-Exos	Three peritract injections (q 2 mo)	20*	60% patient-level & 69. % tract-level closure at 6 mo	No serious AEs	[97]

In addition to perianal fistulas, exosome therapy has shown promise in other challenging wound-healing contexts, including pharyngocutaneous fistulas following total laryngectomy [71] and postsurgical colocutaneous fistulas [72]. When delivered via thermoresponsive hydrogels or direct injection, exosomes have been shown to reduce inflammation, increase angiogenesis, and accelerate tissue regeneration. Their ability to modulate immune responses and promote repair processes highlights their versatility in managing complex wounds and fistulas across various anatomical sites [72].

The therapeutic benefits of exosomes are dose dependent, with studies indicating that moderate doses (e.g., 10 µg/100 µL) yield optimal outcomes, whereas higher concentrations may not provide additional benefits owing to receptor saturation or counteractive inflammatory effects (Fig. 6) [54]. This underscores the importance of precise dosing in clinical applications. Moreover, exosome therapy offers a cell-free alternative that circumvents the ethical and safety concerns associated with stem cell transplantation, such as malignant transformation or immune rejection [37, 52].

Fig. 6.

Clinical and immunological outcomes of ADSC-Exo therapy in fistula treatment. The left panel shows improved fistula closure rates in animals treated with ADSC-Exos (~ 70%) compared with controls (~ 30%). The middle panel depicts cytokine modulation, demonstrating increased anti-inflammatory IL-10 levels and decreased proinflammatory TNF-α levels following therapy. The bottom panel highlights disease state changes, where untreated tissue exhibits active NF-κB signaling (red), whereas treated tissue shows inhibited NF-κB activity (green), reflecting reduced inflammation and tissue repair

EVs derived from ADSCs have also demonstrated therapeutic potential in fistula healing. In a porcine model, local delivery of ADSC-EVs via a thermoresponsive Pluronic F-127 hydrogel resulted in complete fistula healing, reduced fibrosis, decreased inflammation, and enhanced angiogenesis compared with those of hydrogels alone or untreated controls [73].

Similarly, the amniotic fluid-derived MSC (AF-MSC) secretome and exosomes exhibit anti-inflammatory effects in IBD models. In human intestinal subepithelial myofibroblasts (SEMFs), AF-MSC-Exos reduced the expression of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and TLR-4 while increasing the expression of anti-inflammatory cytokine IL-10, suggesting their potential as a cell-free therapy for IBD [37].

These findings collectively highlight the therapeutic potential of MSC-Exos and EVs in fistula healing and IBD, offering a safe, effective, and minimally invasive alternative to conventional treatments. However, the translation of EV-based therapies into clinical practice is subject to complex regulatory oversight under the ATMP framework, which governs their manufacturing, quality control, and pharmacovigilance [68, 74]. Additionally, economic modeling for EV-based therapeutics remains scarce, raising uncertainties about large-scale cost-effectiveness and health system integration [69].

Action mechanism of ADSC and MSC-Exos in fistula healing

MSCs may regulate perianal fistulas by suppressing T-cell activity, balancing inflammatory cytokines (TGF-β, TNF-α, and IL-6/7/13), and secreting IL-10 to promote tissue repair in perianal fistulizing patients with Crohn’s disease (Figs. 5 and 7, and 8); however, the mechanisms involved remain unclear [75, 76]. Phase II/III trials have demonstrated the efficacy of MSCs through the release of anti-inflammatory cytokines (adipose/bone marrow-derived), which promote vascular repair and re-epithelialization, with local administration being safe for the treatment of refractory perianal fistulizing Crohn’s disease (Fig. 5) [9, 76, 77] and murine models confirming sustained anti-inflammatory effects [78]. While MSCs show promise (optimal dose of 3 × 10⁷ cells/mL) with high efficacy and few adverse events in patients with Crohn’s fistula [79], further studies are needed to validate their clinical application [9, 76, 78, 79].

Fig. 7.

Therapeutic Mechanisms of ADSC and MSC-Exos in Inflammatory Bowel Disease. The figure illustrates the therapeutic effects of ADSCs and MSC-Exos in IBD. Key mechanisms include epithelial healing via the modulation of TNFα, IL-1β, and M2 macrophage polarization, alongside the suppression of proinflammatory factors (e.g., elevated TNFα and IL-1β in ulceration). Contrasts between disease state markers (IL-10, TGFβ) and therapeutic interventions are highlighted

Fig. 8.

Mechanisms of Action of ADSC and MSC-Exos in Perianal Fistulas. This figure summarizes the therapeutic mechanisms of ADSCs and MSC-Exos in promoting the healing of perianal fistulas. Key processes include immune modulation (T-cell regulation, cytokine balance, macrophage polarization to the M2 phenotype), tissue regeneration (angiogenesis, re-epithelialization, and reduced fibrosis), and activation of signaling pathways (HIF-1α/TGF-β/Smad). The figure highlights how ADSCs and MSC-Exos restore immune balance, reduce inflammation, and enhance structural repair in fistulous tracts

IL-10 is a key anti-inflammatory cytokine that plays a pivotal role in immune regulation within the gut. MSCs engineered to overexpress IL-10 (IL-10-MSCs) or exosomes enriched with IL-10 can suppress inflammatory cell infiltration, reduce Th2 cytokine production, and inhibit proinflammatory cytokines such as IL-5 and IL-13. The immunosuppressive effect of IL-10 is mediated through its action on CD4 + T cells, downregulating their proinflammatory responses and promoting a tolerogenic environment. Blockade of IL-10 reverses these benefits, underscoring its central role in mediating the therapeutic effects of MSCs and their exosomes [80].

Autophagy-enhanced MSC-derived EVs reduce renal fibrosis in vitro by suppressing IL-1 and TNF-α expression (Figs. 2 and 5) [81], suggesting that autophagy induction may increase MSC immunosuppression, EV efficacy, and survival, thereby improving autoimmune disease therapy, including perianal fistulizing Crohn’s disease. Additionally, EV-nanofiber-hydrogel composites promote fistula healing through macrophage polarization and angiogenesis, indicating promising therapeutic potential for perianal fistulizing CD treatment [82]. These findings position autophagy-modulated MSCs and their secretion as promising therapeutic strategies that warrant further investigation [81, 82].

In summary, MSC-Exos represent a breakthrough in regenerative medicine for fistula treatment, combining high efficacy with an excellent safety profile. Their ability to modulate key signaling pathways, restore microbial and metabolic balance, and promote tissue repair positions them as a transformative therapy for refractory perianal and other complex fistulas. Future research should focus on standardizing production protocols, improving delivery methods and expanding clinical trials to confirm their long-term benefits across diverse patient populations.

Challenges and future directions

The use of ADSCs and MSC-Exos for fistula treatment presents notable challenges, particularly with respect to safety and therapeutic efficacy. A major concern is the potential tumorigenicity of ADSCs, as they may differentiate into undesirable tissues, such as calcified tissues, or even promote tumor development. The influence of the local microenvironment can worsen these risks, leading to unintended differentiation and possibly aiding tumor metastasis. While exosomes are considered to carry a lower risk, there is still a lack of conclusive long-term safety data, and further studies with larger cohorts and extended follow-up periods are needed. Another significant issue is the short half-life of systemically administered exosomes, which limits their therapeutic potential. This limitation underscores the need for localized delivery methods and the development of biomaterials to increase exosome retention at the target site [83]. Early research into the use of ADSC-Exos in biomaterials, such as chitosan hydrogels, as well as the use of genetic engineering techniques and chemical modifications for fistula treatment, represents not only a future research direction but also a major therapeutic strategy in cell-free therapy. Thus, this field is still in its infancy and requires substantial further investigation.

Many clinical studies, especially early-phase and pilot trials, have enrolled a limited number of participants. For example, phase I studies of MSC-Exos often include fewer than 10 patients, which restricts the statistical power and generalizability of the findings [37]. Even in larger meta-analyses, most individual studies included only 5–33 patients, with a few exceptions, such as the Panés et al. trial (212 patients) [79]. Several trials lacked control groups or used nonrandomized, open-label designs, increasing the risk of bias and limiting the ability to attribute observed effects solely to the intervention. Many early ADSC studies were single-arm studies or compared them with historical controls rather than with contemporaneous placebo groups [84]. Many studies have reported outcomes at 3–6 months, with fewer providing long-term data on the durability of fistula closure and recurrence rates. This limits the understanding of sustained efficacy and late adverse events [79]. While most trials report favorable safety profiles, the small sample sizes and short follow-up periods may underrepresent rare or delayed complications, such as tumorigenicity or ectopic tissue formation [84]. Most studies enrolled adults with complex, refractory fistulas and excluded those with significant comorbidities or concurrent therapies, potentially limiting their applicability to broader patient populations [84].

There is still no consensus clinical dose for MSC-Exos. Early trials delivered between 1 and 5 micrograms of total protein per fistula tract and up to 100 micrograms per treatment session. The only controlled animal study in complex fistulas showed a bell-shaped response: 10 micrograms in 100 µl outperformed both lower and higher doses, suggesting rapid receptor saturation or negative feedback at higher levels. Biodistribution work revealed that intravenously injected vesicles clear from plasma within minutes and accumulate mainly in the liver, spleen and lung, repeating first-pass entrapment long-term with whole MSCs. Experimental homing techniques, including peptide display, magnetic loading and antibody conjugation, improve uptake in vitro but have not yet produced meaningful enrichment in vivo, and none of the chemical methods have reached the good-manufacturing-practice grade. Rigorous dose-finding studies that relate local exosome concentrations to pharmacodynamic markers such as cytokine shifts or fistula closure are therefore still lacking. Exosome cargo can trigger innate immune sensors. Vesicle-associated DNA or RNA and heat-shock proteins activate Toll-like receptors, and exosomal DNA produces type I interferon responses in autoimmune models. Tissue factor-bearing vesicles from tumors or inflammatory sources accelerate thrombin generation; similar procoagulant activity has been measured in some MSC-Exo preparations and occurs after MSC infusion. Whole adipose-derived MSCs can form unwanted tissue, and their vesicles may transfer oncogenic microRNAs or profibrotic proteins such as TGF-beta. However, long-term surveillance beyond five years after local injection is still lacking. Open questions include whether complement or thrombosis pathways are activated by repeated dosing and whether anticoagulation should be performed with systemic delivery. The benefit is strongly model- and strain dependent. Most DSS- or TNBS-induced colitis models improve after MSC treatment, yet PI3K-gamma knockout mice deteriorate, developing deeper ulcers and dysbiosis. The cell source, passage number and priming conditions matter: umbilical cord MSCs often outperform bone marrow-derived MSCs, and preconditioning with interleukin-6 or hypoxia markedly alters their efficacy. Outcome measures vary between laboratories, from macroscopic scores to cytokine panels and microbiota analysis—making meta-analysis difficult. Therefore, translation should rely on head-to-head comparative studies that use harmonized protocols and common endpoints. Mesenchymal stem cells are classified as advanced-therapy medicinal products in the European Union and follow the IND/BLA pathway in the United States. Exosome products do not yet belong to a defined regulatory class; authorities are still debating whether they fall under existing biological rules or require a new framework. To date, no exosome therapy has received marketing authorization. For cell products, release criteria are mostly limited to viability and surface markers, and there is no single functional potency assay that predicts clinical activity. For exosomes, the situation is even less standardized: size distribution, particle number, cargo profiling and simple in vitro bioassays are used case by case. Donor-to-donor variability, senescence beyond passage five, freeze–thaw loss of key enzymes and heterogeneity introduced by isolation methods all compromise batch consistency. Sterility testing is complicated by nanosize, and high-volume GMP purification of vesicles is available at only a handful of facilities worldwide. The cost is still large; one dose of darvadstrocel is priced at approximately sixty-five thousand euros, and current exosome production costs are even higher, with no reimbursement code yet in place.

Research should prioritize elucidating the precise mechanisms of action of ADSCs, particularly their interactions with the immune system and local tissues, to mitigate the risks of unintended differentiation and tumorigenesis. Improving administration protocols, such as the timing, dosage, and combination with other therapies, is crucial for maximizing therapeutic outcomes. The development of advanced biomaterials to improve the stability and delivery of exosomes and address logistical challenges such as cell viability and cost will be essential for scaling these therapies for broader clinical use. These advancements are key to establishing ADSC-based treatments as practical and widely accepted methods for fistula therapy.

Gene editing is rapidly redefining next-generation cell products for Crohn’s-related perianal fistulas. CRISPR/Cas9 has been used to knock in immunoregulatory genes (PD-L1, IL-10) and chemokine receptors (CXCR4) in MSCs, creating hypoimmunogenic lines that home more efficiently to fistula tracts and require lower doses to achieve closure [85]. Knockout of proapoptotic mediators such as BAX further prolongs graft survival. In parallel, “synthetic exosome mimetics” are gaining traction. High-pressure extrusion of MSC membranes results in the generation of cell-derived nanovesicles at ~ 100-fold greater yields than natural exosome secretion does, while exosome–liposome hybrids permit stoichiometric loading of CRISPR plasmids or small RNAs and show enhanced endosomal escape in vitro [86]. These platforms aim to combine the biorecognition of vesicles with the scalability of liposomes, addressing one of the greatest manufacturing constraints in the field [87].

Systemically infused vesicles are cleared within minutes; therefore, multiple localization strategies are in development. Loading of exosomes with superparamagnetic iron oxide nanoparticles enables “dual targeting”: passive homing via exosomal surface proteins plus active guidance by an external magnetic field, resulting in five-to-sixfold greater accumulation at wound sites in preclinical models [88]. Compared with bolus injection, shear-thinning hydrogels formed from oxidized alginate or catechol-functionalized chitosan protect vesicles from enzymatic degradation and release them in response to local mechanical stress; one study reported prolonged vesicle residence (>14 days) and a 40% increase in fistula closure [89]. Microfluidic “EV-on-a-chip” devices are being designed to isolate, load and concentrate vesicles in a closed system, potentially enabling bedside preparation with reduced operator variability [90].

EV-based products currently lack a dedicated regulatory category. Potency assays are largely surrogate, size distribution, surface marker, and simple macrophage-migration readout methods, and none have yet reliably correlated with clinical efficacy [91]. Batch consistency is still problematic: donor-to-donor variability, passage-related senescence, and freeze‒thaw loss of enzymatic cargo can alter vesicle bioactivity by an order of magnitude. The number of GMP-compliant facilities capable of producing >10¹⁴ particles per lot is limited, and sterility testing is complicated by vesicle size and lipid content. Regulatory agencies in the EU and USA are therefore requesting tighter characterization panels (protein + RNA cargo, procoagulant activity, and endotoxin levels) before first-in-human dosing, which may lengthen development timelines but is essential for product comparability [92].

Mesenchymal stem cells are classified as ATMPs in the European Union and follow the IND/BLA pathway in the United States. Exosome-based products currently lack a dedicated regulatory category, and agencies are actively evaluating whether these products should be regulated under existing biologic frameworks or a novel classification [68, 69, 74]. Manufacturing challenges—such as donor–to-donor variability, batch inconsistency, limited GMP-grade production facilities, and the absence of validated potency assays—further complicate regulatory approval. From an economic standpoint, the high production and delivery costs, coupled with a lack of established reimbursement pathways, raise significant concerns regarding the cost-effectiveness and large-scale clinical implementation of these therapies [69]. The incorporation of robust health economic models and regulatory harmonization will be critical for the sustainable integration of ADSC- and MSC-Exo–based therapies into mainstream clinical practice.

Future directions are expected to shape the next generation of MSC and exosome therapeutics. Gene‑editing is being applied to knock out classical HLA antigens or to insert immune‑modulatory genes such as PD‑L1, IL‑10 or CXCR4. This strategy could produce off‑the‑shelf MSCs that evade rejection, home more efficiently to fistula tracts and require lower doses to achieve closure. Researchers are building biomimetic lipid nanoparticles that carry the membrane proteins of natural exosomes but can be manufactured at far higher yields and loaded with defined cargo. Food‑grade vesicles, for example, those isolated from bovine milk, are also being explored as orally administered carriers for anti‑inflammatory small RNAs. Research is moving toward integrated regimens that combine MSC-Exos with other agents. Examples include smart hydrogels that release vesicles together with antibiotics or growth factors and viral vectors that simultaneously silence several inflammatory targets, while exosomes deliver pro‑healing signals. These combination approaches aim to increase response rates well beyond today’s ceiling of approximately 60% while also addressing cost, scalability and safety constraints. Together, genome‑engineered MSCs, synthetic exosome mimetics and rational combination platforms stand for the most promising avenues for improving efficacy and bringing cell‑free or enhanced‑cell therapies for intestinal fistulas into routine clinical practice.

Conclusion

Fistula-in-ano (FIA), especially in patients with Crohn’s disease, remains challenging despite advancements in surgical techniques. The emergence of ADSCs has led to a significant shift toward regenerative therapies, showing promise in accelerating fistula healing and improving patient outcomes. MSC-Exos further enhance this potential by promoting tissue repair, reducing inflammation, and supporting wound healing. However, critical questions remain that necessitate further research and clinical trials. The precise mechanisms by which ADSCs facilitate fistula closure—whether through tissue regeneration, immunomodulation, or a combination of both—are not fully understood. Additionally, the optimal conditions for ADSC differentiation, the most effective delivery methods, and the long-term viability of these cells after injection require clarification. Standardizing treatment protocols, including the type, dosage, and frequency of ADSC administration, is also crucial to ensure consistent and reproducible outcomes. Moreover, economic challenges, including the excessive cost of ADSC preparation and the need for scalable production methods for MSC-Exos, must be addressed to make these therapies accessible on a larger scale. Given these unresolved issues, we strongly encourage the scientific community to pursue large-scale, randomized controlled trials and targeted studies to refine and optimize ADSC therapy for FIA [98, 99]. Only through rigorous scientific investigation can the full therapeutic potential of ADSCs be realized, paving the way for a new standard in the treatment of perianal fistulas and ultimately enhancing the quality of life for patients with Crohn’s disease and related conditions.

Manufacturing and engineering hurdles persist. Natural vesicle yields are low, batch-to-batch variability is high, and senescence after passage five reduces product potency. Emerging solutions, such as CRISPR-edited “off-the-shelf” MSC lines and high-throughput synthetic exosome mimetics, offer scalability but still require validation under good manufacturing-practice conditions. Delivery and retention remain problematic. Systemically infused vesicles are cleared from circulation within minutes. Magnetic nanoparticle labeling, shear-thinning injectable hydrogels and microfluidic “EV-on-a-chip” devices have improved site-specific deposition in preclinical models, yet none have been rigorously evaluated in human fistulas. Dose‒response relationships are also undefined; bell-shaped curves seen in animals highlight the need for careful pharmacokinetic‒pharmacodynamic studies, and analytical gaps impede progress. Exosome products have no harmonized potency assays; current release criteria (size, particle count, surface markers) correlate poorly with clinical activity. Because a dedicated regulatory framework does not yet exist, developers face uncertain approval pathways and reimbursement prospects. The clinical trial design is suboptimal. Most studies to date are single-center, unblinded and underpowered, with heterogeneous endpoints such as external-orifice closure or MRI indices. Future phase III trials must incorporate molecular biomarkers—phosphorylated Akt, nuclear β-catenin, and phosphorylated Smad2/3—along with objective imaging so that pathway engagement can be linked directly to patient outcomes.

Over the next five years, five actions are essential. Manufacturing can be standardized by adopting gene-edited master cell banks and defining a minimal release panel that includes cargo profiling and procoagulant activity. Delivery can be optimized by clinically testing magnetically guided vesicles and smart hydrogels in large-animal fistula models before moving to adaptive human trials. Harmonize endpoints by agreeing on composite measures that combine MRI fistula activity scores with validated patient-reported outcomes. Securing regulatory clarity through early engagement with authorities to shape a fit-for-purpose pathway for acellular vesicle therapies. Health-economic modeling should be included in late-phase trials so that cost-effectiveness is understood before market launch. ADSCs and MSC-Exos have progressed from conceptual promise to demonstrable clinical benefit, yet four key barriers, manufacturing consistency, targeted delivery, regulatory certainty and robust trial design, must be overcome before they can become affordable, standard-of-care treatments for Crohn’s perianal fistulas.

Abbreviations

ADSCs

Adipose-derived stem cells

MSCs

Mesenchymal stem cells

MSC-Exos

Mesenchymal stem cell-derived exosomes

ADSC-Exos

Adipose-derived stem cell-derived exosomes

GI

Gastrointestinal

CD

Crohn’s disease

IBD

Inflammatory bowel disease

EVs

Extracellular vesicles

SVF

Stromal vascular fraction

PRP

Platelet-rich plasma

PDAI

Perianal Disease Activity Index

TNF-α

Tumor necrosis factor-alpha

IL-10

Interleukin-10

VEGF

Vascular endothelial growth factor

TGF-β

Transforming growth factor-beta

MRI

Magnetic resonance imaging

IDO

Indoleamine 2,3-dioxygenase

UC-MSCs

Umbilical cord-derived mesenchymal stem cells

AF-MSCs

Amniotic fluid-derived mesenchymal stem cells

LIFT

Ligation of the intersphincteric fistula tract

EMA

European Medicines Agency

Author contributions

Mohsen Sheykhhasan: Conceptualization, supervision, writing – original draft, project administration. Piao Yang: Data curation, Formal analysis. Seyyed Mohammad Yaghoubi: Figure preparation. Saeedeh Zare Jalise: Writing – review & editing, final approval. Naresh Poondla: Investigation, Visualization. Maryam Taghavi Narmi: Methodology, Writing – review & editing. Nikoo Baghal Darbandi: Writing – review & editing. All the authors critically reviewed and approved the final manuscript.

Funding

This research received no specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

All data analyzed in this review are derived from publicly available studies cited in the references. No new datasets were generated.

Declarations

Ethics approval and consent to participate

Not applicable. This is a review article analyzing previously published studies; no new human or animal subjects were involved.

Consent for publication

Not applicable. The manuscript contains no individual person’s data requiring consent.

Competing interests

The authors declare that they have no competing financial or nonfinancial interests related to this work.