Hurdles to overcome for mesenchymal stem cell translation from bench to bedside

Abstract

Mesenchymal stem or stromal cells (MSCs) are among the most extensively studied cell populations in regenerative medicine due to their multipotent differentiation potential, secretion of trophic factors, and immunomodulatory effects. Over the past two decades, preclinical studies have demonstrated encouraging results across musculoskeletal, cardiovascular, neurological, and immune-mediated disorders. However, the translation of MSCs from the laboratory to routine clinical practice remains hindered by unresolved scientific, technical, and regulatory challenges. This review provides a critical appraisal of these hurdles, organized across three major stages of translation: In vitro research, in vivo animal studies, and clinical application. In vitro issues include the heterogeneity of isolation techniques, replicative senescence during expansion, genetic and epigenetic instability, and the need for xeno-free, standardized culture platforms. In vivo challenges arise from poor cell survival, low engraftment rates, off-target migration, and microenvironmental influences that shape therapeutic outcomes. Clinical translation introduces additional complexity, including inter-patient variability, large-scale manufacturing difficulties, stringent regulatory compliance, high production costs, and the absence of harmonized potency assays. Solutions under exploration include the use of automated bioreactors, biomimetic scaffolds, hypoxic preconditioning, extracellular vesicle-based therapies, and international standardization efforts. Addressing these hurdles through multidisciplinary collaboration is essential for MSC-based therapies to become reliable, safe, and accessible regenerative treatments.

Key Words: Regenerative medicine; Clinical translation; Stem cell expansion; Scaffolds; Immunomodulation; Good manufacturing practice

Core Tip: Regenerative medicine has been practiced in a variety of settings for decades. It is a multidisciplinary branch of medicine that aims at replacing degenerated cells or regenerating new human cells, tissues, or organs that have been lost or damaged due to the effects of aging, illness, or congenital defects. The main objective of regenerative medicines is to restore normal functions of the cells or tissues in patients affected by degenerative disorders. Mesenchymal stem cells (MSCs) have the potential to cure the most critical regions or take over the function of a wounded organ that is considered beyond repair using conventional therapies. In the last two decades, thousands of in vitro, in vivo, and clinical trials have been conducted using MSCs. Though the regenerative potential of MSCs has been extensively studied, there are still a lot of challenges that need to be addressed for their successful translation from bench to bedside.

INTRODUCTION

Regenerative medicine represents an interdisciplinary field aimed at restoring normal tissue and organ function that has been impaired by trauma, degenerative disease, or aging. At the center of this effort lies the therapeutic promise of stem cells, particularly mesenchymal stem or stromal cells (MSCs)[1,2]. MSCs are non-hematopoietic, multipotent cell capable of differentiating into osteoblasts, chondrocytes, adipocytes, and other lineages[3,4]. In addition to their differentiation capacity, MSCs exert powerful paracrine effects by secreting cytokines, chemokines, and extracellular vesicles (EVs) that modulate inflammation, enhance angiogenesis, and promote endogenous repair mechanisms[5,6]. These secretory functions allow MSCs to orchestrate a pro-regenerative microenvironment even in the absence of long-term engraftment or differentiation, broadening their therapeutic utility across a wide spectrum of diseases[7,8].

Indeed, MSCs have been explored not only in orthopedic and musculoskeletal disorders but also in cardiovascular, hepatic, neurological, and immune-mediated pathologies[9-11]. The preclinical literature documenting MSC efficacy is vast and compelling. In rodent and large-animal models, MSCs have been shown to improve fracture healing, regenerate articular cartilage, limit infarct size following myocardial ischemia, ameliorate liver fibrosis, and prevent severe immune complications such as graft-vs-host disease[12-16]. These findings have catalyzed rapid clinical translation: As of today, thousands of MSC-related clinical trials are registered worldwide, exploring indications as diverse as osteoarthritis, Crohn’s disease, spinal cord injury, acute respiratory distress syndrome, systemic lupus erythematosus and so on[17,18]. Despite this intense global activity, the clinical impact of MSC therapies has been modest relative to initial expectations. The vast majority of clinical trials remain in early phases, with variable and sometimes inconclusive outcomes. Only a few products have achieved regulatory approval, and these are restricted to narrow indications (Table 1)[19-21].

Table 1 Mesenchymal stem cell-based products that have received market approval from major regulatory agencies.

Product name	Developer company	Cell type	Indication	Regulatory body	Country	Year	Ref.
Ryoncil™ (remestemcel-L)	Mesoblast	Allo-BM-MSC	Pediatric SR-aGvHD	United States FDA	United States	2024	[20]
Cupistem®	Anterogen	Auto-AD-MSCs	Perianal Crohn’s fistula	MFDS	South Korea	2012	[19]
Cartistem®	Medipost	UC-MSC	Knee OA	MFDS	South Korea	2012	[19]
Temcell HS®	JCR Pharmaceuticals	Allo-BM-MSC	SR-aGvHD	PMDA	Japan	2015	[19,20]
Amimestrocel (conditional approval)	Platinum Life Excellence Biotech	UC-MSC	SR-aGvHD with gastrointestinal tract involvement in 14 years of age and older	NMPA	China	2025	[21]
Cellgram AMI	Pharmicell	Auto-BM-MSC	AMI	MFDS	South Korea	2011	[19]
NeuroNata-R	Corestem	Auto-BM-MSC	ALS	MFDS	South Korea	2014	[19]
Stempeucel	Stempeutics Research	Allo-BM-MSC	CLI	DCGI	India	2016	[19]
MesestroCell	Cell Tech Pharmed	Auto-BM-MSC	OA	IFDA	Iran	2018	[19]
Stemirac	Nipro Corp	Auto-BM-MSC	SCI	PMDA	Japan	2018	[19]

Allo-BM-MSC: Allogeneic bone marrow-derived mesenchymal stem cells; SR-aGvHD: Steroid-refractory acute graft vs host disease; FDA: Food and Drug Administration; Auto-AD-MSCs: Autologous adipose derived mesenchymal stem cell; MFDS: Ministry of Food and Drug Safety; UC-MSC: Umbilical cord-derived mesenchymal stem cell; OA: Osteoarthritis; PMDA: Pharmaceuticals and Medical Devices Agency; NMPA: National Medical Products Administration; Auto-BM-MSC: Autologous bone marrow-derived mesenchymal stem cell; AMI: Acute myocardial infarction; ALS: Amyotrophic lateral sclerosis; CLI: Critical limb ischemia; DCGI: Drugs Controller General of India; IFDA: Iran Food and Drug Administration; SCI: Spinal cord injury.

The reasons for this translational bottleneck are multifactorial. At the in vitro level, variability in MSC isolation techniques, donor heterogeneity, and culture practices influence cell quality and potency[22]. Expansion under suboptimal conditions can accelerate genetic drift and senescence, undermining reproducibility and safety[23-25]. At the in vivo level, challenges include poor survival and engraftment of transplanted cells, differences between controlled laboratory models and the complexity of human disease, and uncertainty regarding long-term safety[26,27]. Finally, at the clinical and commercial levels, large-scale manufacturing, quality control, regulatory approval, patient selection, and cost-effectiveness present formidable barriers that must be systematically addressed before MSC therapies can achieve widespread adoption[28].

The persistence of these hurdles has led to increasing recognition that a more critical and integrative perspective is necessary. Enthusiasm for MSCs has often outpaced rigorous mechanistic understanding, and inconsistent methodologies have clouded interpretation of trial results. To move the field forward, it is essential to delineate the full spectrum of translational barriers, identify the underlying causes, and highlight innovative strategies that are being developed to overcome them. Only through such a comprehensive assessment can the promise of MSC-based therapies be converted into reproducible clinical reality. Hence, the aim of this review is to provide a comprehensive analysis of the barriers facing MSC therapy, categorized into three domains: In vitro challenges (standardization of isolation and culture, genetic/epigenetic stability, and biomimetic systems), in vivo challenges (translation from controlled systems to complex organisms, tissue-specific differences, microenvironmental influences, and long-term safety), and clinical challenges (manufacturing, logistics, regulatory approval, patient heterogeneity, and commercialization). For each challenge, potential strategies to overcome limitations are critically evaluated, with a view toward guiding the future of MSC translation.

CHALLENGES IN VITRO STUDIES

The first stage of MSC translation involves their derivation, culture, and expansion in controlled laboratory conditions. While in vitro studies have provided essential knowledge regarding MSC biology, they also represent a critical bottleneck because the quality of cells generated at this stage profoundly affects downstream preclinical and clinical outcomes. Below, the major categories of in vitro hurdles are discussed in detail.

Isolation and expansion techniques

The process of isolating MSCs is foundational yet fraught with variability. Bone marrow aspiration remains the classical source, but yields decline with donor age and the procedure itself is invasive, often producing heterogeneous mixtures of stromal and hematopoietic cells[29,30]. Adipose tissue offers a minimally invasive alternative with abundant yields; however, enzymatic digestion protocols using collagenase raise concerns regarding residual enzyme contamination and non-good manufacturing practice (GMP) compliance[31,32]. Umbilical cord-derived MSCs, especially from Wharton’s jelly, have gained popularity due to their neonatal origin, high proliferative index, and immunoprivileged status. However, variability between cord processing methods limits reproducibility across laboratories[33,34].

Several techniques exist to enrich for MSCs during isolation. Density gradient centrifugation remains common to isolate MSCs from bone marrow aspirate, although it fails to guarantee purity[35,36]. Explant culture allows MSCs to migrate from tissue fragments, preserving their phenotype but yielding small numbers over long culture periods. While, enzymatic digestion increases yield but risks damaging surface epitopes[33,35]. More advanced methods include immunoselection using surface markers such as CD271, Stro-1, and CD146, which enrich for subpopulations with superior clonogenicity and differentiation potential[37,38]. Nevertheless, these approaches are expensive and technically demanding, complicating their integration into routine manufacturing.

Large-scale expansion presents further challenges. Clinical applications often require 10⁷-10⁹ cells per patient, necessitating extensive passaging[39,40]. Repeated culture drives replicative senescence characterized by flattened morphology, diminished clonogenic potential, reduced expression of immunomodulatory molecules, and secretion of pro-inflammatory cytokines as part of a senescence-associated secretory phenotype[41]. Such functional deterioration undermines therapeutic potency. Strategies under investigation include limiting passage numbers, employing three-dimensional (3D) bioreactor systems that expand cells more efficiently, and using pharmacological agents such as antioxidants to delay senescence[40,42-44].

Media composition, culture practices, and environment

Media formulation exerts profound effects on MSC quality. Fetal bovine serum has historically served as the gold standard supplement because of its nutrient richness, but its use is increasingly untenable for clinical applications. Risks include xenogeneic pathogen transmission, immunogenic reactions, and substantial batch-to-batch variability[45,46]. Alternatives such as human platelet lysate and human serum have shown strong potential, with clinical studies demonstrating comparable or superior proliferation rates compared to fetal bovine serum-cultured MSCs[26,45,47]. Chemically defined serum-free formulations eliminate biological variability altogether, although high costs and source-specific optimization remain obstacles[48,49].

Culture practices have also great impact on MSC fate. Seeding density plays a critical role in maintaining proliferation and differentiation potential. Studies reported that excessively low density leads to no proliferation, while high density induces premature senescence[50,51]. Passage number has also a strong correlation with functional decline of MSCs. Hence, early-passage cells (P1-P5) are generally recommended for therapeutic applications[52-54]. Media replacement schedules could further influence outcomes. While frequent complete changes replenish nutrients and reduce reactive oxygen species accumulation, they may inadvertently remove autocrine and paracrine factors important for MSC maintenance[55-57]. Gradual or partial media exchange could be proposed as a compromise, maintaining signaling cues while minimizing metabolic stress.

The culture environment extends beyond media composition. Oxygen tension plays a pivotal role during in vitro expansion of MSCs. In vivo, MSCs reside in hypoxic niches (2%-8% O₂), but conventional culture exposes them to atmospheric levels (approximately 21% O₂), accelerating oxidative stress and senescence[24,58]. Culturing under physiological hypoxia has been shown to extend proliferative lifespan, enhance immunomodulation, and preserve differentiation potential[59,60]. Mechanical cues such as substrate stiffness, shear stress, and cyclic stretch also modulate MSC behavior. For instance, rigid substrates promote osteogenesis, whereas softer matrices encourage neurogenic or adipogenic differentiation[61-64]. Advanced bioreactor platforms simulate these mechanical conditions, offering scalable and physiologically relevant environments.

The in vivo origin of MSCs and the perivascular niche

MSCs were first identified by Friedenstein et al[65] in the 1960s and, around 30 years later, renamed characterized by Caplan[66]. MSCs are multipotent cells capable of differentiating into osteoblasts, adipocytes, and chondrocytes[67-69]. They are found in nearly all tissues and can migrate to sites of injury when required[70,71]. However, it is unclear whether they are resident within tissues or replenished from the bone marrow[72]. Furthermore, the in vivo origin of MSCs remains uncertain as well. A landmark study by Crisan et al[73,74] revealed that many MSC-like cells originate from perivascular compartments, primarily pericytes and adventitial cells, found throughout human tissues. These perivascular cells, when cultured, show clonogenicity and multilineage differentiation, supporting the perivascular-origin hypothesis of MSCs[73].

Pericytes arise from multiple embryonic lineages that vary by tissue type. Their exact developmental origins remain incompletely defined, with evidence indicating diverse sources across organs[75]. In the cephalic region and thymus, pericytes originate from the neuroectoderm[75-78]. In the lung, heart, liver, and gut, they primarily derive from the mesothelium[75,77,79]. In most other organs, pericytes are of mesodermal origin[80,81]. This developmental diversity suggests that pericytes are a heterogeneous population whose origin reflects the embryological context of each tissue[77].

While it is traditionally considered that MSCs are mesoderm-derived, similar cells from ectodermal origins, such as dental pulp stem cells, also exhibit MSC-like features defined by Dominici et al[69]. Regardless of the origin and tissue source, MSCs show multi-differentiation, strong immunomodulatory and therapeutic potential[82]. Although their core properties are alike, their sensitivity and responsiveness to inflammation or regeneration may vary depending on their tissue of origin[72,83]. Source selection, isolation tactics, and efforts to replicate in vivo potency during therapeutic application after in vitro expansion would all benefit from a better mechanistic understanding of these native identities, including niche signals that maintain quiescence, mobilization cues following injury, and lineage-priming.

Genetic and epigenetic stability

Beyond extrinsic culture factors, intrinsic genetic and epigenetic mechanisms also determine MSC behavior. Prolonged culture induces telomere shortening and genomic instability, which may manifest as chromosomal aberrations or aneuploidy[84,85]. Though such alterations do not undergo malignant transformation, however, it raises concern regarding both safety and efficacy for clinical application[86]. At the transcriptional level, prolonged passaging downregulates stemness-associated genes including sex-determining region Y-box 2, octamer-binding protein 4, and NANOG, diminishing self-renewal and multipotency[87].

Epigenetic drift represents another layer of instability. DNA methylation changes can silence stemness genes or activate differentiation pathways prematurely[88]. Histone modifications, such as increased H3K9 methylation, promote transcriptional repression and senescence, while acetylation of H3K27 is associated with proliferation and renewal[89,90]. Non-coding RNAs, particularly microRNAs (miRNAs), are emerging as critical regulators: MiR-21 supports proliferation and immunomodulation, miR-1246 has roles in paracrine activity, and dysregulation of miR-92a impairs angiogenesis[91-93]. Disruption of these epigenetic regulators during in vitro expansion may therefore critically undermine therapeutic function of MSCs. To mitigate these risks, several approaches are under exploration. Hypoxic culture reduces oxidative DNA damage and preserves telomere length[94,95]. Genetic and epigenetic monitoring, including karyotyping, telomere length assessment, and methylation profiling, are increasingly recommended as part of GMP quality control frameworks[96-104] (Table 2).

Table 2 Methods to detect genetic stability of in vitro expanded mesenchymal stem cells.

Assay	What it detects	Strengths	Limitations	Suggested use/frequency	Ref.
Conventional karyotype (G-banding)	Large chromosomal abnormalities	Low cost; detects numerical and structural chromosomal abnormalities	Low resolution (approximately 5-10 Mb), not sufficient to predict full genetic stability, laborious	At master cell bank and before clinical release	[96-98]
FISH	Low mosaicism, minor structural abnormalities	Rapid, sensitive, easy data interpretation	The chromosomal aberration being searched for must be known beforehand	Not a screening technique, complementary to karyotyping for further proof of genotypic stability	[96-98]
M-FISH	Overall view of all chromosomes in a single assay	High resolution (approximately 1.5 Mb)	Highly expensive, difficult to analyze and interpret data	Not to be used to replace karyotyping	[96,99]
a-CGH	Copy number variations	Higher resolution than karyotype (≤ 50 kb), medium cost, no need for metaphases, requires only the genomic DNA	Cannot detect balanced translocations, inversion and intragenic rearrangement; need of experienced cytogenetic specialist	At defined passage thresholds and for new donor lines or when recurrent abnormalities are found	[96-98]
WGS	Single-nucleotide changes and copy number alterations	Highest resolution; comprehensive	Costly; complex analysis; uncertain clinical significance for many variants	As confirmatory test for master bank or if abnormalities suspected	[100]
Telomere length (qPCR)	Telomere attrition	Simple surrogate for replicative senescence	Not definitive for malignancy risk	Periodic, after a certain number of passages	[101]
DNA methylation profiling	Epigenetic drift; aging signatures	Useful for senescence, potency	Interpretation still evolving	For research and characterization; consider for potency correlation	[102-104]

FISH: Fluorescence in situ hybridization; M-FISH: Multiplex/multicolor fluorescence in situ hybridization; a-CGH: Array comparative genomic hybridization; WGS: Whole-genome sequencing; qPCR: Quantitative polymerase chain reaction.

Biomimetic scaffold designs

Conventional two-dimensional plastic culture lacks the complexity of the native extracellular matrix, leading to altered cell morphology and loss of functional properties[105]. Biomimetic scaffolds provide a more physiologically relevant microenvironment, supporting cell adhesion, survival, and lineage-specific differentiation[106,107]. Natural scaffolds derived from collagen, fibrin, hyaluronic acid, and chitosan offer biocompatibility but often suffer from variability and limited mechanical strength[108]. Synthetic scaffolds such as polylactic acid, polycaprolactone, and polyethylene glycol allow precise control over stiffness and degradation kinetics but require biochemical modification to support cell adhesion[109,110].

Hybrid scaffolds combine the advantages of natural and synthetic materials, while advanced fabrication techniques such as electrospinning and 3D bioprinting enable precise control of architecture and spatial distribution of bioactive cues[110]. For example, scaffolds incorporating bone morphogenetic proteins and high stiffness are being tailored for bone regeneration, while soft, aligned hydrogel scaffolds are designed to support neural regeneration. Gradients in scaffold stiffness or biochemical signaling further enable engineering of complex, multi-lineage tissues[111,112].

The translational potential of scaffolds depends not only on their biological performance but also on their manufacturability and regulatory approval. GMP-compliant scaffold production requires reproducibility, sterility, and biocompatibility testing. Ultimately, biomimetic scaffolds not only serve as tools for MSC expansion but also as delivery vehicles in vivo, enhancing engraftment, survival, and paracrine activity in therapeutic contexts.

CHALLENGES IN VIVO STUDIES

While in vitro studies provide controlled insights into MSC biology, their translation into in vivo contexts introduces far greater complexity. Once transplanted into living organisms, MSCs encounter host immune responses, dynamic microenvironments, and mechanical challenges that can profoundly alter their fate. Understanding and overcoming these barriers are essential for bridging preclinical promise with clinical efficacy.

Translational gaps within in vitro and animal models

One of the most persistent hurdles in MSC research is the discrepancy between robust results observed in vitro and inconsistent outcomes in vivo. In vitro, MSCs demonstrate high proliferation, differentiation, and paracrine secretion. However, following transplantation, survival rates drop precipitously. Studies show that within hours to days of systemic infusion, a majority of cells are cleared from circulation or undergo apoptosis due to anoikis, hypoxia, and immune-mediated attack[113,114]. This low engraftment contrasts sharply with the large therapeutic effects sometimes observed, suggesting that paracrine signaling rather than direct tissue integration is the primary mechanism of action[114,115].

Animal models, while indispensable, do not always recapitulate human physiology. Rodent immune systems differ in sensitivity and responsiveness, often exaggerating or underestimating MSC effects. Moreover, scaling cell doses from rodents to humans is non-linear; a dose that is therapeutic in a 25-g mouse does not translate proportionally to a 70-kg human[116]. Large animal models, such as swine or nonhuman primates, provide closer approximations but are expensive and logistically challenging[117]. The reliance on reductionist models partly explains why many therapies succeed in rodents yet falter in clinical trials.

Efforts to bridge these gaps include the use of humanized mouse models that harbor human immune cells and more faithfully replicate host-donor interactions. In addition, advanced organ-on-chip systems are being developed to complement animal testing, offering platforms that integrate human cells, microvascular perfusion, and physiological mechanical stresses. These innovations hold promise for better predicting in vivo performance of MSCs prior to costly clinical translation.

Source-specific therapeutic efficacy

MSC origin exerts significant influence on in vivo outcomes. Bone marrow-derived MSCs remain the most studied due to their historical precedence and strong osteogenic and chondrogenic differentiation capacity[68,118]. They have demonstrated efficacy in models of bone defect repair and osteoarthritis, but their limited proliferative potential and age-dependent decline restrict scalability. Adipose-derived MSCs are abundant and proliferative, making them attractive for high-volume applications such as wound healing and ischemic injury[119,120]. However, they exhibit weaker osteogenic differentiation, which may limit utility in orthopedic applications[68,118].

Umbilical cord-derived MSCs, particularly from Wharton’s jelly, display high proliferative capacity, immune privilege, and a robust paracrine secretome[121]. These properties make them particularly suited for allogeneic use, where immune rejection is a concern[122,123]. Placenta-derived MSCs offer angiogenic and immunomodulatory benefits, showing promise in vascular and inflammatory conditions[124]. Dental pulp and synovium-derived MSCs provide lineage-specific advantages: Dental pulp MSCs excel in neural and vascular regeneration[125], while synovial MSCs are highly chondrogenic and effective for cartilage repair[126].

Tailoring MSC source selection to specific disease contexts has emerged as a critical determinant of therapeutic success. For example, adipose-derived MSCs may be ideal for ischemic heart disease where vascular regeneration is required, whereas bone marrow-derived MSCs remain superior for bone and cartilage repair. However, systematic head-to-head clinical comparisons remain rare, and most studies employ heterogeneous definitions and protocols, complicating direct comparison[68,118-120,127-134] (Table 3).

Table 3 Detailed comparison of the therapeutic potential of mesenchymal stem cells derived from various sources.

Source	Preferential lineage-specific differentiation	Typical best-fit indications	Pros	Cons	Ref.
Bone marrow	Strong osteogenic and chondrogenic	Bone defects, osteoarthritis, non-union bones	Extensively studied; good for bone and cartilage repair	Lower yield; donor age effect; invasive harvest procedure	[68,118,127]
Adipose tissue	Cardiomyocytes, skeletal muscle cells, neurons, hepatocytes, and tenocytes	Wound healing, ischemic injury, soft tissue repair	High yield of cells from liposuction; abundant tissue source; proliferative; useful for soft tissue regeneration	Lower osteogenic and chondrogenic differentiation compared to bone marrow	[119,120,127,128]
Umbilical cord/Wharton’s jelly	Osteogenic, chondrogenic, adipogenic, vascular, neuronal	Immune modulation, systemic inflammatory disorders, arthritis, cardio- and cerebrovascular	Neonatal origin, high proliferation, immune-privileged, trophic activity, antifibrotic, good for allogeneic off-the-shelf use	Variable processing methods; donor bank logistics	[127,129]
Synovial fluid	Tissue-biased properties (e.g., chondrogenic, neurogenic)	Cartilage repair	Useful for lineage-specific repair	Lower availability; niche-specific handling	[127,130,131]
Dental pulp	Odontoblasts, osteoblasts, endothelial cells, and nerve cells	Neuronal, vascular and odontogenic disorders	Highly proliferative; can be derived from deciduous teeth as well	Tiny primary tissue that gives lower cell yield	[127,132-134]

Microenvironmental influence and mode of delivery

Once introduced into host tissues, MSCs encounter diverse microenvironments that shape their behavior[135]. The inflammatory milieu is particularly influential. In conditions of acute inflammation, exposure to cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon-γ can enhance MSC immunomodulatory functions by stimulating the secretion of anti-inflammatory mediators like prostaglandin E2, indoleamine 2,3-dioxygenase, and IL-10[135,136]. Conversely, chronic inflammation or high levels of oxidative stress may induce apoptosis or senescence, reducing efficacy[137,138].

Mechanical properties of the host tissue also regulate MSC fate through mechanotransduction pathways. Rigid microenvironments encourage osteogenesis, whereas softer, elastic tissues promote adipogenesis or neurogenesis. This context dependence necessitates careful consideration of delivery site and mode[139-141]. Delivery strategies strongly influence engraftment and efficacy. Intravenous administration, while minimally invasive, results in the majority of cells being trapped in the pulmonary capillary bed, limiting systemic biodistribution[142]. This pulmonary first-pass effect has been harnessed in some contexts, such as acute lung injury, but represents a barrier in others[142]. Local injection into target tissues enhances retention and site-specific activity but is invasive and often associated with poor cell survival due to limited oxygen and nutrient supply[143]. Scaffold- or hydrogel-based delivery systems represent a promising compromise, providing structural support, mimicking extracellular matrix cues, and sustaining local paracrine signaling[144-146].

Long-term fate and safety

Long-term outcomes remain a major uncertainty in MSC transplantation. The majority of infused MSCs disappear within weeks, raising questions about the durability of their therapeutic effects[113,114]. While paracrine signaling may initiate repair cascades that persist beyond MSC survival, the lack of long-term engraftment limits their potential for direct tissue replacement[114].

Safety concerns are paramount. Over two decades of clinical use demonstrate that MSC therapy has a favorable short- to mid-term safety profile across various diseases and delivery routes (intravenous, intra-arterial, intra-articular, intrathecal, and intramuscular)[147,148]. Comprehensive systematic reviews and meta-analyses involving thousands of patients show consistent safety, with no increase in overall or serious adverse events, or in mortality, compared with controls, indicating a low short-term risk[148]. Studies revealed that only transient fever was consistently linked to MSC treatment, while arrhythmia and injection-site reactions showed minor, non-significant trends. All other adverse events displayed no meaningful association with MSC administration[148].

Although MSCs are generally considered safe and non-tumorigenic, rare reports have raised concerns about ectopic tissue formation or malignant transformation, particularly following extensive in vitro manipulation. The immunoprivileged nature of MSCs also raises the possibility of immune escape in the event of malignant transformation[149]. These risks underscore the importance of stringent genomic monitoring during culture and careful long-term surveillance following transplantation.

Advanced tracking technologies are being developed to study MSC fate in vivo. Imaging modalities such as magnetic resonance imaging, positron emission tomography, and bioluminescence allow non-invasive monitoring of cell survival and migration[150-152]. Genetic labeling with reporter constructs enables precise lineage tracing, although safety concerns limit their use in clinical contexts[153]. The integration of such tools into preclinical and clinical studies will be essential for clarifying the long-term behavior of transplanted MSCs.

Strategies to overcome in vivo challenges

Several approaches have been developed to improve MSC survival and functionality in vivo. Preconditioning, or “licensing”, of MSCs with hypoxia, cytokines, or pharmacological agents primes cells for hostile environments by upregulating survival and immunomodulatory pathways[113,154,155]. Genetic engineering or culture condition could help overexpression of C-X-C chemokine receptor type 4 that could enhances homing to injured tissues, while anti-apoptotic genes such as Bcl-2 improve survival[156-158]. Additionally, MSCs can be engineered to secrete therapeutic proteins, extending their repertoire beyond native capabilities[159].

Biomaterial-based delivery platforms, including hydrogels and scaffolds, improve engraftment by providing mechanical support and localized trophic signaling[106,109,112]. Encapsulation strategies can shield MSCs from immune attack while enabling controlled release of paracrine factors[160,161]. Co-delivery with supportive cells, such as endothelial progenitor cells, further enhances survival and functional integration[162,163].

An alternative approach gaining momentum is the use of EVs derived from MSCs. These vesicles, carrying proteins, lipids, and nucleic acids, mediate many of the paracrine effects attributed to MSCs[164,165]. EV-based therapies bypass the risks associated with live-cell transplantation, such as immune rejection and tumorigenicity, while offering scalability and stability[166]. Early preclinical data support their efficacy in cardiovascular, neurological, and musculoskeletal models, positioning EVs as a promising next-generation therapy[167-169].

CHALLENGES IN CLINICAL TRANSLATION

The leap from preclinical promise to clinical application represents the most formidable stage in the translational pathway of MSCs. Despite extensive investigation in vitro and in vivo, the transition into human patients exposes a new array of challenges that span biological variability, manufacturing scalability, regulatory complexity, and economic sustainability. This section critically examines the hurdles faced during clinical translation and outlines emerging strategies to overcome them.

Bridging preclinical and clinical studies

One of the first barriers lies in the translation of findings from animal models to human patients. While small animal models such as mice and rats provide mechanistic insights, their immune systems, metabolic rates, and tissue repair capacities differ substantially from those of humans. As a result, MSCs that demonstrate efficacy in rodents often fail to reproduce similar outcomes in clinical trials[116]. Large animal models such as pigs, dogs, and nonhuman primates offer closer physiological relevance but are costly, ethically challenging, and logistically complex[117,170]. Consequently, preclinical evidence often overestimates the magnitude and durability of clinical benefits.

Furthermore, dosing regimens established in animal studies rarely translate directly to human contexts. Whereas rodent experiments may use cell doses equivalent to millions of MSCs per kilogram of body weight, such quantities are neither practical nor safe in human applications[171]. Extrapolating an optimal therapeutic dose for humans remains uncertain, as there is no consensus on whether efficacy depends primarily on absolute cell number, total secretory output, or duration of exposure[172]. The absence of standardized potency assays compounds this uncertainty, hindering rational trial design.

Variability in clinical trial outcomes

Clinical trials investigating MSC therapy have produced mixed results. While some studies report significant improvements in conditions such as graft-vs-host disease, osteoarthritis, and Crohn’s disease, others demonstrate only marginal or no benefit compared to standard care[82,173,174]. This variability reflects a confluence of factors. Patient characteristics, including age, comorbidities, immune status, and disease stage, significantly influence responsiveness to MSCs[82]. Older patients often exhibit reduced MSC efficacy, likely due to systemic inflammatory environments and impaired endogenous repair mechanisms[175,176].

Trial heterogeneity further complicates interpretation. MSCs are derived from diverse sources, including bone marrow, adipose tissue, and umbilical cord, each have unique properties[68,119,124]. Differences in isolation techniques, expansion protocols, passage numbers, and storage conditions introduce additional variability[177,178]. Delivery routes, ranging from intravenous infusion to local injection, further diversify outcomes[142,143]. Without harmonized standards for cell characterization and potency testing, direct comparisons between trials are nearly impossible.

An additional concern is the placebo effect, which is particularly strong in regenerative medicine and musculoskeletal disorders. Patients often report subjective improvements in pain and function, complicating the interpretation of trial endpoints[179]. Objective biomarkers of MSC activity are lacking, making it difficult to distinguish true biological effects from placebo responses. Hence, there is an urgent need for reliable biomarkers to guide patient stratification and early response assessment in MSC therapy. Promising candidates include circulating inflammatory cytokines (e.g., IL-6, TNF-α), chemokines, exosomal miRNA signatures, and functional immune readouts such as T-cell activity. Preliminary data from rheumatic diseases suggest that elevated IL-6 and TNF-α may predict poor therapeutic response or help identify patients most likely to benefit from MSC-based immunomodulation[180,181]. However, these findings need prospective validation in randomized clinical trials. Future studies should incorporate biomarker endpoints particularly cytokine and EV profiles to enable adaptive patient selection and establish surrogate markers for MSC efficacy[72].

Manufacturing and supply chain challenges

A critical step in clinical translation is the production of MSCs at a scale and quality suitable for therapeutic use. Manufacturing must adhere to GMP standards, which mandate stringent control over every step of the process including tissue procurement, isolation, expansion, cryopreservation, and delivery. These requirements ensure safety and reproducibility but substantially increase cost and complexity[182,183].

The expansion of MSCs to clinically relevant doses is resource-intensive. Conventional two-dimensional culture flasks are laborious, while large-scale bioreactor systems provide greater efficiency but require significant capital investment and technical expertise[184,185]. Cryopreservation introduces further challenges: While it enables batch production and long-term storage, the freeze-thaw process can impair cell viability, alter surface marker expression, and reduce immunomodulatory potency[186]. Optimizing cryoprotectants and thawing protocols remains an area of active research.

Supply chain logistics represent another bottleneck. MSCs must be transported under tightly controlled temperature and sterility conditions, often across long distances from centralized production facilities to hospitals[187]. Any deviation in cold chain management risks compromising cell viability. Decentralized manufacturing at the point of care has been proposed, but standardizing quality control across multiple sites presents its own challenges[188]. A hybrid model involving regional hubs with robust distribution networks may balance scalability and quality assurance.

Regulatory and ethical complexities

Regulatory agencies such as the United States Food and Drug Administration, the European Medicines Agency, and Japan’s Pharmaceuticals and Medical Devices Agency classify MSCs as advanced therapy medicinal products, subjecting them to rigorous evaluation for safety, efficacy, and manufacturing consistency[189]. However, regulatory frameworks vary across regions, creating inconsistencies that hinder global collaboration and commercialization[190]. Harmonizing international standards is therefore a priority to facilitate multicenter trials and global approval pathways.

A persistent regulatory challenge lies in defining potency assays for MSCs. Unlike small molecules or monoclonal antibodies, MSCs exert therapeutic effects through multifaceted mechanisms, including immunomodulation, angiogenesis, and trophic support. Capturing this complexity in a single assay is not feasible. Instead, regulators encourage a panel of functional tests such as T-cell suppression assays, cytokine secretion profiles, and differentiation capacity to serve as surrogates[191-193]. Yet, these remain poorly standardized, leading to delays in approval.

Ethical considerations also come to the forefront in MSC therapy. Donor tissue sourcing, particularly from perinatal tissues such as umbilical cord and placenta, requires informed consent and careful ethical oversight. Autologous MSC therapies raise fewer ethical concerns but are limited by donor variability and the time required for expansion. Allogeneic therapies promise off-the-shelf availability but necessitate stringent donor screening and raise concerns about long-term immunogenicity[194,195]. Public perception and trust are fragile; cases of unregulated “stem cell clinics” offering unproven treatments have damaged credibility, emphasizing the need for robust ethical and regulatory oversight[196].

Economic and commercialization barriers

Even if MSC therapies clear scientific and regulatory hurdles, economic realities may limit widespread adoption. Manufacturing costs are high, with estimates ranging from $10000 to $30000 per patient dose depending on cell source and expansion method[19]. Without reimbursement strategies, access will be restricted to affluent healthcare systems and patients, exacerbating global health disparities. Demonstrating cost-effectiveness is therefore critical. Long-term follow-up studies that capture reductions in hospitalizations, surgeries, or disability are essential to justify reimbursement by payers.

Commercialization also requires addressing intellectual property and market competition. Patent protections around MSC sources, manufacturing methods, and delivery systems are often fragmented, creating barriers for new entrants. Moreover, the rise of alternative regenerative modalities, including induced pluripotent stem cells and EV-based therapies, threatens to outpace MSC commercialization unless scalability and affordability are improved. Partnerships between academia, industry, and healthcare systems are likely essential to create sustainable business models.

Future strategies for clinical translation

To overcome these hurdles, several innovative strategies are being pursued. Standardization of isolation, expansion, and potency assays across laboratories would reduce variability and enhance reproducibility. Automation and closed-system bioreactors promise to reduce contamination risks, labor costs, and variability, thereby improving scalability. Cryopreservation protocols are being optimized with novel cryoprotectants and controlled thawing systems to preserve potency.

On the regulatory front, international collaborations such as the International Society for Cell & Gene Therapy are working to harmonize definitions and guidelines, enabling smoother global approval pathways. Adaptive trial designs, incorporating biomarkers of response, may accelerate evaluation and regulatory approval. From a therapeutic perspective, MSC-derived EVs represent a cell-free alternative that circumvents many manufacturing and safety concerns[197]. Early studies suggest that EVs may replicate the paracrine effects of MSCs with greater scalability and stability[198,199]. Ultimately, the successful clinical translation of MSCs will depend on a combination of scientific innovation, regulatory clarity, ethical integrity, and economic feasibility. With coordinated efforts across stakeholders, MSC-based therapies have the potential to move beyond experimental promise and become an integral component of mainstream regenerative medicine.

FUTURE PERSPECTIVES

The field of MSC-based therapy is poised at a transformative juncture. Although significant hurdles remain, emerging technologies and evolving paradigms suggest new strategies to overcome current barriers. One promising avenue is the development of MSC-derived EVs and exosomes as cell-free therapies[198,199]. These nanoscale vesicles recapitulate many of the paracrine benefits of MSCs, including immunomodulation, angiogenesis, and tissue repair, while avoiding the risks associated with live cell transplantation such as immune rejection or tumorigenicity[166]. Early preclinical data support their efficacy across cardiovascular, neurological, and musculoskeletal disorders, and clinical translation efforts are already underway[200-202].

Another frontier is genetic and metabolic engineering of MSCs to enhance therapeutic potency. CRISPR-Cas9 and other editing platforms allow precise modification of genes related to survival, homing, or secretion of therapeutic factors. Engineering MSCs to overexpress chemokine receptors like C-X-C chemokine receptor type 4, or anti-apoptotic proteins such as Bcl-2, could demonstrate improved engraftment and functional outcomes[156,203]. Metabolic modulation, such as optimizing mitochondrial function or glycolytic pathways, may further refine their resilience in hostile microenvironments[55,204].

Biomaterials and bioengineering innovations offer complementary opportunities. Advanced hydrogels, 3D bioprinted scaffolds, and smart biomaterials capable of releasing growth factors or providing mechanical support are being integrated with MSC therapy to enhance survival and site-specific effects[109,110]. These platforms may also allow controlled delivery, reducing the need for repeated administrations.

On the translational side, standardization and harmonization of protocols remain urgent. The adoption of universal potency assays, shared biobanks, and international trial frameworks will improve reproducibility and accelerate approval pathways. In parallel, automation and closed-system bioreactors will reduce costs and variability, making MSC therapies more economically viable. Future clinical trial designs should incorporate precision medicine principles, stratifying patients by age, immune status, or disease subtype to maximize response rates.

Finally, ethical and societal considerations must remain central. Ensuring equitable access, preventing exploitation by unregulated stem cell clinics, and fostering public trust through transparency will be critical to the responsible development of MSC therapies. Engagement with policymakers, patient advocacy groups, and healthcare providers will help ensure that MSC-based therapies are not only effective but also accessible and ethically sound.

CONCLUSION

MSCs represent one of the most intensively studied platforms in regenerative medicine, offering multipotent differentiation capacity and robust paracrine activity. Despite decades of research, their translation from bench to bedside remains challenged by biological variability, limited engraftment, manufacturing bottlenecks, and regulatory complexity. However, advances in genetic engineering, biomaterials, and cell-free strategies such as EV therapies are reshaping the landscape. With concerted efforts toward standardization, scalability, and ethical oversight, MSC-based interventions may soon progress from experimental promise to reliable clinical reality (Figure 1). The future of regenerative medicine will likely see MSCs integrated into multifaceted therapeutic strategies, serving as both direct cellular agents and as sources of novel biologics, ultimately broadening the horizons of human healing and repair.

Figure 1 Challenges encountered during the translation of mesenchymal stem cell therapy from bench to bedside, and suggested solutions to overcome them. MSC: Mesenchymal stem cell; GMP: Good manufacturing practice.

ACKNOWLEDGEMENTS

The authors would like to thank Mahfuzul Islam and Ariful Islam for their support and reviewing this manuscript.