Adipose-derived mesenchymal stem cells: Source-dependent heterogeneity, translational challenges, and emerging precision strategies

Abstract

Adipose-derived mesenchymal stem cells (ADSCs) have emerged as an important cell source in regenerative medicine because of their accessibility, abundance, multilineage differentiation potential, and paracrine activity. However, ADSCs are not biologically uniform, and their properties are strongly influenced by donor-related factors, anatomical origin of adipose tissue, and technical procedures used for cell isolation, expansion, and characterization. This review summarizes current advances in defining the source-dependent characteristics of ADSCs, with particular emphasis on donor age, metabolic status, adipose depot specificity, isolation methods, culture conditions, and source-related molecular and functional heterogeneity. Evidence from transcriptomic, epigenetic, immunophenotypic, and secretome studies indicates that ADSCs from different sources may differ substantially in proliferation and differentiation capacity, immunomodulatory activity, and therapeutic performance. Major challenges remain in translating these findings into clinical practice, including donor variability, inconsistent manufacturing workflows, lack of standardized potency assays, and insufficient integration of source-stratified strategies into product development. Emerging directions such as single-cell and multi-omics profiling, cell-free secretome-based therapeutics, and source-aware manufacturing frameworks may improve precision and reproducibility in ADSC-based therapies. A clearer understanding of ADSC source dependency will be essential for optimizing donor selection, improving product consistency, and advancing the safe and effective clinical translation of regenerative medicine applications.

Key Words: Adipose-derived mesenchymal stem cells; Source dependency; Donor heterogeneity; Adipose tissue depot; Secretome; Regenerative medicine

Core Tip: Adipose-derived mesenchymal stem cells are a heterogeneous cell population whose biological and therapeutic properties are strongly influenced by donor background, adipose tissue origin, and manufacturing conditions. This review summarizes source-dependent differences in proliferation, differentiation, immunomodulation, and secretome composition, and highlights key challenges in standardization and clinical translation. Emerging approaches, including single-cell profiling, multi-omics analysis, and cell-free secretome-based therapy, may improve the precision and reproducibility of adipose-derived mesenchymal stem cell-based regenerative strategies.

INTRODUCTION

Adipose-derived mesenchymal stem cells (ADSCs) represent an important class of adult multipotent stem cells that have attracted broad attention in regenerative medicine, tissue engineering, and immunomodulation because of their accessibility, abundance, and multilineage differentiation potential[1]. ADSCs can be isolated from multiple adipose tissue depots, including subcutaneous, visceral, perirenal, epididymal, and infrapatellar fat pads, and cells derived from these distinct sites may display different biological properties[2,3]. Increasing evidence indicates that ADSC heterogeneity is shaped by donor-related variables, including age, sex, metabolic status, and health conditions, as well as by tissue-specific microenvironmental cues and differences in isolation and culture procedures[4]. This source dependency is reflected in variations in proliferation and differentiation capacity, immunophenotype, secretome composition, and therapeutic efficacy, which together complicate standardization and clinical translation[5,6].

ADSCs are now recognized as a heterogeneous rather than uniform cell population, and their functional diversity is influenced by both anatomical origin and donor background[1,4-6]. ADSCs derived from different adipose depots show differential expression of stemness markers, pluripotency-related genes, and lineage-specific differentiation programs[1,7]. For example, epididymal and inguinal fat-derived ADSCs have shown stronger adipogenic and osteogenic differentiation, whereas subcutaneous fat-derived ADSCs may exhibit greater chondrogenic potential[1,7]. Donor age also modulates ADSC biology, although the magnitude of this effect is not fully consistent across studies, with some reports showing declines in proliferation and differentiation and others showing more modest changes[8-11]. Donor sex further contributes to functional variability, and female-derived ADSCs have shown enhanced immunosuppressive effects compared with male-derived cells in some in vitro studies[12,13].

The culture microenvironment also critically influences ADSC behavior, especially through oxygen tension and culture dimensionality[14,15]. Hypoxic preconditioning and three-dimensional (3D) culture systems have been shown to enhance proliferation, stemness maintenance, and differentiation potential while also shifting the secretome toward a more regenerative and immunomodulatory profile[14,15]. The ADSC secretome, including soluble mediators and extracellular vesicles such as exosomes, mediates many of the therapeutic effects of these cells, including angiogenesis promotion, immunomodulation, and tissue repair[16-18]. The composition and efficacy of this secretome are themselves influenced by culture conditions and require careful optimization for translational use[19,20]. Clinically, ADSCs have been explored in a range of settings, including ischemic heart disease, osteoarthritis, wound repair, metabolic disorders, and autoimmune conditions[18,20-22]. These disease settings contribute substantially to global health burden, further underscoring the translational importance of improving regenerative strategies[23]. Their therapeutic value derives from both multilineage differentiation and paracrine activity[24-26]. However, reproducible outcomes remain difficult to achieve because of source-related heterogeneity, culture-induced senescence, and protocol variability[27-29]. To address these limitations, current efforts focus on multi-omics profiling, selection of functional subpopulations such as CD271-positive ADSCs, and optimization of culture systems to improve cell quality and efficacy[30-32]. Figure 1 provides a conceptual overview of source-dependent ADSC heterogeneity and its translational implications.

Figure 1 Source-dependent heterogeneity of adipose-derived mesenchymal stem cells and its translational implications. A: Multiple determinants contribute to adipose-derived mesenchymal stem cell (ADSC) source dependency, including donor-related variables such as age, metabolic status, and sex, as well as adipose tissue depot, isolation method, and culture conditions; B: These source-related factors shape the biological and functional heterogeneity of ADSCs, which is reflected in differences in proliferation, differentiation potential, immunophenotype, immunomodulation, secretome composition, extracellular vesicle output, and transcriptomic/epigenetic features; C: This heterogeneity has important translational implications for donor selection, potency assessment, cell bank standardization, source-stratified therapy, secretome-based therapy, and precision translation. ADSCs: Adipose-derived mesenchymal stem cells; 3D: Three-dimensional.

DONOR INDIVIDUAL FACTORS AFFECTING ADSCS CHARACTERISTICS

Age and aging-related changes

Donor age is an important determinant of ADSC biology, particularly with respect to proliferation and differentiation capacity[8-11]. ADSCs isolated from younger donors generally show greater clonogenicity and higher proliferation rates than those derived from older donors, which tend to enter replicative senescence earlier[8,9,33]. At the molecular level, aged ADSCs display characteristic senescence-related changes, including telomere shortening, increased senescence-associated β-galactosidase activity, upregulation of p16INK4a and p21, and activation of DNA damage response pathways[33,34]. These alterations are associated with reduced regenerative capacity and changes in secretory behavior[33,34].

Aging is also accompanied by shifts in lineage commitment, and aged ADSCs often show reduced osteogenic differentiation, which may contribute to age-related bone loss and marrow adiposity[8,10,11]. By contrast, adipogenic differentiation may be relatively enhanced in older cells, which is consistent with increased fat accumulation during aging[10,11]. However, the effect of age on ADSC differentiation is not completely uniform across studies, likely because of differences in donor health status, tissue origin, and experimental design[10,11]. Transcriptomic and proteomic analyses further indicate that aging ADSCs undergo broad molecular remodeling, including downregulation of genes involved in proliferation and differentiation together with a pro-inflammatory transcriptional shift, although the corresponding proteomic alterations appear less consistent[34,35]. Importantly, transplantation of ADSCs from aged donors may induce adverse effects in recipients, including physical dysfunction, which highlights the clinical relevance of donor age[36]. Multi-omics studies have also identified stemness-related gene clusters that decline with donor age and passage number, suggesting potential biomarkers for selecting ADSCs with better regenerative potential[30].

Metabolic status and disease background

Metabolic disorders such as obesity and type 2 diabetes mellitus markedly affect ADSC phenotype and function through changes in the adipose tissue microenvironment[37]. ADSCs isolated from obese donors show altered proliferation, increased secretion of pro-inflammatory cytokines such as interleukin-6 and monocyte chemoattractant protein-1, and mitochondrial dysfunction, all of which impair regenerative capacity[37,38]. Excessive lipid accumulation in preadipocytes during obesity may also compromise adipogenic differentiation and contribute to dysfunctional adipose tissue remodeling[37]. ADSCs from patients with type 2 diabetes mellitus similarly show reduced migratory ability, weaker support for angiogenesis, and greater susceptibility to apoptosis and senescence under hyperglycemic conditions[37,39]. These abnormalities are linked to chronic inflammation and extracellular matrix remodeling in diabetic adipose tissue, which can imprint a pathological “memory” on ADSCs and thereby modify their biological behavior[37,39].

Evidence from weight-discordant monozygotic twins further supports the importance of metabolic status, because increased donor weight has been associated with enhanced adipogenic differentiation, increased inflammatory marker expression, and reduced angiogenic potential even under shared genetic background[5]. Altered microRNA expression has also been reported in ADSCs from obese individuals, and downregulation of miR-155 has been linked to dysregulated secretion of insulin-like growth factor-1 and interleukin-10, connecting metabolic syndrome with secretome remodeling and systemic inflammation[40]. Metabolic disease additionally affects ADSC energy metabolism, and shifts in glycolysis, oxidative phosphorylation, and reactive oxygen species production can influence proliferation, differentiation, and therapeutic behavior[41]. Collectively, these findings indicate that obesity and diabetes impair ADSC biology by promoting pro-inflammatory phenotypes, weakening differentiation and angiogenic function, and altering secretory profiles, thereby potentially limiting the efficacy of autologous ADSCs in metabolically diseased patients[37-41].

DIFFERENCES IN ADIPOSE TISSUE ANATOMICAL SITE ORIGINS

Comparison of subcutaneous and visceral fat-derived ADSCs

ADSCs isolated from subcutaneous fat depots, such as abdominal and gluteal fat, and those derived from visceral depots, such as the omentum and mesentery, differ in gene expression and functional properties[3,4]. Visceral fat-derived ADSCs have been reported to display a more angiogenesis- and inflammation-related molecular profile than subcutaneous ADSCs[3,4]. This phenotype may favor neovascularization and tissue remodeling, but it is also often associated with a more pronounced inflammatory background[3,4]. By contrast, subcutaneous fat-derived ADSCs may show a phenotype more favorable for osteogenic differentiation[1,3,4]. Visceral ADSCs, on the other hand, may display greater adipogenic differentiation efficiency, which is consistent with the metabolic activity and lipid storage function of visceral fat[3,4].

These depot-specific differences are likely driven by differences in developmental origin and local microenvironmental cues[4]. Transcriptomic and proteomic analyses indicate that visceral ADSCs preferentially express genes associated with angiogenesis and inflammation, whereas subcutaneous ADSCs more strongly express genes related to osteogenesis and extracellular matrix remodeling[3,4]. The inflammatory milieu of visceral fat may therefore predispose visceral ADSCs to a pro-inflammatory phenotype, which could influence their therapeutic safety and efficacy[4,37]. In addition, differences in cytokine and growth factor secretion between these depots may alter their immunomodulatory properties and regenerative performance[4,37]. Together, these findings indicate that adipose tissue origin should be considered carefully when selecting ADSCs for regenerative applications, because depot-specific properties may substantially influence cell behavior and clinical outcome[3,4,37].

Specificity of different subcutaneous fat depots

Even within subcutaneous adipose tissue, ADSCs derived from different anatomical regions may show functional heterogeneity[5]. Cells isolated from abdominal, gluteal, and thigh depots can differ in proliferation and differentiation potential[1,5]. Some studies suggest that ADSCs from different subcutaneous depots are not biologically equivalent and may vary in their therapeutic suitability[1,5]. This regional variation may be related to local blood flow, mechanical stress, innervation, and intrinsic developmental programs of adipocyte progenitors[5].

Regional metabolic differences among subcutaneous depots may also influence ADSC functional performance[5]. Microenvironmental factors such as mechanical loading and neural input may further affect ADSC secretome composition, immunomodulatory activity, and lineage commitment[5]. These observations suggest that anatomical precision should be considered when selecting subcutaneous adipose tissue as an ADSC source[5].

INFLUENCE OF ISOLATION AND CULTURE METHODS ON DEFINING ADSCS CHARACTERISTICS

Comparison of enzymatic digestion and mechanical separation methods

Isolation method is an important determinant of ADSC yield and phenotype[37]. Enzymatic digestion and mechanical separation are the two principal approaches used to isolate ADSCs[37]. Enzymatic digestion, most commonly using collagenase, is widely employed because it yields relatively large numbers of viable cells[37]. However, variables such as enzyme type, concentration, digestion time, and temperature can affect surface receptor integrity, initial cell viability, and later functional properties[37]. Excessive collagenase concentration or prolonged digestion may over-disrupt extracellular matrix components, thereby altering stromal vascular fraction composition and affecting ADSC phenotype and differentiation potential[37].

In contrast, mechanical separation methods, including ultrasound- or shear force-based approaches, impose less biochemical stress and may better preserve native cellular subpopulations and niche-related information[37]. This may help retain functional heterogeneity, although mechanical methods usually yield fewer cells and may introduce a greater proportion of non-target cells, which can complicate downstream applications[37]. Different isolation methods may therefore influence the composition of the stromal vascular fraction and the subsequent biological behavior of ADSCs[37]. These methodological effects are highly relevant when interpreting source-dependent characteristics, because non-standardized isolation procedures make it difficult to distinguish intrinsic biological differences from isolation-induced variation[37]. Isolation protocols should therefore be selected according to the intended research or clinical use, balancing cell yield, viability, and preservation of heterogeneity[37].

Challenges in standardizing culture conditions

Culture conditions strongly influence ADSC phenotype, proliferation, differentiation, and paracrine activity, and remain a major source of variability across studies[6,14,15]. One important source of inconsistency is the use of fetal bovine serum, because batch-to-batch variation in growth factors, hormones, and other undefined components can affect ADSC proliferation, differentiation, and immunophenotype[6]. This variability reduces reproducibility and limits clinical translation[6]. To address this problem, chemically defined media and human platelet lysate-based media have been proposed as alternatives and have shown promise in supporting more consistent ADSC expansion[6].

Oxygen tension is another key determinant of ADSC function[14,15]. Physiological hypoxia, typically 2%-5% O₂, more closely resembles the in vivo niche and has been shown to shift ADSC metabolism from oxidative phosphorylation toward glycolysis, thereby promoting stemness maintenance, proliferation, and paracrine secretion[14]. Hypoxic culture can also alter differentiation trajectories and immunomodulatory activity, often enhancing therapeutic potential[14,15]. However, no consensus has yet emerged regarding the optimal oxygen conditions or media formulations for ADSC culture[6,14]. As a result, ADSC preparations generated in different laboratories often display divergent functional profiles, and culture-induced phenotypic drift may obscure intrinsic donor- or depot-specific traits[29,42]. The lack of universally accepted chemically defined culture systems also limits direct comparison across studies, making rigorous characterization under standardized, serum-free, and physiologically relevant conditions essential for distinguishing true source-dependent properties from culture artifacts and for producing clinically compliant ADSC products with more predictable efficacy[6,14].

SOURCE-DEPENDENT MOLECULAR AND FUNCTIONAL CHARACTERIZATION

Transcriptomic and epigenetic features

Multi-omics analyses have revealed marked heterogeneity within ADSCs across different adipose tissue sources[3]. This heterogeneity is reflected in differences in the abundance and activity of ADSC subpopulations, which are regulated by tissue origin and local microenvironmental factors[3]. For example, ADSCs isolated from subcutaneous and visceral depots display distinct transcriptomic profiles that correspond to different functional capacities and lineage biases[3]. These findings support the view that source-dependent molecular programs contribute substantially to ADSC diversity[3].

Epigenetic regulation also plays a central role in source-dependent ADSC characteristics[43]. DNA methylation and histone modifications are thought to provide a mechanism through which ADSCs retain memory of their tissue of origin[43]. ADSCs from obese donors or from particular anatomical depots show distinct DNA methylation patterns that influence chromatin accessibility of genes involved in differentiation and functional commitment[43]. Multi-omics analyses integrating transcriptomic, methylomic, and proteomic data have further identified stemness-related gene clusters associated with donor age and cell passage, highlighting the dynamic epigenetic regulation of ADSC biology[30]. In addition, post-transcriptional and RNA-based regulatory mechanisms may contribute to source-dependent ADSC heterogeneity[3,44]. Aging also reshapes the ADSC transcriptomic landscape, and older donor-derived ADSCs display reduced expression of genes related to proliferation and differentiation, whereas corresponding proteomic changes appear less consistent, suggesting substantial post-transcriptional complexity[34]. Overall, these findings indicate that the molecular identity and functional potential of ADSCs are closely linked to tissue source and epigenetic state, both of which should be considered in regenerative applications[3,30,34,43].

Immunophenotype and secretome analysis

Although ADSCs consistently express canonical mesenchymal stem cell markers such as CD73, CD90, and CD105, expression of other markers, including CD34, CD146, and CD271, varies according to tissue source and donor background[37]. This variability likely reflects the presence of distinct functional states or subpopulations within ADSC preparations[37]. For example, CD146 expression has been reported to be higher in ADSCs from leaner donors than in cells from heavier co-twins, and this difference correlates with angiogenic and adipogenic potential[5]. Such findings suggest that immunophenotypic differences are biologically meaningful rather than purely technical[5,37].

The secretome, consisting of soluble factors and extracellular vesicles, is central to ADSC therapeutic activity[15,37,45]. Source-dependent differences strongly influence its composition, and ADSCs from inflamed or diseased adipose tissue tend to secrete more pro-inflammatory mediators, whereas ADSCs from healthier tissue sources are relatively enriched in anti-inflammatory and reparative factors[37]. Proteomic analyses of conditioned media and exosomes have shown that ADSCs from different sources produce distinct profiles of cytokines, growth factors, and bioactive molecules that regulate immune responses, angiogenesis, and tissue repair[45]. Culture conditions also modify secretome output, and ADSCs cultured under hypoxic conditions or in 3D bioreactor systems show enhanced secretion of vascular endothelial growth factor and monocyte chemoattractant protein-1, thereby promoting angiogenesis and immunomodulatory activity[15]. Donor sex may likewise influence secretome-mediated immune regulation, with female-derived ADSCs producing higher levels of anti-inflammatory mediators such as indoleamine 2,3-dioxygenase 1 in some settings[12]. These findings indicate that both immunophenotypic variability and secretome heterogeneity are central to ADSC functional identity and should be incorporated into source characterization and therapeutic design[5,12,15,37,45]. The major source-dependent factors influencing ADSC biological characteristics are summarized in Table 1.

Table 1 Source-dependent factors influencing the biological characteristics of adipose-derived mesenchymal stem cells.

Factor	Representative condition/source	Main affected properties	Representative findings	Ref.
Donor age	Young vs aged donors	Proliferation, senescence, osteogenesis	Aging is associated with reduced proliferation, earlier senescence, and impaired osteogenesis	[8-10,33,34]
Metabolic status	Obesity	Inflammatory phenotype, mitochondrial function, secretome	Obesity is associated with inflammatory activation and reduced regenerative capacity	[5,37,38,40]
Disease background	Type 2 diabetes mellitus	Migration, angiogenesis, senescence	Diabetic ADSCs show impaired migration and angiogenic support and increased senescence susceptibility	[37,39,41]
Sex	Female vs male donors	Immunomodulation, transcriptomic profile	Sex-related differences may influence immunomodulatory and molecular features	[12,13,32]
Adipose tissue depot	Subcutaneous vs visceral fat	Molecular profile, osteogenic/adipogenic tendency	Visceral ADSCs show a more inflammatory profile, whereas subcutaneous ADSCs may favor osteogenesis	[1,3,4,37]
Subcutaneous regional depot	Abdominal vs gluteal/thigh depots	Proliferation, differentiation potential	Different subcutaneous depots are not functionally equivalent	[1,5]
Isolation method	Enzymatic digestion	Yield, phenotype	Higher cell yield, but phenotype may be affected by digestion conditions	[37]
Isolation method	Mechanical separation	Heterogeneity preservation	Better preservation of native subpopulations, but lower yield	[37]
Culture conditions	Hypoxia	Metabolism, stemness, secretome	Hypoxia may enhance stemness and paracrine activity	[14,15]
Culture conditions	2D vs 3D culture	Functional state, secretome potency	3D culture may improve regenerative secretome features	[14,15]
Epigenetic regulation	DNA methylation/tissue memory	Molecular memory, differentiation	Epigenetic regulation contributes to source-dependent heterogeneity	[30,43]
Secretome heterogeneity	Healthy vs diseased source	Cytokines, growth factors, EV composition	Diseased sources tend to generate more pro-inflammatory secretomes	[15,37,45]

ADSCs: Adipose-derived mesenchymal stem cells; 2D: Two-dimensional; 3D: Three-dimensional; EV: Extracellular vesicle.

CHALLENGES AND FUTURE DIRECTIONS IN CLINICAL TRANSLATION

Standardization of donor selection and cell bank establishment

Clinical application of ADSCs requires rigorous standardization of donor selection and cell banking procedures to ensure reproducible and effective cell products[30,46,47]. Donor-related variables such as age, body mass index, metabolic status, and adipose tissue harvest site all influence ADSC proliferation, differentiation potential, immunomodulatory activity, and secretome composition[5,8,10,11,37]. ADSCs from older donors may show reduced proliferative ability and altered osteogenic differentiation, although some reports suggest that age-related effects on differentiation are not uniform[8,10,11]. Body mass index and metabolic comorbidities such as hypertension and coronary artery disease have also been associated with differences in mesenchymal cell number and viability within the stromal vascular fraction[5,37]. In addition, adipose tissue origin, such as subcutaneous vs visceral fat, confers distinct transcriptomic and metabolomic characteristics that influence regenerative and immunomodulatory properties[3].

These differences indicate that donor screening standards should include age, body mass index, metabolic status, and precise tissue harvest site[5,8,10,11,37]. Such criteria may help identify donor populations that yield more reproducible and functionally superior ADSCs[5,37]. However, establishing clinical-grade ADSC cell banks introduces further complexity, because cryopreservation and thawing may exacerbate or conceal pre-existing donor-dependent differences and can affect cell viability, proliferation, differentiation, and secretome composition[30,46]. Optimization of cryopreservation conditions, including cryoprotectant formulation and freezing-thawing parameters, is therefore essential for preserving cell quality[46]. Another important requirement is the development of potency assays tailored to ADSCs, and these assays should incorporate source-dependent functional readouts, including cytokine secretion, immunosuppressive indices, and differentiation markers, to serve as more reliable predictors of therapeutic efficacy[6]. Integration of multi-omics analyses may further facilitate identification of biomarkers that reflect both potency and source-specific characteristics, thereby supporting generation of high-quality ADSC cell banks and improving the reproducibility and safety of clinical application[30,47].

Source selection strategies for disease-specific therapies

Because ADSCs from different donors and adipose depots are biologically heterogeneous, source selection should be aligned with the target disease context[6,22]. In a precision medicine framework, ADSCs would ideally be selected according to the functional properties most relevant to the intended therapeutic indication[6,22]. For example, in bone defect repair, ADSCs from younger donors and subcutaneous depots with stronger osteogenic potential may be advantageous because these cells show enhanced mineralization and osteogenic marker expression[8,10,48]. In contrast, treatment of autoimmune or inflammatory disorders may require ADSCs with stronger immunomodulatory activity and a more anti-inflammatory secretome profile[6,22]. Donor sex may also be relevant to source selection, because female-derived ADSCs have shown greater suppression of peripheral blood mononuclear cell proliferation than male-derived cells in some studies, suggesting sex-dependent differences in immunomodulatory potency[12,13].

To address limitations associated with less favorable ADSC sources, several preconditioning strategies have been explored[14,15]. Hypoxic culture, cytokine priming, and 3D culture systems have all been reported to enhance ADSC proliferation, differentiation, and secretome potency[14,15]. Hypoxic preconditioning has been reported to enhance angiogenic factor secretion and regenerative activity[49]. Similarly, 3D bioreactor culture under hypoxia with platelet lysate supplementation can generate secretomes with stronger anti-inflammatory and pro-regenerative activity[15]. These approaches may allow functional rescue of ADSCs from suboptimal donors or depots and thereby broaden the therapeutic applicability of both autologous and allogeneic products[14,15]. Ultimately, basic findings on ADSC source dependency must be validated in clinical studies, and future trials should incorporate donor-related and tissue source-related variables as stratification factors to determine how they affect efficacy and safety outcomes[50]. Standardized potency assays and multidimensional characterization of ADSC products used in trials will also be important for linking source-dependent features with therapeutic response, refining source selection strategies, and improving product quality[50]. In addition, high-resolution transcriptomic approaches may provide further insight into source-dependent functional heterogeneity and differentiation trajectories[51].

EMERGING DIRECTIONS FOR PRECISION TRANSLATION OF ADSCS

Single-cell and multi-omics strategies for source stratification

Recent advances in high-resolution molecular profiling are providing a more refined framework for stratifying ADSC sources before clinical use[52-54]. Conventional bulk analyses are often insufficient to resolve the cellular hierarchy, differentiation trajectories, and functional subpopulations that coexist within adipose-derived stromal cell preparations[52,53]. By contrast, single-cell and integrative multi-omics approaches allow investigators to identify depot-specific molecular programs, cell-state transitions, and candidate potency-associated signatures that are not readily detectable at the bulk level[54,55]. These methods may therefore help distinguish intrinsic source-related heterogeneity from variability introduced during tissue processing and ex vivo expansion[53-55].

From a translational perspective, these approaches are particularly valuable because they can support the development of source-stratified manufacturing strategies[52,54,55]. Adipose tissue obtained from different depots, donors, and collection procedures is intrinsically heterogeneous, and this variability is further amplified by non-uniform processing workflows[55]. In parallel, recent reviews of ADSC clinical studies have emphasized that high-resolution transcriptomic methods are becoming increasingly important for clarifying mechanisms of action and predicting therapeutic responses[52]. Together, these findings suggest that future ADSC production pipelines may benefit from combining donor screening with single-cell and multi-omics profiling to identify cell populations most suitable for specific disease indications[53-55].

Cell-free therapeutic translation of the ADSC secretome

Cell-free therapeutic strategies based on ADSC-conditioned medium, extracellular vesicles, and exosomes are emerging as an important extension of ADSC-based regenerative medicine[56-59]. Increasing evidence indicates that many beneficial effects previously attributed to transplanted mesenchymal stromal cells are mediated largely through secreted bioactive factors rather than long-term engraftment of the cells themselves[56,57,60]. In this context, ADSC secretome products have gained particular attention in wound repair, skin regeneration, and inflammatory tissue injury because of their pro-angiogenic, anti-inflammatory, antiapoptotic, and matrix-remodeling effects[57-59]. Reviews of ADSC secretome-based therapy have also highlighted their potential advantages as ready-to-use cell-free therapeutics for cutaneous wound healing and broader skin regeneration applications[57,58].

At the same time, secretome-based therapy remains highly sensitive to upstream biological and manufacturing variables[57-60]. The heterogeneity of mesenchymal stem cell-derived extracellular vesicles is strongly influenced by tissue or cell source, medium composition, culture dimensionality, hypoxia, and downstream isolation and storage procedures[56]. Recent systematic and narrative reviews likewise emphasize that standardization, large-scale production, and clinical validation remain major barriers to implementation of mesenchymal stem cell- and ADSC-derived secretome products[57,59,60]. Thus, while secretome-based therapy may reduce some of the constraints associated with live-cell transplantation, it does not remove the need for strict control of source-dependent biological variability[59,60].

Manufacturing readiness, clinical translation, and the “living drug” concept

Another important direction is the integration of ADSC source selection into scalable manufacturing and regulatory frameworks[52,54,61-65]. Reviews addressing mesenchymal stem cell-based advanced therapy medicinal products have repeatedly highlighted that donor variability, disease background, tissue source, and process-related heterogeneity remain major barriers to product consistency and clinical reproducibility[54]. More recent work has also described the historical evolution of adipose-derived products from tissue transfer to stromal vascular fraction, expanded ADSCs, and cell-free derivatives, emphasizing the need for more standardized manufacturing logic across these platforms[63,65]. In this context, adipose-derived cellular therapeutics are increasingly being viewed as dynamic biological products rather than static cell suspensions, leading to the concept of adipose tissue-derived cells as “living drugs”[65].

Clinical translation is likewise moving toward more indication-specific deployment of adipose-derived products[52,62-64]. Recent reviews and disease-focused studies have described ongoing progress in synovial regeneration, diabetes-related applications, chronic wound repair, and broader regenerative medicine uses, but they also stress that few ADSC-based interventions have achieved full approval because standardization and in vivo mechanisms remain incompletely defined[52,61]. Future progress will therefore depend on combining source-aware donor selection, omics-guided potency assessment, scalable cell-based or cell-free manufacturing, and disease-specific clinical trial design[64,65]. The major translational challenges in ADSC-based therapy and potential strategies to address them are summarized in Table 2.

Table 2 Major translational challenges in adipose-derived mesenchymal stem cell-based therapy and potential strategies to address them.

Challenge	Main problem	Potential strategy	Ref.
Donor heterogeneity	Major variability in phenotype and function	Source-aware donor screening	[5,6,8,12,37]
Depot-specific variability	Different depots yield distinct ADSCs	Match source to therapeutic indication	[1,3,4,37]
Regional variability	Subcutaneous depots are not equivalent	Improve anatomical precision in tissue collection	[1,5]
Isolation inconsistency	Isolation methods alter downstream cell behavior	Standardize processing workflows	[37]
Culture-induced drift	Serum, oxygen, and culture format reshape phenotype	Use defined media and standardized culture systems	[6,14,15,29]
Cryopreservation variability	Freezing and thawing affect cell quality	Optimize banking and post-thaw validation	[30,46]
Lack of potency assays	Functional release criteria remain unclear	Develop source-aware potency markers	[6,30,47,50]
Incomplete molecular stratification	Bulk analyses miss functional subpopulations	Apply single-cell and multi-omics profiling	[52-55]
Secretome heterogeneity	Secretome products are highly source- and process-dependent	Standardize production and characterization	[56-60]
Manufacturing inconsistency	Scale-up and reproducibility remain limited	Introduce source-aware manufacturing and QC systems	[54,63,65]
Limited trial stratification	Clinical trials often insufficiently stratify source/product features	Incorporate source-related variables into trial design	[50,52,61,62,64]

ADSCs: Adipose-derived mesenchymal stem cells; QC: Quality control.

CONCLUSION

The biological and therapeutic characteristics of ADSCs are profoundly influenced by their source. This source dependency is shaped by donor-related variables such as age and metabolic status, by the anatomical origin of adipose tissue, and by the methods used for cell isolation and preparation. As a result, ADSC heterogeneity has important implications for both mechanistic research and clinical translation. Recent advances in omics technologies, including transcriptomics, epigenomics, proteomics, and single-cell profiling, have improved understanding of this heterogeneity by identifying distinct subpopulations and molecular memory mechanisms that regulate ADSC behavior. These findings highlight the importance of integrating multidimensional molecular data with functional evaluation to better define source-dependent ADSC variability. At the same time, major challenges remain. Donor-related and tissue-related factors interact with technical variables to shape ADSC phenotype and function, while discrepancies in isolation, expansion, characterization, and potency testing continue to limit reproducibility and comparability across studies. Future progress will require standardized operating procedures, source-specific quality control systems, and predictive models that combine molecular signatures with functional assays. In parallel, strategies such as preconditioning and cell-free secretome-based approaches deserve further evaluation to improve consistency and therapeutic performance. A more rigorous and source-aware framework for ADSC characterization and standardization will be essential for advancing their safe, effective, and precise clinical translation.

ACKNOWLEDGEMENTS

The authors thank their colleagues for helpful discussions during the preparation of this manuscript.