Advancing human adipose-derived mesenchymal stem cell-derived exosomes for androgenetic alopecia: Appraisal and methodological recommendations

Abstract

Fu et al investigated human adipose-derived mesenchymal stem cell exosomes for androgenetic alopecia, identifying a stable set of 232 proteins and proposing the CDC42-Wnt/β-catenin- glycogen synthase kinase 3β signaling axis. These findings support the potential for clinical translation. To further strengthen clinical relevance, we recommend: (1) More comprehensive exosome characterization, including key marker analysis; (2) Reporting both particle count and protein concentration, and establishing dose-response relationships; (3) Direct validation of CDC42’s role through knockout or overexpression experiments; (4) Multi-level evaluation of Wnt/β-catenin signaling; (5) Enhanced biochemical validation in animal models; (6) Quantitative analysis of microneedle delivery parameters and retention; and (7) Early development of a streamlined quality control and safety framework. These methodological advancements will help exosome-based therapies better align with emerging regulatory standards and clinical guidelines. By addressing these aspects, future research can facilitate the safe, effective, and reproducible application of exosome-based treatments in clinical practice.

Key Words: Androgenetic alopecia; Extracellular vesicles; Human adipose-derived mesenchymal stem cell-derived exosomes; CDC42; Wnt/β-catenin signaling; Quality control; Dermal papilla cells

Core Tip: Key challenges for translating human adipose-derived mesenchymal stem cell-derived exosomes in androgenetic alopecia include: Incomplete exosome marker information; lack of standardized reporting for particle counts and protein content; absence of direct CDC42 function validation; limited depth in Wnt/β-catenin pathway analysis; insufficient biochemical data in animal models; undefined microneedle delivery parameters; limited safety profiling; and lack of integrated quality control and potency release criteria.

TO THE EDITOR

Fu et al[1] provided a thoughtful evaluation of human adipose-derived mesenchymal stem cell-derived exosomes for androgenetic alopecia, identifying a core set of 232 proteins and proposing the CDC42-Wnt/β-catenin-glycogen synthase kinase 3β (GSK3β) axis related to dermal papilla cell responses. Their approach, including stable cargo profiling and microneedle-assisted delivery, offers a promising foundation for clinical application. Further improvements in exosome characterization, dose measurement, mechanism validation, animal model rigor, delivery methods, and quality control could enhance reproducibility and clarity. A comprehensive overview of current limitations and recommended improvements for each procedural aspect is provided in Table 1.

Table 1 Procedural assessment and recommendations for human adipose-derived mesenchymal stem cell-exosome research.

Procedures	Current limitations	Recommended improvements	Priority level
Exosome characterization	Incomplete marker information; limited use of negative controls; possible contamination from serum supplements	Include key markers (Alix, TSG101, syntenin), broader negative controls; clarify serum supplementation and collection methods to avoid contamination	Medium
Dose normalization	Only total protein reported; lack of particle count, particle-to-protein ratio; no dose-response analysis; batch variability not addressed	Report both particle count and protein concentration; include particle-to-protein ratio, yield per cell, batch variability; establish dose-response curves with multiple doses; normalize exposure by particle number per area in animal studies	High
Mechanism validation	No direct validation of CDC42 function; mechanism inferred only from changes after treatment; limited depth in Wnt/β-catenin pathway analysis	Demonstrate CDC42’s direct role via knockdown/overexpression and exosome content analysis; assess effects on dermal papilla cells and β-catenin localization; perform rescue experiments and detailed pathway analysis, including target gene expression and reporter activity	High
In vivo model	Phenotypic changes only; limited biochemical measurements; lack of validity safeguards (randomization/blinding); short follow-up duration	Add biochemical measurements (skin/serum dihydrotestosterone, androgen receptor target genes); implement randomization and blinding; extend follow-up to cover a full hair cycle for durability assessment	Medium
Delivery optimization	Microneedle delivery parameters undefined (needle length, density, passes, pressure, intervals); no tracking of distribution or retention	Specify microneedle parameters (length, density, passes, pressure, interval); use imaging techniques to track exosome distribution and optimize dosing	Medium
QC	No minimal QC panel defined; lack of clear release criteria; insufficient disclosure of key variables (size, marker intensity, endotoxin, etc.)	Define a streamlined QC panel early; select key markers from the 232-protein set; disclose criteria (particle size, particle-to-protein ratio, marker intensity, endotoxin level, sterility, residual bovine protein) to ensure reproducibility	Medium
Safety evaluation	Limited safety profiling; no integrated framework; regulatory requirements not fully addressed	Evaluate safety via cytokine panels, pro-fibrotic markers, hemolysis, coagulation, local irritation scores; develop an integrated quality control and safety framework to meet regulatory standards	Low

QC: Quality control.

EXTRACELLULAR VESICLE CHARACTERIZATION AND DOSE NORMALIZATION

Current exosome identification relies on morphology, size, and selected markers, with calnexin as a negative control[2]. Adding markers such as Alix, TSG101, and syntenin, along with broader negative controls, can improve accuracy[2]. Clarifying serum supplementation and collection methods helps assess possible contamination[2]. Reporting only total protein is insufficient, as it does not fully capture the complexity and variability of exosome preparations. This approach overlooks critical parameters such as particle number, particle-to-protein ratio, yield per cell, and batch-to-batch consistency, which are essential for accurate dose standardization and reproducibility[3]. Without these data, it becomes challenging to compare results across experiments or to establish reliable dosing protocols, potentially limiting the translational relevance of the findings. To address these limitations, we recommend adopting a more comprehensive and quantitative approach to exosome characterization and dose normalization. Specifically, both particle count (using nanoparticle tracking analysis or similar technologies) and total protein mass should be routinely reported for each batch. Calculating and disclosing the particle-to-protein ratio and yield per cell will provide a clearer picture of exosome quality and enable more precise inter-study comparisons. Furthermore, documenting batch variability by reporting these metrics for multiple independent preparations can help ensure consistency and reproducibility. Establishing dose-response curves using a range of exosome concentrations is also essential. This allows for the determination of key pharmacological parameters such as the minimum effective dose and EC50, which are critical for optimizing therapeutic efficacy and minimizing off-target effects[3]. In animal studies, normalizing exosome exposure by particle number per unit area and cumulative load will enhance the comparability of results across different experimental conditions and models. Implementing these practices will significantly improve the rigor and transparency of dose standardization, ultimately strengthening the reliability and translational potential of exosome-based therapies.

MECHANISTIC RIGOR: CDC42 NECESSITY AND WNT/Β-CATENIN DEPTH

Support for a CDC42-Wnt/β-catenin-GSK3β axis presently depends on post-treatment expression changes. Demonstrating CDC42 as a transferred, functionally necessary cargo rather than a secondarily induced mediator would reduce ambiguity[4]. However, the current mechanistic evidence is mainly correlative and indirect, leaving open whether CDC42 is truly a transferred, essential exosomal cargo or simply upregulated by other components. The investigation into the Wnt/β-catenin pathway also lacks detailed analysis of downstream events, which may weaken the study’s mechanistic rigor and overall impact.

To substantially address these limitations and elevate the scientific robustness of the manuscript, we recommend a staged experimental approach. First, quantifying intra-exosomal CDC42 per particle would provide direct evidence of its transfer via exosomes. Next, generating CDC42-depleted exosomes by donor cell siRNA or CRISPR knockdown, followed by assessment of their ability to support dermal papilla cell proliferation, migration, and β-catenin nuclear localization, would clarify the necessity of CDC42. Rescue experiments, involving re-expression of CDC42 or supplementation with recombinant CDC42 alongside appropriate adsorption controls, would further establish functional sufficiency. Additionally, mapping the temporal dynamics of β-catenin localization (cytosolic vs nuclear), GSK3β phosphorylation status, canonical target gene transcription (such as AXIN2, LEF1, and cyclin D1), and TOP/FOP reporter activity would enable a more detailed dissection of pathway activation[5].

IN VIVO MODEL AND MICRONEEDLE DELIVERY

The testosterone propionate model mainly describes phenotypic changes. Adding biochemical measurements, such as skin or serum dihydrotestosterone and androgen receptor target genes, along with robust validity measures like randomization and blinding, can improve reliability[6]. Extending follow-up to cover a full hair cycle will assess durability[6]. For microneedle delivery, specifying needle length, density, number of passes, pressure, and treatment intervals is essential for reproducibility[7]. Imaging techniques can help track distribution and optimize dosing[7].

QUALITY CONTROL AND STRUCTURAL CHARACTERIZATION

Defining a minimal quality control panel early can ensure consistency in production. The 232-protein set can be reduced to a few key markers covering major functions. Criteria such as particle size, particle-to-protein ratio, marker intensity, endotoxin level, sterility, and residual bovine protein should be clearly disclosed to facilitate reproducibility[8].

POTENCY AND SAFETY FRAMEWORK

Functional potency can be assessed by measuring dermal papilla cell proliferation, migration, and β-catenin nuclear translocation at standardized particle-to-cell ratios[8,9]. Linking potency to structural variables may help identify surrogate markers for future testing[9]. Safety evaluation should include cytokine panels, pro-fibrotic markers, hemolysis, coagulation, and local irritation scores to meet regulatory requirements[10].

FUTURE DIRECTIONS

Future priorities include: (1) Standardized exosome characterization and dosing; (2) Direct validation of CDC42 and Wnt pathway mechanisms; (3) Robust animal models with clear validity safeguards; (4) Optimized microneedle delivery parameters; and (5) Integrated quality control and safety frameworks.

CONCLUSION

Microneedle-delivered human adipose-derived mesenchymal stem cell-derived exosomes show promise as a regenerative therapy for androgenetic alopecia, supported by stable cargo data and a proposed signaling pathway[1]. Progress toward clinical use will depend on standardized characterization, dose normalization, mechanism validation, rigorous animal studies, quantitative delivery, and clear quality control and safety measures.

ACKNOWLEDGEMENTS

We thank the authors for their valuable work and the opportunity to comment on this important topic.