Protocol defines product: Navigating function, safety and translation of induced pluripotent stem cell-derived mesenchymal stem cells

Abstract

Induced pluripotent stem cell-derived mesenchymal stem cell (iMSC) represent an important next step in regenerative medicine. By deriving mesenchymal stem cell-like cells from renewable induced pluripotent stem cell sources, researchers aim to overcome the donor variability, limited scalability, and senescence that have long constrained adult mesenchymal stem cell therapies. The recent study published in World Journal of Stem Cells by Ababneh et al illustrates how differences in derivation protocols can yield iMSC populations that are functionally and metabolically distinct despite meeting minimal mesenchymal stem cell criteria. This reinforces a key translational lesson: Protocol defines product. Moving forward, successful clinical translation will depend on aligning differentiation strategy with therapeutic purpose, integrating safety evaluation throughout development, and adopting manufacturing processes compatible with good manufacturing practice standards. Beyond marker expression, emphasis must now shift toward potency, standardisation, and indication-relevant critical quality attributes. Through such alignment, iMSC technologies may finally achieve reproducibility, safety, and therapeutic reliability.

Key Words: Induced pluripotent stem cell-derived mesenchymal stem cell; Induced pluripotent stem cell; Mesenchymal stem cell; Good manufacturing practices; Potency assay; Critical quality attribute; Clinical translation

Core Tip: This editorial highlights the emerging principle that in induced pluripotent stem cell-derived mesenchymal stem cell (iMSC) development, protocol defines product. Drawing on recent comparative data, it underscores how different derivation routes yield functionally distinct iMSCs despite meeting minimal mesenchymal stem cell criteria. The piece advocates aligning differentiation methods with therapeutic mechanisms, embedding genomic safety early, and designing good manufacturing practices-ready, comparable manufacturing processes. By integrating biological insight with process discipline, iMSC technology can achieve reproducibility, potency, and safety, thereby turning conceptual promise into clinically reliable cell therapies.

This editorial refers to “Impact of differentiation protocols on the functionality of mesenchymal stem cells derived from induced pluripotent stem cells” by Ababneh et al, 2025; https://doi.org/10.4252/wjsc.v17.i12.110564.

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INTRODUCTION

Over the past two decades, the field of mesenchymal stem cell (MSC) clinical research has advanced remarkably yet inconsistently[1]. Despite the breadth of clinical investigations, variability across donors, tissues, and culture systems continues to blur outcomes. The rise of induced pluripotent stem cell (iPSC)-derived MSC (iMSC) offers a potential solution; a renewable, well-characterised source capable of extensive proliferation without senescence[2,3]. This approach combines the biological appeal of MSCs with the manufacturing scalability of iPSCs, allowing developers to envision a more standardised living biologic. Crucially, this concept is no longer merely theoretical; the successful completion of a phase I clinical trial utilizing an iMSC (CYP-001) for acute graft-vs-host disease has unequivocally demonstrated that such scalable platforms can yield safe and durable clinical outcomes over a two-year follow-up[4].

The study by Ababneh et al[5] published in the World Journal of Stem Cells arrives timely at this juncture. By directly comparing multiple iMSC differentiation routes under unified laboratory conditions, they demonstrate that while phenotypic convergence may occur, functional equivalence cannot be assumed. While the head-to-head design strengthens attribution of functional differences to derivation protocol, it is important to recognise certain boundaries of the study. Ababneh et al[5] evaluated a limited number of iPSC donor lines under research-grade conditions, without direct comparison to good manufacturing practices (GMP)-adapted manufacturing workflows. As such, the extent to which specific protocol-dependent features generalise across broader donor panels, alternative reprogramming strategies, or clinical-grade systems remains an open and important question (Figure 1).

Figure 1 Conceptual pathway showing how differentiation protocols imprint distinct phenotypic and functional identities on induced pluripotent stem cell-derived mesenchymal stem cell, ultimately influencing translational suitability. The framework emphasises the central principle: Protocol defines product. iPSC: Induced pluripotent stem cell.

TRANSLATING IMSCS: FROM BIOLOGICAL INSIGHT TO MANUFACTURING DISCIPLINE

The observations of Ababneh et al[5] crystallise a larger truth in regenerative medicine: Translating cell products into safe and effective therapies requires a continuum of alignment, from biology, process design to manufacturing.

Potency linked to purpose

Not all MSC therapies act through the same mechanism of action[6], and not all iMSCs express it equally. Potency should therefore be defined by intended clinical function[7], rather than by standard minimal criteria[8] (Table 1). For immune disorders, assays reflecting immunosuppression (e.g., T-cell suppression) remain relevant; for vascular or tissue repair, angiogenic or trophic assays may be more appropriate. Importantly, iMSC potency may also vary with metabolic state, secretome composition, or epigenetic imprint[3,9]. For complex indications such as degenerative diseases where multiple pathways are engaged, composite potency assays may be required. In practice, such composite assays might combine measurements of trophic factor secretion (e.g., vascular endothelial growth factor or hepatocyte growth factor release)[10], matrix remodelling activity[11], and anti-inflammatory effects[12]. For musculoskeletal or neurodegenerative conditions, integrating functional readouts such as protection from apoptosis, enhancement of matrix synthesis, or modulation of inflammatory cytokine profiles may better reflect therapeutic relevance. Rather than relying on a single surrogate marker, these multi-parameter approaches acknowledge the inherently pleiotropic nature of iMSC-mediated repair. The field is moving toward defining such mechanism-specific critical quality attributes (CQAs)[13,14], ensuring that laboratory measures correspond to clinical outcomes rather than convenient surrogates.

Table 1 Evolving focus in the evaluation of mesenchymal stem cell products.

Evaluation focus	Traditional MSC approach	Next-generation iMSC perspective
Cell identity	ISCT minimal criteria (CD73/CD90/CD105, tri-lineage differentiation)	ISCT + transcriptomic, metabolic, and functional profiling
Potency	Generic immunosuppression or differentiation assays	Indication-specific assays aligned with mechanism of action
Safety	Karyotype, sterility, mycoplasma (often late-stage)	Integrated genomic surveillance, residual pluripotency and tumorigenicity testing from early development
Manufacturing	Often donor-dependent, Matrigel or foetal bovine serum	Xeno-free, chemically defined, good manufacturing practices-compatible and comparable
Reproducibility	Limited by donor and passage variability	Enabled through renewable induced pluripotent stem cells source + defined process control

While traditional mesenchymal stem cell assessments emphasised minimal phenotypic criteria, induced pluripotent stem cell-derived mesenchymal stem cell development increasingly requires function- and safety-based qualification. MSC: Mesenchymal stem cell; iMSC: Induced pluripotent stem cell-derived mesenchymal stem cell; ISCT: International Society for Cell & Gene Therapy.

Safety integrated early and continuously

Because iMSCs originate from pluripotent sources, they inherit both opportunities and risks. Rigorous early-phase testing should be viewed as foundational rather than optional[15]. When stringent manufacturing processes and quality controls are applied to completely exclude residual undifferentiated iPSCs, the clinical safety profile is highly reassuring[4]. Indeed, systemic delivery of clinical-grade iMSCs has recently demonstrated an absence of teratoma formation, therapy-related malignancies, or other serious adverse events in a two-year human clinical cohort[4]. To achieve and guarantee this level of safety, modern surveillance tools such as high-resolution karyotyping, chromosomal microarray and genome-wide methylation profiling must be utilized to enable continuous quality monitoring throughout cell line establishment and expansion. Embedding these assays into development pipelines builds regulator and clinician confidence long before the clinical trial stage. A risk-based quality management system (QMS) must explicitly account for ‘epigenetic memory’ or donor-specific DNA methylation signatures that may persist despite reprogramming[16]. Mapping and monitoring these epigenetic scars are vital for ensuring the long-term genomic stability, immunomodulatory consistency, and lineage fidelity of the final cellular product[17].

Manufacturing with foresight and comparability

Manufacturing choices, including matrix, medium, and passaging strategy, profoundly influence iMSC identity. Many academic protocols rely on Matrigel, feeder layers, or undefined sera, which are incompatible with GMP production. Transitioning to xeno-free, chemically defined systems not only facilitates compliance but also enhances reproducibility across production sites. However, process transition alone does not ensure equivalence.

Comparability studies must demonstrate that the GMP-adapted product is biologically and functionally consistent with the original research material[18]. Evaluations should span morphology, transcriptome, secretome, and potency assays. Increasingly, comparability frameworks are being standardised by anchoring assessments to predefined CQAs rather than platform-specific parameters[19]. Establishing reference lots, harmonised assay panels, and acceptance ranges allows products manufactured in different bioreactors or facilities to be evaluated against a common benchmark. When combined with risk-based comparability strategies and well-defined QMS, this approach enables controlled evolution of manufacturing platforms without compromising product identity or clinical intent[19]. This principle aligns perfectly with the United States Food and Drug Administration’s guidance on flexible Chemistry, Manufacturing, and Controls requirements, which facilitates iterative process improvements during development, provided they are anchored to robust analytical comparability and predefined acceptance ranges[20]. Furthermore, an effective QMS[21] that incorporates process analytical technologies, such as the real-time monitoring of metabolite consumption, and secreted cytokines, helps detect drift before it affects final product quality[22,23]. Particularly as clinical manufacturing transitions from static 2D planar cultures to advanced 3D microcarrier-based dynamic bioreactors, integrating artificial intelligence-driven predictive modelling into these process analytical technologies frameworks has proven essential[24]. This automation minimizes batch-to-batch variance and ensures continuous alignment with predefined CQAs at commercial scales[25].

Building toward consistency and scalability

The promise of iMSCs lies in scalability: One qualified iPSC donor line can, in theory, supply limitless batches. Yet true consistency demands disciplined process control. Master and working cell banks must be fully characterised, with defined release criteria covering identity, sterility, mycoplasma, adventitious agent testing, and genomic stability[18,26]. Embedding in-process controls, such as cell morphology scoring, metabolite thresholds, and growth kinetics, ensures early detection of variability. Developers should also establish pre-defined acceptance ranges for CQAs and set up corrective-action protocols to sustain reproducibility across batches and facilities[14,26].

Aligning with evolving standards and community transparency

The International Society for Cell & Gene Therapy (ISCT) continues to drive consensus efforts to standardize nomenclature[27] and establish rigorous reporting criteria for primary mesenchymal stromal/stem cell clinical trials[13]. Building on this foundation, and recognizing the unique developmental trajectory and regulatory challenges of pluripotent sources, the joint Japanese Society for Regenerative Medicine-ISCT iPSC Committee has extended these efforts to establish Quality by Design principles, stringent CQAs, and process comparability frameworks specifically for iPSC-derived therapies[28]. Parallel to this, the International Society for Stem Cell Research has established recommendations to enhance reproducibility in basic human stem cell research[29]. Engagement with such initiatives not only aligns research with regulatory expectations but helps shape the language by which iMSCs will be evaluated. Transparency is equally important. Publishing process details, comparability data, and potency rationales, including those showing negative or neutral results, accelerates collective progress. Reproducibility is a shared responsibility, and open reporting transforms isolated findings into community standards. Taken together, these principles emphasise that GMP is not a regulatory burden but a scientific discipline that transforms creative differentiation into reliable production. The translation of iMSCs will succeed not by eliminating variability but by understanding, measuring, and controlling it.

QUESTIONS STILL WORTH EXPLORING

While translational frameworks are taking shape, Ababneh et al[5] also highlight areas of continuing discovery.

Metabolic nuance

The finding that some iMSC lines exhibit lower mitochondrial potential yet reduced oxidative stress suggests adaptive metabolic rewiring, perhaps toward glycolysis or enhanced mitophagy. Understanding whether this shift represents beneficial resilience or latent fragility requires targeted metabolic flux and mitochondrial quality-control studies.

Surface markers and meaning

Variability in CD90 and CD105 expression raises a deeper question: Are these merely descriptive labels, or do they signify functional subsets relevant to immunomodulation or migration[30,31]? Single-cell multi-omics study may reveal whether these markers correspond to specific potency mechanisms or to transcriptomic reprogramming[32]. Recognising these uncertainties reminds us that variation is not only a challenge to control, but also a window into underlying biology that can guide refinement of manufacturing and quality standards.

BEYOND MINIMAL CRITERIA: LOOKING AHEAD WITH PRECISION

The next phase of iMSC development will depend not on producing another “differentiation success story” but on achieving reproducibility and clinical relevance. Validation across donors and contexts, mechanism-based potency testing, and GMP-aligned process control will define the benchmarks of progress[13]. The appealing idea that a single, immortal iPSC donor could eliminate variability must be balanced by realism: Consistency will arise not from assumptions of stability, but from disciplined qualification and transparent process documentation[17]. In practice, protocol defines product as much as biology itself.

CONCLUSION

The study by Ababneh et al[5] marks an important step in the maturation of regenerative medicine. It reminds us that a cell’s identity is more than its markers, for it carries the imprint of every choice made along its developmental path. Understanding that story reflects the field’s own evolution, moving from empirical exploration toward engineered precision. The comparative insights discussed here also highlight what remains to be done. Translational confidence in iMSC platforms will ultimately depend on extending such analyses across diverse iPSC donors, reprogramming methods, and explicitly GMP-adapted processes. This includes multi-donor validation, head-to-head GMP bridging runs, and formal process comparability frameworks using shared reference cell banks, predefined equivalence margins, and orthogonal CQAs. Acknowledging these limitations does not diminish the importance of protocol comparisons; rather, it positions them as the necessary foundation for building reproducible, regulator-ready, and clinically credible iMSC products. As iMSC technology continues to mature, the guiding principle remains clear yet humble: Each step of the process shapes not just the product, but also our collective ability to translate science into healing.