Letter to the Editor: Advancing induced pluripotent stem cell-derived mesenchymal stem cells - insights from comparative differentiation protocols and emerging innovations

Table 1 Summary of recent induced pluripotent stem cell-to-mesenchymal stem cells differentiation protocols (2024-2025)

Protocol type	Key features	Advantages	Limitations	Proposed solution	Ref.
Sequential GF induction	Stepwise use of FGF2, BMP4, PDGF, EGF; 2-3 weeks duration	High yield (> 85% MSCs); scalable	Donor variability; GF cost	Use AI-optimised minimal GF cocktails and pooled GMP-grade GF suppliers	[18]
AI-guided optimization	Machine learning predicts media/GF combos; integrates CRISPR	Reduced heterogeneity; 20%-30% efficiency gain	Requires computational resources	Share trained models and datasets via consortia to lower barrier and validate generalisability	[5]
Suspension-based	ROCK inhibitor + StemMACS media; feeder-free	GMP-compatible; stimulates angiogenesis	Lower adipogenic potential	Combine 3D microcarrier conditions or post-differentiation niche conditioning to restore adipogenic capacity	[24]
Mesodermal-focused	Activin A + CHIR99021 for primitive streak; FGF2 for MSCs	Enhanced skin regeneration applications	Protocol-specific senescence	Combine small-molecule-driven mesodermal induction (e.g., CHIR99021/Wnt activation) with minimal essential growth factor sets to reduce cost and variability	[25]
Gene-edited hybrid	CRISPR for pluripotency knockdown; GF supplementation	Tumor-safe; tailored potency	Ethical/regulatory hurdles	Adopt a Quality-by-Design framework for gene-edited iMSCs, including off-target genome analysis, transcriptomic comparability to parental iMSCs, and long-term culture stability testing	[6]

GF: Growth factor; FGF2: Fibroblast growth factor 2; BMP4: Bone morphogenetic protein 4; PDGF: Platelet-derived growth factor; EGF: Epidermal growth factor; MSC: Mesenchymal stem cell; AI: Artificial intelligence; GMP: Good Manufacturing Practice; iMSCs: Induced pluripotent stem cell-derived mesenchymal stem cells.

Table 2 A three-tier potency assay

Tier	Assay category	Specific metrics (thresholds)
Phenotypic core	Surface + lineage transcription	CD73/CD90/CD105 ≥ 95%
		RUNX2 ≥ 2.5-fold vs BM-MSC (qPCR)
		PPARG ≤ 0.5-fold
Functional potency	Proliferation, migration, stress resilience	CFU-F ≥ 50/103 cells
		Transwell migration index ≥ 0.7 (24 hours)
		ROS ≤ 60% BM-MSC baseline (DCFDA)
Translational predictors	3D microenvironment + AI modeling	3D spheroid invasion ≥ 70% (72 hours, Matrigel)
		AI-predicted batch consistency (R2 ≥ 0.85 via scRNA-seq input)

RUNX2: Runt-related transcription factor 2; BM-MSC: Bone marrow mesenchymal stem cell; qPCR: Quantitative polymerase chain reaction; PPARG: Peroxisome proliferator-activated receptor gamma; CFU-F: Colony forming units-fibroblast; ROS: Reactive oxygen species; DCFDA: 2’,7’-dichlorofluorescin diacetate; AI: Artificial intelligence; scRNA-seq: Single-cell RNA sequencing.