Multilayered control of retinal stem/progenitor cell fate in the single-cell and organoid era: Developmental blueprints and regenerative opportunities

Table 6 Two-tier, omics-enabled quality control framework linking multi-omics resources to Good Manufacturing Practice release criteria for retinal organoids and organoid-derived products

QC domain (CQA)	Essential for clinical translation?	Essential multi-omics criteria (process dev/periodic lot qualification)	Minimal routine GMP lot-release panel (examples; targeted assays)	Engineering → CMC translation output	Key evidence/precedent	Ref.
Identity & composition (intended lineage; correct cell-type stoichiometry)	Yes (CQA definition + re-qualification)	scRNA-seq cell-type composition; “composition envelope” across lots; reference-mapping to human retina atlas (maturity/trajectory score)	Targeted marker panel (flow/qPCR/IF): Lineage markers + subtype markers; morphology metrics where applicable	Converts atlas cell states into measurable CQAs; derives reduced marker sets; flags drift early	Retinal organoids vs adult retina single-cell reference; organoid heterogeneity quantified at scale	[143]
Purity/off-target tissues (non-retinal CNS, mesenchymal, RPE contamination etc.)	Yes	scRNA-seq off-target fraction; stress/reactive state detection (hypoxia/ISR/gliosis modules)	Release: Residual pluripotency (OCT4/TRA-1-60/NANOG) negative; proliferation (Ki67) limits; off-target marker negatives; viability & total cell number	Sets acceptance criteria for “allowed impurities”; links drift to process parameters (media/patterning/selection)	Organoid variability across systems supports need for systematic QC	[144]
Maturity/developmental congruence (photoreceptor/RPE functional readiness)	Yes for qualification; not per-lot mandatory	Reference mapping to fetal/adult retina trajectories; optional scATAC/multiome for competence state; optional spatial for lamination	Release: Maturity-linked targeted markers (e.g., phototransduction/synaptic readiness proxies) + predefined in-process timepoints	Defines “Goldilocks” maturity window; prevents under-/over-mature lots	HA conditioning improves photoreceptor maturation and uniformity (supports measurable maturation CQAs)	[110]
Potency (mechanism-linked biological activity)	Yes	Omics used to select potency mechanisms (pathway engagement signatures) and to justify assay choice; optional proteomics/metabolomics to connect transcript → function	Release: Validated potency assay(s) aligned to mechanism (e.g., RPE phagocytosis/TEER; photoreceptor light-response surrogates + integration-sensitive proxies)	Bridges omics biomarkers → potency assay design; supports assay justification	FDA potency guidance emphasizes mechanism-linked potency tests; lifecycle potency assurance	[145]
Safety/genetic stability (tumorigenicity risk, genome integrity)	Yes	Genomic characterization strategy (karyotype/CNV; WCB/MCB characterization); optional WGS where justified	Release: Sterility/mycoplasma/endotoxin; viability; residual pluripotency negative; proliferation limits; stability post-thaw	Links bank characterization to release and long-term follow-up	Cell substrate characterization expectations; ATMP quality requirements in trials	[146]
Comparability (manufacturing changes)	Yes when changes occur	Re-map lots with scRNA composition + maturity score; stress signature comparison; optional multiome/spatial if MoA-critical	Release: Same validated panel + bridging study endpoints	Provides quantitative “sameness” evidence after changes	FDA comparability guidance for CGT products	[162]

QC: Quality control; CQA: Critical quality attribute(s); scRNA-seq: Single-cell RNA sequencing; GMP: Good Manufacturing Practice; CMC: Chemistry, manufacturing, and controls; qPCR: Quantitative polymerase chain reaction; IF: Immunofluorescence; ISR: Integrated stress response; OCT4 Octamer-binding transcription factor 4 (POU5F1); TRA-1-60: Tumor rejection antigen 1-60; Ki67: Marker of cellular proliferation (Ki-67 antigen); CNS: Central nervous system; RPE: Retinal pigment epithelium; scATAC: Single-cell assay for transposase-accessible chromatin; HA: Hyaluronic acid; TEER: Transepithelial electrical resistance; FDA: Food and Drug Administration; CNV: Copy number variation; WCB: Working cell bank; MCB: Master cell bank; WGS: Whole-genome sequencing; ATMP: Advanced therapy medicinal product; CGT: Cell and gene therapy; MoA: Mechanism of action.