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

Table 3 Müller glia reprogramming “minimal experimental standard” - non-negotiable evidence hierarchy for conversion claims

Evidence tier	What must be demonstrated	Minimum required controls	Readouts that count as “orthogonal”	Pass/fail interpretation rule	Typical artifact this prevents
Tier 1: Genetic lineage tracing	Converted neurons are MG-origin	MG-specific inducible CreERT2; quantify recombination efficiency	Reporter-independent validation	No tracing = claim not interpretable	Mis-assigned cellular origin
Tier 2: Vector specificity/promoter leakage control	Transgene expression is cell-type restricted	AAV-GFAP leakage tests; alternative promoters; no-virus controls	Spatial mapping of transgene vs cell identity	Leakage unresolved = conversion invalid	“Apparent conversion”
Tier 3: Single-cell identity triangulation	True fate switch vs stress mimicry	Injury-only vs intervention; batch controls	scRNA ± ATAC; stress signatures; GRN congruence	Marker-only = insufficient	Stress-induced pseudo-neurons
Tier 4: Morphology + protein-level confirmation	Neuronal morphology consistent	Blinded morphometrics; layer localization	Immunostaining + morphology metrics	Partial markers without morphology = weak	Marker contamination
Tier 5 Physiology + circuit-level function	Functional maturation & integration	Electrophysiology controls; synapse evidence	Patch clamp; stimulus responses; connectivity proxies	No function = not therapeutic	Immature “neuron-like” cells
Tier 6: Contextual reproducibility	Robust across injury paradigms	Excitotoxic vs mechanical injury	Same validation stack across models	Single-context only = fragile	Context-dependent artifacts

MG: Müller glia; CreERT2: Cre recombinase-estrogen receptor T2 fusion protein; AAV: Adeno-associated virus; GFAP: Glial fibrillary acidic protein; scRNA: Single-cell RNA; ATAC: Assay for transposase-accessible chromatin; GRN: Gene regulatory network.