Regenerative-system dysfunction in organ fibrosis: Maladaptive regeneration, fibrotic memory, and therapeutic reprogramming

Table 5 Experimental platforms for distinguishing association, causality, and memory

Platform	Primary contribution/causal inference	Memory or reversibility requirements	Main limitation
Single-cell/single-nucleus RNA sequencing	Identifies cell states and heterogeneity; not causal by itself[8,37]	Longitudinal sampling can suggest persistence; exposure history and a functional test are needed	Sampling bias, dissociation effects, and lack of spatial context
Spatial transcriptomics	Maps tissue architecture and candidate interactions; not causal alone[65,66]	Can localize retained niches, but cannot alone exclude continuing stimulus	Resolution, analytical inference, and tissue-sampling limitations
Multiome/chromatin profiling	Links regulatory and transcriptional states; stronger inference requires perturbation[48]	Test retained programs after withdrawal and selectively perturb the candidate state	A chromatin signature is not itself proof of memory
Lineage tracing/barcoding	Establishes origin and fate, not causal necessity without an additional intervention[28]	Combine with withdrawal/re-exposure and appropriate functional perturbation[52]	Label specificity, recombination efficiency, and mostly experimental-animal use
Inducible perturbation/Perturb-seq	Tests candidate regulatory contributions when controls and endpoints are appropriate[29]	Perturb an already retained state after injury reduction; assess later function and re-exposure	Perturbation during ongoing injury may identify an injury pathway rather than memory
Organoids/expandable epithelial cultures	Experimentally tractable human-derived models[50,51]	Add and validate injury, washout, recovery, and rechallenge; this is a proposed extension, not automatic platform capability	Incomplete immune, vascular, and mechanical niches; variable maturation
Ex vivo diseased ECM/tissue systems	Tests matrix-conditioned behavior and niche instruction[34]	Transfer away from diseased ECM or remove residual stimuli to test cell-autonomous retention	Continued ECM exposure is continuing niche instruction, not proof of intrinsic memory
Longitudinal human sampling	Establishes clinical relevance and candidate biomarker trajectories[9,20]	Document injury reduction, recovery interval, and confounders; couple associations to functional models	Limited repeat sampling and no routine direct genetic causal test

Functional tissue recovery must be measured separately from cell-state or molecular normalization. Platform availability does not guarantee a valid memory test. Direct evidence depends on the exposure, resolution, transfer/rechallenge, and perturbation design described in “Definition and conceptual boundaries”. ECM: Extracellular matrix.