Lung organoids for preclinical evaluation of stem cell therapies: Opportunities, evidence, and translational challenges

Table 1 The lung organoid platform as feasibility evidence for preclinical stem cell validation

Assessment dimension	Study type	Key findings	Evidence level	Limitations	Ref.
Immunomodulatory mechanism	Murine lung organoid-macrophage co-culture	Bone marrow-derived MSCs suppressed LPS-induced proinflammatory cytokines and NF-κB activation in alveolar and interstitial macrophages	Direct (murine organoid)	Not validated in human organoids; culture medium incompatibility limits generalizability	[18]
Long-term safety monitoring (tumorigenicity, genetic stability)	Theoretical framework	Genomic approaches (WGS, karyotyping, scRNA-seq) proposed for assessing oncogenic risk of candidate cells	Theoretical (proof-of-principle)	No prospective study	[65,81,82]

Evidence level definitions: Direct evidence - stem cell or extracellular vesicle therapies directly validated within a specified organotypic system; theoretical evidence - proposed applications for which no directly validated studies have been conducted. MSCs: Mesenchymal stromal cells; LPS: Lipopolysaccharide; NF-κB: Nuclear factor kappa B; WGS: Whole genome sequencing; scRNA-seq: Single-cell RNA sequencing.

Table 2 Comparison of characteristics among lung organoids derived from different stem cell sources

	ASCs	iPSCs	ESCs
Main sources	Autologous lung tissue from patients (AT2 cells, airway basal cells, etc.)	Somatic cell reprogramming (e.g., from fibroblasts, peripheral blood mononuclear cells, etc.)	Inner cell mass of blastocysts (allogeneic)
Applicable to pulmonary disease scenarios	Airway epithelial reconstruction; local tissue repair	Modeling of developmental disorders; autologous transplantation following gene correction	Modeling of early lung development (e.g., congenital anomalies); investigation of key signaling pathways (FGF, Wnt)
Cost	Due to limitations in biopsy tissue accessibility and amplification capacity, the cost of personalized sample acquisition is high	Reprogramming and directed differentiation techniques are complex, with high costs associated with reagents and quality control materials	High, associated with acquisition, specialized maintenance, and ethical compliance procedures
Advantage	Short cultivation cycle, high maturity; preserves tissue specificity and epigenetic characteristics	Nearly unlimited amplification capacity; multi-lineage differentiation potential	Nearly unlimited amplification capacity; multi-lineage differentiation potential; serves as a vital tool for early-stage lung development research
Limitations	Cannot differentiate into non-epithelial lineages; may harbor disease-associated genetic alterations	Reprogramming carries a tumorigenic risk; there are significant inter-batch variations in cell maturity	Ethical constraints; immune rejection risk in allogeneic settings; teratoma formation potential
Focus of organoid validation	Assessment of local epithelial regenerative potential; functional validation prior to autologous transplantation	Differentiation efficiency and maturity monitoring; long-term genomic stability tracking	Monitoring of differentiation efficiency and maturity; evaluation of teratoma risk; basic developmental research

The comparative assessment in this table is a qualitative synthesis of published literature and expert interpretation, rather than a validated consensus ranking or meta-analysis conclusion. ASC: Adult stem cell; iPSC: Induced pluripotent stem cell; ESC: Embryonic stem cell; AT2: Alveolar type 2; FGF: Fibroblast growth factor.

Table 3 Applications of lung organoids in evaluating stem cell therapy

Disease type	Source/organoid type	Stem cell/EV therapy	Evaluation metrics	Key findings	Study design/sample size	Ref.
Pulmonary fibrosis	-	-	-	No published studies have been identified that directly evaluate MSC- or MSC-EV-based therapy in pulmonary fibrosis lung organoid models; current evidence is primarily derived from animal models and 2D culture experiments	-	-
COPD/emphysema	Mouse-derived/Lung epithelial progenitor cell-derived organoids	hUC-MSC-EVs; hUC-MSCs	Number/size of organoids; AT2/AT1 marker balance; collagen deposition; inflammatory infiltration	hUC-MSC-EVs reduce inflammatory infiltration and collagen deposition, restore the normal number and size of organoids, and rebalance the AT2/AT1 ratio	Murine organoids; n = 3-5 mice per group; single exposure model	[58]
Acute lung injury	Mouse-derived/Lung organoid-macrophage co-culture model	MSCs (bone marrow origin)	Macrophage pro-inflammatory function; organoid morphology	MSCs mitigate LPS-induced acute lung injury by inhibiting the pro-inflammatory function of macrophages	Murine organoid-macrophage co-culture; n = 3 independent experiments; 2 organoid donors + ≥ 3 MSC donors	[18]
Lung cancer	Human-derived/iPSCs-derived bronchial organoids (BLO); patient-derived LCOs	PSC-MSC-derived EVs (loaded with cisplatin)	LDH release; CCK8 metabolic activity; apoptosis-related genes (e.g., P53)	The empty EVs themselves exhibit cytotoxicity toward both LCO and BLO, suggesting that MSC-EVs may exert non-specific effects	Human iPSCs-derived organoids; n = 3-4 technical replicates; 2 independent differentiations	[33]

Except for the pulmonary fibrosis row (for which no published studies were identified), all other therapies involving stem cells or stem cell-derived extracellular vesicles listed in this table have been directly validated within lung organoid systems. EV: Extracellular vesicle; MSC: Mesenchymal stromal cell; 2D: Two-dimensional; COPD: Chronic obstructive pulmonary disease; hUC-MSC-EVs: Human umbilical cord mesenchymal stem cell-derived extracellular vesicles; hUC-MSCs: Human umbilical cord mesenchymal stem cell; AT2: Alveolar type 2; LPS: Lipopolysaccharide; iPSCs: Induced pluripotent stem cells; BLO: Branched lung organoid; LCO: Lung cancer organoid; PSC: Pluripotent stem cell; LDH: Lactate dehydrogenase.

Table 4 Comparison of the advantages and limitations among lung organoids, lung-on-a-chip systems, animal models, and two-dimensional cell cultures

Comparison dimensions	Animal model	2D cell culture	Lung organoids	Lung-on-a-chip
Human-derived physiological fidelity	Moderate	Low	High	Medium to high
Cost	Low	Low	Medium to high	Moderate
Throughput	Low	High	High	Moderate
Level of operational difficulty	Medium to high	Low	Low	Low
Reproducibility and standardization	Moderate	High	Moderate	Moderate
Applicability of stem cell therapy	Moderate	Low	High	High
Core limitations	Significant species differences; ethical controversies	Lack of a physiological gradient; absence of multicellular interactions or an immune microenvironment	No physiological mechanical stress; lacks functional vascular and nervous systems	High technical complexity; incomplete vascularization
Classic applications	Research on systemic disease mechanisms; in vivo analysis; systemic toxicity assessment	Early drug screening; basic research on cellular signaling pathways; large-scale cytotoxicity testing	Disease modeling; PDO drug sensitivity testing; research on stem cell differentiation and repair mechanisms; establishment of a precision medicine biobank	The relationship between mechanics and disease; organ-organ interactions; assessment of inhaled nanoparticle toxicity

The comparative assessment results presented in this table reflect the authors’ qualitative synthesis of existing literature and expert interpretations, rather than representing a validated consensus ranking or the conclusions of a meta-analysis. The ratings (‘Low’, ‘Moderate’, ‘High’) are descriptive generalizations based on the volume and directness of available evidence for each platform and should be interpreted as relative indicators rather than absolute performance metrics. These ratings may vary depending on specific experimental contexts, disease models, and research questions. They should be regarded as indicative references, not as quantitative benchmarks. PDO: Patient-derived organoid.