Spatial heterogeneity of lineage-committed muscle stem cell subpopulations challenges bipotency assumptions in Thai-French crossbred cattle.

BACKGROUND AND AIM: Muscle-derived stem cells (MDSCs) are promising cellular resources for cultured meat because skeletal muscle contains progenitors that can generate myogenic and adipogenic tissues. However, whether the apparent dual-lineage differentiation of CD29-positive MDSCs reflects intrinsic bipotency or heterogeneous, lineage-committed subpopulations remains unclear. This study aimed to establish a simplified protocol for isolating CD29-positive MDSCs from adult Thai-French crossbred cattle, characterize satellite cell (SC) and fibro-adipogenic progenitor (FAP) subpopulations, and determine whether their spatial composition governs differentiation outcomes.

MATERIALS AND

METHODS

Skeletal muscle samples were obtained from three adult male Pon Yang Kham Thai-French crossbred cattle. Five independent tissue samples were used to validate cell isolation, while three spatial isolates (BNS1-BNS3) from distinct chuck roll locations of one donor were used for detailed characterization. Muscle-derived cells were isolated by enzymatic digestion, filtration, red blood cell lysis, and short-term culture enrichment. CD29-positive cells were characterized and separated into CD56-positive SCs and CD56-negative FAPs using fluorescence-activated cell sorting. Proliferation, immunofluorescence, lipid accumulation, and quantitative gene expression analyses were performed to assess lineage-specific differentiation.

RESULTS

The protocol yielded 5.13 ± 1.23 × 10⁵ viable cells/g tissue and >95% CD29-positive enrichment. Spatial isolates showed pronounced variation in SC:FAP composition, ranging from 4.3:95.1 to 96.0:3.8. Both subpopulations exhibited approximately 6-8-fold proliferative capacity during early passages. SCs formed multinucleated myotubes and expressed Myf5, MyHC, and MyoG following myogenic induction. FAPs accumulated intracellular lipids and showed increased CIDEC expression following adipogenic induction. Unfractionated CD29-positive populations displayed reciprocal lineage-directed responses under myogenic and adipogenic conditions, indicating that differentiation behavior was determined by the composition and selective induction of pre-existing lineage-biased subpopulations.

CONCLUSION

CD29-positive MDSCs from adult Thai-French crossbred cattle represent spatially heterogeneous mixtures of lineage-committed SCs and FAPs rather than a uniformly bipotent progenitor population. The simplified isolation approach provides an accessible source of expandable bovine progenitor cells, while the marked spatial heterogeneity highlights the need for subpopulation-aware cell sourcing and quality control to improve reproducibility in cultured meat production.