Source-dependent perlecan heparan sulfate chains in dECM modulate stem cell fate and multilineage differentiation

Perlecan, a heparan sulfate (HS) proteoglycan enriched in cell-derived decellularized extracellular matrix (dECM), carries N-terminal HS chains that act as co-receptor reservoirs for FGF-2, but whether this bioactivity is source-dependent is unknown.

Passage-6 infrapatellar fat pad-derived stem cells (IPFSCs) were expanded on dECM from unmodified (dE) or SV40-immortalized (s-dE) IPFSCs, with or without heparinase III (HPA) to ablate HS chains, yielding five groups: PL (plastic), dE, dE(H), s-dE, and s-dE(H). Stemness, surface markers, and trilineage differentiation were assessed by RT-qPCR, histology, immunohistochemistry, and colorimetric assays over 21-day induction periods.

HPA selectively cleaved HS chains from matrix-resident perlecan without disrupting the core protein. Both dECM substrates suppressed adipogenic potential. HPA markedly enhanced adipogenesis in both groups. Chondrogenic and osteogenic responses were strikingly source-dependent: dE(H) preconditioning impaired chondrogenic differentiation, while s-dE(H) preconditioning conferred superior chondrogenic output-including the most prominent sGAG deposition and the largest pellet size among the groups examined-and exclusively promoted osteogenic matrix mineralization.

These findings indicate that perlecan-anchored HS chains are context-dependent regulators of stem cell fate, whose effects are modulated by dECM cellular origin, likely via structural differences in HS chains that differentially modulate FGF and BMP signaling. Targeted manipulation of matrix HS architecture offers a tunable strategy for dECM-based preconditioning in musculoskeletal regenerative medicine.