Temporal Layering Of Extracellular Matrix Programs Defines Osteogenic Maturation In Human Mesenchymal Stromal Cells
Abstract
Osteogenic differentiation of human mesenchymal stromal/stem cells (MSCs) is commonly described as a progression from lineage commitment to extracellular matrix (ECM) maturation and mineralization. However, the temporal organization of these transcriptional programs and their relative discriminatory power remain insufficiently resolved. We re-analyzed the public RNA-seq time-course dataset GSE129036, comprising 43 samples from four donors collected over 0–504 h following osteogenic induction. Early (0–24 h) and Late (168–504 h) stages were compared using donor-adjusted differential expression analysis, pathway enrichment, ECM-focused temporal profiling, and candidate prioritization. Data-derived ECM and mineralization panels were further benchmarked against literature-derived gene sets using dimensionality reduction, clustering metrics, temporal ordering, and sigmoidal plateau modeling.
Early and Late stages showed clear transcriptomic separation, with 7,338 significantly differentially expressed genes. Enrichment analyses revealed broad pathway reprogramming, while ECM-focused analyses showed marked stage-dependent remodeling across matrisome categories. A compact late-stage ECM candidate panel highlighted genes associated with collagen fibrillogenesis, matrix remodeling, and ECM organization. Data-derived ECM and mineralization panels outperformed literature-derived gene sets in stage discrimination, demonstrating improved clustering quality and stronger stage-dependent expression structure. Temporal modeling showed that ECM and mineralization programs initiated in overlapping time windows but exhibited distinct activation architectures. ECM-associated genes displayed progressive activation and earlier plateau behavior, whereas mineralization genes showed more heterogeneous and delayed stabilization. These findings support a model in which osteogenic maturation is governed by temporally layered transcriptional programs rather than a single late-stage transition. Data-derived ECM and mineralization panels showed improved internal stage discrimination relative to literature-derived gene sets within this dataset, with higher clustering quality and stronger stage-dependent expression structure.
Keywords
mesenchymal stem cells;, osteogenic differentiation;, extracellular matrix;, mineralization;, RNA-sequencing;, temporal transcriptomics;
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