Mechanoepigenetic Targeting of Histone Methyltransferase EZH2 Increases Potential of Scalable Manufacturing in Human Derived Primary Mesenchymal Stromal Cells

Manufacturing clinical grade mesenchymal stromal cells (MSCs) remains a major bottleneck for cell-based therapies, as extensive in-vitro expansion on standard tissue-culture plastic (TCP) drives loss of stemness, reduced immunomodulatory activity, and diminished therapeutic efficacy. Although substrate stiffness is known to influence MSC fate through mechanotransduction, the epigenetic mechanisms linking mechanical stress to progressive phenotypic drift remain poorly defined. A mechano-epigenetic pathway is identified in this work, centered on the histone methyltransferase EZH2 - that governs chromatin remodeling and loss of stemness during serial passaging. Multi-omics, high-resolution imaging, and functional assays show that MSCs expanded on mechanically stiff TCP accumulate H3K27me3 repressive chromatin mark, lose SWI/SNF -ARID1A chromatin- remodeling foci, and exhibit an altered chromatin accessibility profile. Pharmacological inhibition of EZH2 with GSK343 selectively reduced H3K27me3, restored ARID1A-containing SWI/SNF organization, and preserved MSC morphology and expression of canonical stemness markers (CD73, CD90, CD105) even at later passage. ATAC-seq analysis revealed that GSK343 rebalanced chromatin accessibility, reopening TEAD/YAP-responsive regulatory regions while repressing accessibility at lineage- priming and senescence-associated sites. RNA-seq demonstrated that GSK343 maintained transcriptional programs associated with immunomodulation, migration, and trophic signaling, while suppressing hyperproliferative and senescence-associated genes that are characteristic of late-passage MSCs. Proteomic profiling of MSC secretomes further showed that GSK343 attenuated pro-fibrotic ECM factors and senescence- linked proteins while enhancing angiogenic and reparative mediators. Functionally, conditioned media from GSK343-treated MSCs significantly increased primary chondrocyte proliferation, demonstrating preserved therapeutic potency. Together, these findings establish EZH2 as a central mediator of stiffness-induced epigenetic drift in human MSCs and demonstrate that EZH2 inhibition can maintain the stemness without compromising their expansion ability. This work provides a foundational strategy for mechano-epigenetic engineering of MSCs and highlights EZH2 inhibition as a scalable, manufacturing-compatible approach to preserve potency for regenerative medicine applications. SIGNIFIANCE STATEMENTPrimary mesenchymal stem/ stromal cells (MSC) derived from bone marrow are currently under numerous clinical trials for regenerative medicine and cancer treatment. Despite showing tremendous potential for their differentiation, trophic and immunomodulatory properties, their efficacy in clinical endpoint is limited. The traditional cell culture condition in cell culture flask limits their clinical translation by changing the MSC phenotype with serial passaging, a critical step required for harvesting a clinically appropriate number of cells. This work discovered the precise epigenetic mechanism involving histone methylation responsible for such changes in MSC phenotype. By inhibiting the histone methylation by GSK343, this study showed that MSC phenotype can be maintained over serial passaging as demonstrated by microscopy, multi-omics, and in vitro functional assays.