Biology subjects

Kaonis, S.

Publications and source records attributed to Kaonis, S..

6 recordsLinked to original sources

bioRxiv2025

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.

bioengineering↗

bioRxiv2023

Hyperoxia Induced Chromatin Remodeling in Mesenchymal Stromal Cells

Chromatin is a highly dynamic entity of the eukaryotic cell nucleus. New evidence is emerging in support of the notion that chromatin can locally and globally rearrange itself to adapt with the cellular microenvironmental changes. Such changes include oxidative stress such as supraphysiological oxygen level, found in hyperoxia. Although it is known that hyperoxia can result in DNA damage and alterations in cell function, it is not well understood how the chromatin architecture changes under such a condition and what the functional significance of such change entails. In this work we developed an imaging-based technique to visualize and characterize nanoscale chromatin remodeling under hyperoxia, created via hydrogen peroxide treatment. We found high spatiotemporal variability of remodeling in different chromatin domains such as the euchromatin, heterochromatin and interchromatin. Chromatin remodeling was hindered by the GSK126 mediated inhibition of methyltransferase EZH2, which regulates the chromatin compaction. Epigenetic modifications and DNA damage under hyperoxia was investigated, which was found affected by the pretreatment of GSK126. The developed techniques and findings inform us with new mechanistic insights of chromatin remodeling which might lead to new intervention strategies to target genotoxic hyper-oxidative stress, which is common in degenerative diseases and aging, and for cell therapy in regenerative medicine.

biophysics↗

bioRxiv2023

Chromatin Remodeling and Epigenetic Modifications in Planar Cell Migration

The cell nucleus plays a critical role in cell migration by its deformability under forces, by acting as a piston that activates mechanosensitive channels, and also by serving as a ruler tailoring cell response to spatial constraints. Cell nuclear mechanics determine the mechanobiology of static and migrating cells. Here, we report that intranuclear chromatin architecture plays a previously unknown role during cell migration on planar substrates. By inhibiting histone deacetylation, which compacts chromatin, and a methyltransferase, which affects chromatin remodeling and chromatin compaction, we showed that cell migration speed and persistence drastically slowed wound closure efficiency in an in vitro scratch wound assay. Chromatin remodeling was visualized and quantified during cell migration, which may be intertwined with nuclear mechanics and shape. Inhibition disrupts remodeling and other unknown roles in chromatin, thus affecting the speed and persistence of cell migration. Interestingly, cytoskeletal stress fiber formation and cell shape were not visibly affected by chromatin modifications, suggesting an exclusive nuclear mechanobiological role in cell migration. These findings provide new insights into how aging and other degenerative conditions affect the plasticity of chromatin architecture and hence its effect on cell migration efficiency.

biophysics↗

bioRxiv2023

Chromatin Profiling of CBFA2T3-GLIS2 AMLs Identifies Key Transcription Factor Dependencies and BRG1 Inhibition as a Novel Therapeutic Strategy

Oncogenic fusions involving transcription factors are present in the majority of pediatric leukemias; however, the context-specific mechanisms they employ to drive cancer remain poorly understood. CBFA2T3-GLIS2 (C/G) fusions occur in treatment-refractory acute myeloid leukemias and are restricted to young children. To understand how the C/G fusion drives oncogenesis we applied CUT&RUN chromatin profiling to an umbilical cord blood/endothelial cell (EC) co-culture model of C/G AML that recapitulates the biology of this malignancy. We find C/G fusion binding is mediated by its zinc finger domains. Integration of fusion binding sites in C/G- transduced cells with Polycomb Repressive Complex 2 (PRC2) sites in control cord blood cells identifies MYCN, ZFPM1, ZBTB16 and LMO2 as direct C/G targets. Transcriptomic analysis of a large pediatric AML cohort shows that these genes are upregulated in C/G patient samples. Single cell RNA-sequencing of umbilical cord blood identifies a population of megakaryocyte precursors that already express many of these genes despite lacking the fusion. By integrating CUT&RUN data with CRISPR dependency screens we identify BRG1/SMARCA4 as a vulnerability in C/G AML. BRG1 profiling in C/G patient-derived cell lines shows that the CBFA2T3 locus is a binding site, and treatment with clinically-available BRG1 inhibitors reduces fusion levels and downstream C/G targets including N-MYC, resulting in C/G leukemia cell death and extending survival in a murine xenograft model.

cancer biology↗

bioRxiv2022

Characterization of Mechanics Driven Heterogeneity in Mesenchymal Stromal Cells

Mesenchymal stromal or stem cells (MSC) are one of the most promising candidates for a myriad of cell therapy applications because of their multipotency, trophic properties and immunomodulatory properties. Despite showing promises in numerous preclinical and clinical studies, MSC based therapy is not yet a reality for regenerative medicine due their suboptimal outcome at the clinical endpoint. Suboptimal function of MSC is often attributed to the monolayer expansion process on plastic which is a necessary condition to reach the therapeutically relevant number, and also to their response to a fibrotic environment post transplantation. In both scenarios of plastic culture and fibrotic conditions, the mechanical environment experienced by the MSC is completely different from the natural mechanical niche of the MSC. Accordingly, the role of mechanical environment has been shown to be a critical determinant of MSC gene expression and function. In this study we report that human bone marrow derived primary MSC population becomes phenotypically heterogenous when they experience an abnormal mechanical environment, compared to their native environment. Using a newly developed technique to quantify the heterogeneity, we provide the evidence of phenotypical heterogeneity of MSC through high resolution imaging and image analysis. Additionally, we provide mechanistic insight of the origin of such substrate mechanics driven heterogeneity, which is further determined by the cell-cell mechanical communication through the substrate. The outcome of this study might provide mechanism driven design principles to the molecular, cellular and tissue engineering researchers for rational design of MSC culture condition and biomaterials, thus improving their functional outcome.

bioengineering↗

bioRxiv2021

Dedifferentiation alters chondrocyte nuclear mechanics during in vitro culture and expansion

Dedifferentiation of chondrocytes during in vitro passaging before implantation, and post implantation in vivo, is a critical limitation in cartilage tissue engineering. Several biophysical features define the dedifferentiated state including a flattened cell morphology and increased stress fiber formation. However, how dedifferentiation influences nuclear mechanics, and the possible long-term implications of this state, are unknown. In this study, we investigated how chondrocyte dedifferentiation affects the mechanics of the chromatin architecture inside the cell nucleus and the gene expression of the structural proteins located at the nuclear envelope. Through an experimental model of cell stretching and a detailed spatial intranuclear strain quantification, we identified that strain is amplified and distribution of strain within the chromatin is altered under tensile loading in the dedifferentiated state. Further, using a confocal microscopy image-based finite element model and simulation of cell stretching, we found that the cell shape is the primary determinant of the strain amplification inside the chondrocyte nucleus in the dedifferentiated state. Additionally, we found that nuclear envelope proteins have lower gene expression in the dedifferentiated state suggesting a weaker nuclear envelope which can further intensify the intranuclear strain amplification. Our results indicate that dedifferentiation and altered nuclear strain could promote gene expression changes at the nuclear envelope, thus promoting further deviation from chondrocyte phenotype. This study highlights the role of cell shape on nuclear mechanics and lays the groundwork to design biophysical strategies for the maintenance and enhancement of the chondrocyte phenotype during expansion with a goal of successful cartilage tissue engineering. SIGNIFICANCEChondrocytes dedifferentiate into a fibroblast-like phenotype in a non-native biophysical environment. Using high resolution microscopy, intranuclear strain analysis, finite element method based computational modeling, and molecular biology techniques, we investigated how mechanical force causes abnormal intranuclear strain distribution in chondrocytes during the dedifferentiation process. Overall, our results suggest that the altered cell geometry aided by an altered or weakened nuclear envelope structure are responsible for abnormal intranuclear strain during chondrocyte dedifferentiation that can further deviate chondrocytes to a more dedifferentiated state.

biophysics↗