Hyperoxia Induced Alteration of Chromatin Structure in Human Bone Marrow Derived Primary Mesenchymal Stromal Cells

Introduction: : Chromatin in eukaryotic nuclei is a highly dynamic structure that responds to environmental cues, yet the effects of oxidative stress—particularly hyperoxia—on chromatin architecture remain poorly understood. Hyperoxia, characterized by elevated reactive oxygen species (ROS), leads to DNA damage and functional decline in various cell types and is a key contributor to aging and tissue degeneration. Mesenchymal stem cells (MSCs), which are central to regenerative therapies, are especially susceptible to ROS-induced senescence when exposed to oxidative microenvironments both during in vitro expansion and after in vivo transplantation into injured, inflamed tissues.
This project addresses a critical knowledge gap by investigating how hyperoxic stress influences chromatin organization in primary MSCs. We hypothesize that oxidative stress drives chromatin condensation, impairing genome function and cell potency. To test this, we employed a novel nanoscale chromatin imaging platform in combination with ATAC-seq to visualize chromatin remodeling under hyperoxic conditions. Additionally, we evaluated the therapeutic potential of epigenetic modulation via pharmacologic inhibition of EZH2, a key histone methyltransferase, using GSK126 to mitigate stress-induced chromatin compaction and DNA damage.
Our findings uncover a mechanistic link between oxidative stress, chromatin architecture, and epigenetic regulation in MSCs. These insights not only advance our fundamental understanding of nuclear stress responses but also open new avenues for enhancing the resilience of stem cell-based therapies. Ultimately, this work has the potential to inform the development of targeted interventions to preserve MSC function in degenerative disease and inflammatory injury—conditions where oxidative stress is a central pathological feature.

Materials and

Methods: : Cell culture and treatment: Primary human bone marrow-derived MSCs were obtained from Lonza and cultured on tissue culture plastic. Passage 4 cells were treated with the EZH2 inhibitor GSK126 (20 µM) 24 hours post-seeding. After four days of drug treatment, cells were exposed to 500 µM H₂O₂ to model hyper-oxidative stress in tissues and synovial joints where MSC are typically administered.
Live-Cell Imaging: To assess chromatin dynamics in real time, nuclei were stained with NucBlue (live DNA dye), and time-lapse imaging was performed immediately after H₂O₂ exposure using a Zeiss LSM 980 laser-scanning confocal microscope (63× oil objective). A custom image registration algorithm was developed and validated to compute a Chromatin Remodeling Index (CRI, in pixels) per nucleus, generating spatiotemporal maps of chromatin condensation.
ATAC-Seq: Following treatments, cells were trypsinized and processed for ATAC sequencing (Novogene). Peak calling and quantification of differentially accessible chromatin regions were performed. Shared and unique peaks between conditions were identified. Differential accessibility was defined by |log₂ fold change|>0.5 and mean normalized count>5, given the absence of biological replicates.
Immunofluorescence: Cells were fixed, permeabilized, blocked, and stained with antibodies targeting relevant nuclear or DNA damage markers. Imaging was conducted using confocal or super-resolution modes on a Zeiss LSM 980 microscope.
Statistical Analysis: Comparisons between two groups used Student’s t-test. Multi-group comparisons were evaluated using one-way ANOVA followed by Tukey’s post-hoc test, with significance set at p< 0.05. Where appropriate, error bars represent standard deviation. Number of technical or biological replicates are reported in the figure caption.

Results, Conclusions, and Discussions:: Live-cell imaging revealed that hydrogen peroxide (H₂O₂)-induced oxidative stress triggered active chromatin remodeling in primary human MSCs, as quantified by increased Chromatin Remodeling Index (CRI) values (Fig. 1A). Pre-treatment with the EZH2 inhibitor GSK126 significantly attenuated this remodeling response. Euchromatin-rich regions exhibited more persistent CRI changes, suggesting hyperoxia-induced chromatin condensation, particularly in transcriptionally active zones (Fig. 1B). Immunofluorescence staining of H3K9me3, a marker of constitutive heterochromatin, showed elevated levels in H₂O₂-treated controls but was markedly reduced with GSK126 pre-treatment, further supporting the role of EZH2 in mediating stress-induced chromatin compaction (Fig. 1C).
ATAC-seq analysis corroborated these findings. The H₂O₂ group exhibited a loss of chromatin accessibility, with significantly fewer unique peaks compared to control and GSK126-treated cells (Fig. 2A). A heatmap of the top 1000 peaks demonstrated global chromatin closure in the H₂O₂ group (blue signal), while the GSK126 group showed intermediate restoration of open chromatin (Fig. 2B). Differential peak analysis showed that H₂O₂ exposure led to a predominance of downregulated peaks (1,181 down vs. 277 up), while GSK126 pre-treatment reversed this trend (301 up vs. 115 down).
Quantification of DNA damage via γ-H2AX intensity revealed a ~25% reduction following GSK126 pre-treatment (Fig. 3A,B). Super-resolution imaging demonstrated fewer but larger γ-H2AX foci in GSK126-treated cells, indicating spatial consolidation of DNA lesions, suggestive of altered DNA repair dynamics (Fig. 3C–E). H₂O₂ treatment possibly results in DNA damage lesions. When the chromatin condenses, they stay separated, thus making small number of many lesions. On GSK126 pretreatment the DNA damage lesions coalesce in the open chromatin.
Collectively, these data demonstrate that hyperoxic stress drives chromatin condensation and functional impairment in MSCs, while EZH2 inhibition mitigates these effects. We have data to further show that GSK126 treatment does not change the MSC functionality and phenotype (not shown for brevity). This work establishes a live-cell imaging and sequencing-based framework for real-time chromatin remodeling analysis under oxidative stress. By identifying epigenetic modulation as a protective strategy, our findings offer critical mechanistic insight with translational implications—paving the way for enhancing MSC resilience in oxidative microenvironments commonly encountered in aging, inflammation, and regenerative medicine applications.

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