Mesenchymal stem cells (MSCs) are promising candidates for regenerative therapy due to their self-renewal capability, multilineage differentiation potential, and immunomodulatory effects. The molecular characteristics of MSCs are influenced by their location. Recently, epidural fat (EF) and EF-derived MSCs (EF-MSCs) have garnered attention due to their potential benefits to the spinal microenvironment and their high expression of neural SC markers. However, their clinical applications are limited due to cell senescence and limited accessibility of EF. Although many studies have attempted to establish an immortalized, stable SC line, the characteristics of immortalized EF-MSCs remain to be clarified.

To establish and analyze stable immortalized EF-MSCs.

The phenotypes of EF-MSCs were analyzed using optical microscopy. Cell immortalization was performed using lentiviral vectors. The biomolecular characteristics of the cells were analyzed by immunoblotting, quantitative PCR, and proteomics.

The immortalized EF-MSCs demonstrated a significantly extended lifespan compared to the control group, with well-preserved adipogenic potential and SC surface marker expression. Introduction of human telomerase reverse transcriptase genes markedly increased the lifespan of EF-MSCs. Proteomics analysis revealed substantial increase in the expression of DNA replication pathway components in immortalized EF-MSCs.

Immortalized EF-MSCs exhibited significantly enhanced proliferative capacity, retained adipogenic potential, and upregulated the expression of DNA replication pathway components.

Epidural fat contains a population of mesenchymal progenitor cells (MPCs), and this study explores the behavior of these cells on the adjacent dura mater during growth and in response to injury in a p21 knockout mouse model. p21^-/- mice are known to have increased cell proliferation and enhanced tissue regeneration post-injury. Therefore, it is hypothesized that the process by which epidural fat MPCs maintain the dura mater can be accelerated in p21^-/- mice. Using a Prx1 lineage tracing mouse model, the epidural fat MPCs are found to increase in the dura mater over time in both C57BL/6 (p21^+/+ ) and p21^-/- mice; however, by 3 weeks post-tamoxifen induction, few MPCs are observed in p21^-/- mice. These endogenous MPCs also localize to dural injuries in both mouse strains, with MPCs in p21^-/- mice demonstrating increased proliferation. When epidural fat MPCs derived from p21^-/- mice are transplanted into dural injuries in C57BL/6 mice, these MPCs are found in the injury site. It is demonstrated that epidural fat MPCs play a role in dural tissue maintenance and are able to directly contribute to dural injury repair. This suggests that these MPCs have the potential to treat injuries and/or pathologies in tissues surrounding the spinal cord.

Mesenchymal progenitor cells (MPCs) have been recently identified in human and murine epidural fat and have been hypothesized to contribute to the maintenance/repair/regeneration of the dura mater. MPCs can secrete proteoglycan 4 (PRG4/lubricin), and this protein can regulate tissue homeostasis through bio-lubrication and immunomodulatory functions. MPC lineage tracing reporter mice (Hic1) and human epidural fat MPCs were used to determine if PRG4 is expressed by these cells in vivo. PRG4 expression co-localized with Hic1⁺ MPCs in the dura throughout skeletal maturity and was localized adjacent to sites of dural injury. When Hic1⁺ MPCs were ablated, PRG4 expression was retained in the dura, yet when Prx1⁺ MPCs were ablated, PRG4 expression was completely lost. A number of cellular processes were impacted in human epidural fat MPCs treated with rhPRG4, and human MPCs contributed to the formation of epidural fat, and dura tissues were xenotransplanted into mouse dural injuries. We have shown that human and mouse MPCs in the epidural/dura microenvironment produce PRG4 and can contribute to dura homeostasis/repair/regeneration. Overall, these results suggest that these MPCs have biological significance within the dural microenvironment and that the role of PRG4 needs to be further elucidated.

Extracellular vesicles (EVs) are generated and secreted by cells into the circulatory system. Stem cell-derived EVs have a therapeutic effect similar to that of stem cells and are considered an alternative method for cell therapy. Accordingly, research on the characteristics of EVs is emerging. EVs were isolated from human epidural fat-derived mesenchymal stem cells (MSCs) and human fibroblast culture media by ultracentrifugation. The characterization of EVs involved the typical evaluation of cluster of differentiation (CD antigens) marker expression by fluorescence-activated cell sorting, size analysis with dynamic laser scattering, and morphology analysis with transmission electron microscopy. Lastly, the secreted levels of cytokines and chemokines in EVs were determined by a cytokine assay. The isolated EVs had a typical size of approximately 30-200 nm, and the surface proteins CD9 and CD81 were expressed on human epidural fat MSCs and human fibroblast cells. The secreted levels of cytokines and chemokines were compared between human epidural fat MSC-derived EVs and human fibroblast-derived EVs. Human epidural fat MSC-derived EVs showed anti-inflammatory effects and promoted macrophage polarization. In this study, we demonstrated for the first time that human epidural fat MSC-derived EVs exhibit inflammatory suppressive potency relative to human fibroblast-derived EVs, which may be useful for the treatment of inflammation-related diseases.