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Epigenetic reprogramming enhances the therapeutic efficacy of osteoblast-derived extracellular vesicles to promote human bone marrow stem cell osteogenic differentiation.

ABSTRACT: Extracellular vesicles (EVs) are emerging in tissue engineering as promising acellular tools, circumventing many of the limitations associated with cell-based therapies. Epigenetic regulation through histone deacetylase (HDAC) inhibition has been shown to increase differentiation capacity. Therefore, this study aimed to investigate the potential of augmenting osteoblast epigenetic functionality using the HDAC inhibitor Trichostatin A (TSA) to enhance the therapeutic efficacy of osteoblast-derived EVs for bone regeneration. TSA was found to substantially alter osteoblast epigenetic function through reduced HDAC activity and increased histone acetylation. Treatment with TSA also significantly enhanced osteoblast alkaline phosphatase activity (1.35-fold), collagen production (2.8-fold) and calcium deposition (1.55-fold) during osteogenic culture (P ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) exhibited reduced particle size (1-05-fold) (P > 0.05), concentration (1.4-fold) (P > 0.05) and protein content (1.16-fold) (P ≤ 0.001) when compared to untreated EVs. TSA-EVs significantly enhanced the proliferation (1.13-fold) and migration (1.3-fold) of human bone marrow stem cells (hBMSCs) when compared to untreated EVs (P ≤ 0.05). Moreover, TSA-EVs upregulated hBMSCs osteoblast-related gene and protein expression (ALP, Col1a, BSP1 and OCN) when compared to cells cultured with untreated EVs. Importantly, TSA-EVs elicited a time-dose dependent increase in hBMSCs extracellular matrix mineralisation. MicroRNA profiling revealed a set of differentially expressed microRNAs from TSA-EVs, which were osteogenic-related. Target prediction demonstrated these microRNAs were involved in regulating pathways such as 'endocytosis' and 'Wnt signalling pathway'. Moreover, proteomics analysis identified the enrichment of proteins involved in transcriptional regulation within TSA-EVs. Taken together, our findings suggest that altering osteoblasts' epigenome accelerates their mineralisation and promotes the osteoinductive potency of secreted EVs partly due to the delivery of pro-osteogenic microRNAs and transcriptional regulating proteins. As such, for the first time we demonstrate the potential to harness epigenetic regulation as a novel engineering approach to enhance EVs therapeutic efficacy for bone repair.

SUBMITTER: Man K

PROVIDER: S-EPMC8263905 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mesenchymal stem cells (MSCs) are a promising cell population for regenerative medicine applications, where paracrine signalling through the extracellular vesicles (EVs) regulates bone tissue homeostasis and development. MSCs are known to reside in low oxygen tension, which promotes osteogenic differentiation via hypoxia-inducible factor-1α activation. Epigenetic reprogramming has emerged as a promising bioengineering strategy to enhance MSC differentiation. Particularly, the process of hypomethylation may enhance osteogenesis through gene activation. Therefore, this study aimed to investigate the synergistic effects of inducing hypomethylation and hypoxia on improving the therapeutic efficacy of EVs derived from human bone marrow MSCs (hBMSCs). The effects of the hypoxia mimetic agent deferoxamine (DFO) and the DNA methyltransferase inhibitor 5-azacytidine (AZT) on hBMSC viability was assessed by quantifying the DNA content. The epigenetic functionality was evaluated by assessing histone acetylation and histone methylation. hBMSC mineralisation was determined by quantifying alkaline phosphate activity, collagen production and calcium deposition. EVs were procured from AZT, DFO or AZT/DFO-treated hBMSCs over a two-week period, with EV size and concentration defined using transmission electron microscopy, nanoflow cytometry and dynamic light scattering. The effects of AZT-EVs, DFO-EVs or AZT/DFO-EVs on the epigenetic functionality and mineralisation of hBMSCs were evaluated. Moreover, the effects of hBMSC-EVs on human umbilical cord vein endothelial cells (HUVECs) angiogenesis was assessed by quantifying pro-angiogenic cytokine release. DFO and AZT caused a time-dose dependent reduction in hBMSC viability. Pre-treatment with AZT, DFO or AZT/DFO augmented the epigenetic functionality of the MSCs through increases in histone acetylation and hypomethylation. AZT, DFO and AZT/DFO pre-treatment significantly enhanced extracellular matrix collagen production and mineralisation in hBMSCs. EVs derived from AZT/DFO-preconditioned hBMSCs (AZT/DFO-EVs) enhanced the hBMSC proliferation, histone acetylation and hypomethylation when compared to EVs derived from AZT-treated, DFO-treated and untreated hBMSCs. Importantly, AZT/DFO-EVs significantly increased osteogenic differentiation and mineralisation of a secondary hBMSC population. Furthermore, AZT/DFO-EVs enhanced the pro-angiogenic cytokine release of HUVECs. Taken together, our findings demonstrate the considerable utility of synergistically inducing hypomethylation and hypoxia to improve the therapeutic efficacy of the MSC-EVs as a cell-free approach for bone regeneration.

Project description:BackgroundThere is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects.MethodsEVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry.ResultsAlthough Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM.ConclusionsThis study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

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Epigenetic reprogramming enhances the therapeutic efficacy of osteoblast-derived extracellular vesicles to promote human bone marrow stem cell osteogenic differentiation.

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