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Cited by 25Mesenchymal stem cell researchExtracellular vesicles in diseaseBone Tissue Engineering Materials

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Epigenetic Reprogramming via Synergistic Hypomethylation and Hypoxia Enhances the Therapeutic Efficacy of Mesenchymal Stem Cell Extracellular Vesicles for Bone Repair

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Man, Kenny, Brunet, Mathieu Y., Lees, Rebecca, Peacock, Ben, Cox, Sophie C.·2023·International Journal of Molecular Sciences
DOI 10.3390/ijms24087564PMC PMC10142722
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Abstract

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.

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Figure 1 Schematic representation of the investigation of the effects of hypomethylation and hypoxia on the therapeutic potency of hBMSC-derived EVs for bone repair. (A) The influence of AZT and DFO on cell viability and epigenetic function was assessed. (B) The influence of AZT, DFO or AZT/DFO treatment on hBMSCs osteogenic differentiation evaluated by quantifying ALP activity, collagen production and calcium deposition. (C) EVs were isolated from untreated, AZT-treated, DFO-treated and AZT/DFO-treated hBMSCs and characterised in terms of their size, morphology and tetraspanin markers. (D) The effects of AZT/DFO-EVs on hBMSCs osteogenesis and HUVEC angiogenesis. Figure created with BioRender.com. (last assessed on 1 March 2023).
Figure 1 Schematic representation of the investigation of the effects of hypomethylation and hypoxia on the therapeutic potency of hBMSC-derived EVs for bone repair. (A) The influence of AZT and DFO on cell viability and epigenetic function was assessed. (B) The influence of AZT, DFO or AZT/DFO treatment on hBMSCs osteogenic differentiation evaluated by quantifying ALP activity, collagen production and calcium deposition. (C) EVs were isolated from untreated, AZT-treated, DFO-treated and AZT/DFO-treated hBMSCs and characterised in terms of their size, morphology and tetraspanin markers. (D) The effects of AZT/DFO-EVs on hBMSCs osteogenesis and HUVEC angiogenesis. Figure created with BioRender.com. (last assessed on 1 March 2023).CC BY · PubMed Central
Figure 2 The effect of AZT and DFO on the morphology and cell viability of hBMSCs. AZT and DFO caused a time–dose dependent effect on the hBMSC (A) morphology and (B,C) DNA content. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. Scale bar = 50 µm.
Figure 2 The effect of AZT and DFO on the morphology and cell viability of hBMSCs. AZT and DFO caused a time–dose dependent effect on the hBMSC (A) morphology and (B,C) DNA content. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. Scale bar = 50 µm.CC BY · PubMed Central
Figure 3 The effect of AZT/DFO pre-treatment on ALP activity and epigenetic function in hBMSCs. (A) ALP activity in hBMSCs pre-treated with AZT and DFO for 24 or 48 h followed by 14 days osteoinductive culture. The effect of AZT/DFO pre-treatment on hBMSC (B) histone methylation and (C) histone acetylation levels following 3 days in basal conditions. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. ### p ≤ 0.001 vs. untreated control.
Figure 3 The effect of AZT/DFO pre-treatment on ALP activity and epigenetic function in hBMSCs. (A) ALP activity in hBMSCs pre-treated with AZT and DFO for 24 or 48 h followed by 14 days osteoinductive culture. The effect of AZT/DFO pre-treatment on hBMSC (B) histone methylation and (C) histone acetylation levels following 3 days in basal conditions. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. ### p ≤ 0.001 vs. untreated control.CC BY · PubMed Central
Figure 4 The AZT/DFO combined pre-treatment promoted osteogenic differentiation and extracellular matrix mineralisation in hBMSCs. The effects of AZT/DFO on hBMSC (A) ALP activity, (B,C) collagen production and (B,D) calcium deposition. Black staining indicates mineral nodule formation. Scale bar = 200 µm. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05 and ** p ≤ 0.01.
Figure 4 The AZT/DFO combined pre-treatment promoted osteogenic differentiation and extracellular matrix mineralisation in hBMSCs. The effects of AZT/DFO on hBMSC (A) ALP activity, (B,C) collagen production and (B,D) calcium deposition. Black staining indicates mineral nodule formation. Scale bar = 200 µm. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05 and ** p ≤ 0.01.CC BY · PubMed Central
Figure 5 The characterisation of EVs isolated from AZT/DFO-treated hBMSCs. (A) TEM images of isolated EVs. Scale bar = 50 nm. (B) Nano-flow cytometry analysis, depicting the size distribution and concentration of particles. (C) Single-particle phenotyping of hBMSC-derived EVs. EVs were fluorescently labelled with APC-conjugated antibodies specific to CD9, CD63 and CD81. Data are expressed as mean ± SD (n = 3).
Figure 5 The characterisation of EVs isolated from AZT/DFO-treated hBMSCs. (A) TEM images of isolated EVs. Scale bar = 50 nm. (B) Nano-flow cytometry analysis, depicting the size distribution and concentration of particles. (C) Single-particle phenotyping of hBMSC-derived EVs. EVs were fluorescently labelled with APC-conjugated antibodies specific to CD9, CD63 and CD81. Data are expressed as mean ± SD (n = 3).CC BY · PubMed Central
Figure 6 The effects of AZT/DFO-EVs on the general behaviour of hBMSCs. (A) EV cell uptake in hBMSCs following 16 h incubation. (B) Proliferation of hBMSCs treated with EVs after 7 days of basal culture. (C) Histone methylation levels of EV-treated hBMSCs after 3 days basal culture. (D) Histone acetylation levels of EV-treated hBMSCs after 3 days basal culture. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. Scale bar = 100 µm.
Figure 6 The effects of AZT/DFO-EVs on the general behaviour of hBMSCs. (A) EV cell uptake in hBMSCs following 16 h incubation. (B) Proliferation of hBMSCs treated with EVs after 7 days of basal culture. (C) Histone methylation levels of EV-treated hBMSCs after 3 days basal culture. (D) Histone acetylation levels of EV-treated hBMSCs after 3 days basal culture. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. Scale bar = 100 µm.CC BY · PubMed Central
Figure 7 The AZT/DFO-EV treatment promoted osteogenic differentiation and extracellular matrix mineralisation in hBMSCs. The effects of AZT/DFO-EV treatment on hBMSC (A) ALP activity, (B) collagen production, (C) calcium deposition and (D) mineral nodule coverage. (E) Picrosirius red collagen staining and Alizarin red calcium staining of EV-treated MSCs. Black staining indicates mineral nodule formation (white arrows). Scale bar = 200 µm. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
Figure 7 The AZT/DFO-EV treatment promoted osteogenic differentiation and extracellular matrix mineralisation in hBMSCs. The effects of AZT/DFO-EV treatment on hBMSC (A) ALP activity, (B) collagen production, (C) calcium deposition and (D) mineral nodule coverage. (E) Picrosirius red collagen staining and Alizarin red calcium staining of EV-treated MSCs. Black staining indicates mineral nodule formation (white arrows). Scale bar = 200 µm. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.CC BY · PubMed Central
Figure 8 Angiogenic cytokines released from EV-treated HUVECs. The pro-angiogenic cytokine release profile of HUVECs treated with hBMSC-EVs for 3 days. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05 and *** p ≤ 0.001.
Figure 8 Angiogenic cytokines released from EV-treated HUVECs. The pro-angiogenic cytokine release profile of HUVECs treated with hBMSC-EVs for 3 days. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05 and *** p ≤ 0.001.CC BY · PubMed Central
Figure 9 The schematic representation of how the mechanisms of AZT and DFO augment hypomethylation and hypoxia, enhancing the differentiation capacity of hBMSCs and the therapeutic potency of their EVs. Figure created with BioRender.com. (last assessed on 1 March 2023).
Figure 9 The schematic representation of how the mechanisms of AZT and DFO augment hypomethylation and hypoxia, enhancing the differentiation capacity of hBMSCs and the therapeutic potency of their EVs. Figure created with BioRender.com. (last assessed on 1 March 2023).CC BY · PubMed Central

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Metadata

Authors
Man, K., Brunet, M. Y., Lees, R., Peacock, B., & Cox, S. C.
Affiliations
  • University of Birmingham (GB)
  • NanoFCM Co., Ltd., Nottingham NG90 6BH, UK
Corresponding author
Cox, Sophie C.
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2023-04-20
Volume
24
Issue
8
Pages
7564
ISSN
1422-0067 (ISSN-L)
DOI
10.3390/ijms24087564
PMC
PMC10142722
OpenAlex
W4366590089
Cited by
25
References
75
Citations by year
2026: 7 · 2025: 10 · 2024: 6 · 2023: 2
FWCI
4.33
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Molecular Sciences (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • University of Birmingham Research Portal (University of Birmingham) (publishedVersion, cc-by)
Funders
EPSRC (EP/S016589/1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Physical Sciences › Engineering › Biomedical Engineering › Bone Tissue Engineering Materials
Keywords
epigenetic reprogramming, DNA hypomethylation, hypoxia, bone marrow mesenchymal stem cells, azacitidine, deferoxamine, histone acetylation, histone methylation, osteogenic differentiation, bone regeneration, angiogenesis, paracrine signaling, DNA methyltransferases
MeSH terms
Extracellular Vesicles, Hypoxia, Cells, Cultured, DNA, Histones, Humans, Osteogenesis, Cytokines, Epigenesis, Genetic, Mesenchymal Stem Cells, Human Umbilical Vein Endothelial Cells

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