Endothelin-1 enhances the regenerative capability of human bone marrow-derived mesenchymal stem cells in a sciatic nerve injury mouse model
Introduction
Mesenchymal stem cells (MSCs) represent a promising tool for cell therapy as they can be easily isolated and cryo-stored, thus maintaining viability and regenerative capacity with low immunogenicity [1]. However, major challenges remain, including therapeutic efficacy and MSC production, which must be overcome before human MSCs (hMSCs) can be applied in regenerative medicine [[2], [3], [4]]. One of the limitations of current stem-cell-based approaches is that they require the use of substantial cell numbers [5]. Furthermore, clinical applications of hMSCs are hampered by the limited self-renewal ability of these cells in vitro and low activity in vivo [5]. However, this issue may be solved by identifying the priming factors that significantly enhance the regenerative capability of stem cells in vivo. Indeed, improved engraftment and therapeutic efficacy have been reported in MSCs primed by hypoxia, chemical agents, cytokines, or physical factors prior to application [[6], [7], [8], [9], [10], [11]].
In early passages, MSCs are small, spindle-shaped cells with a high proliferative capacity and differentiation potential [12]. During repetitive expansion in vitro, MSCs undergo so-called replicative senescence characterized by enlargement, thereby losing their self-renewal ability and differentiation potency [13,14]. Numerous studies have indicated an increased level of intracellular reactive oxygen species (ROS) as one of the several factors associated with replicative senescence. ROS accumulation plays a role in DNA/RNA modifications that lead to MSC senescence [15]. This observation supports studies aimed at reducing ROS generation, such as treatment of hBM-MSCs with antioxidants or culture under hypoxic conditions [16,17]. However, surplus factors that are mostly regulated by epigenetic phenomena are primarily involved in MSC senescence [10].
Changes in DNA methylation including down-regulation of OCT4 are also associated with replicative senescence [18,19]. Interestingly, iPSC derived MSCs were shown to have erased senescence-related DNA methylation patterns and exhibited epigenetic rejuvenation [19].
OCT4 is known to function in the maintenance of self-renewal properties and has an undifferentiated status in MSCs [20]. During repetitive cell divisions and proliferation in vitro, the expression of OCT4 is down-regulated. Certain conditions, such as serum deprivation or culture under hypoxia, increase OCT4 expression in MSCs [21,22].
Epigenetic changes can be reversed via priming [23,24]. These epigenetic modifications then affect gene expression and thus the therapeutic efficacy of hMSCs [23]. Within mammals, methylation of cytosines at DNA cytosine-phosphate-guanine (CpG) sites account for one of the primary mechanisms of epigenetic variation. Specifically, methylation of CpG sites in promoter regions leads to suppression of transcriptional activation. Furthermore, H3K4me3, H3K9me3, and H3K27me3 dynamic histone methylations were reported to be correlated with transcriptional changes [25,26]. Particularly, H3K4me3 is increasingly detected at active promoter regions and considered as an epigenetic marker of transcription activation [27]. In contrast, H3K27me3 and H3K9me3 are heterochromatin-associated signatures associated with transcriptional suppression [28].
We found that much of endothelin-1 (EDN1) was secreted from MSC clones and exhibited effective therapeutic efficacy in a rat myocardial infarction (MI) model by inducing up-regulation of cadherin 2 and vascular endothelial growth factor (VEGF) expression in MSCs [4]. Although EDN1 has been well-characterized as a 21-amino acid vaso-constricting peptide produced primarily in the endothelium and controls vascular tone [29], we examined its role in altering the epigenetic environment when added as a priming factor to hMSCs to improve their regenerative capability.
Section snippets
hBM-MSC culture and treatment
hBM-MSCs (Lonza, Basel, Switzerland) were grown in Mesenchymal Stem Cell Growth Media (MSCGM; Lonza) at 37 °C in an atmosphere with 5% CO2. hBM-MSCs at passages 7–8 were used in the experiments. The cells were treated with EDN1 (0.025 or 0.25 μg/mL, cat. no. E7764-10UG; Sigma Aldrich) for 24 h. APOBEC1 plasmid (Cat. SC303051; Origene) was transfected into the cells for 24 h using Lipofectamine 2000 and then used for analysis. To knock down APOBEC1, SMARCA4, and SMARCD2, hBM-MSCs were first
EDN1-treated hMSCs enhance neuronal outgrowth from rat spinal cord slice under ex vivo co-culture conditions
To determine the therapeutic applicability of EDN1-treated hMSCs, we performed ex vivo co-culture containing spinal cord slices from rats with naïve hMSCs or EDN1-treated hMSCs.
We evaluated neurite outgrowth by immuno-histochemical staining of neuro-filament-M (NF-M), a major component of the neuronal cytoskeleton. Pre-treatment of hMSCs with EDN1 (0.025 or 0.25 μg/mL) for 24 h induced significantly greater neuronal outgrowth of rat spinal cord slices than that exhibited by naïve hMSCs (Fig. 1a
Mechanism of improvement of the regenerative potential of hMSCs by EDN1
In the current study, hMSCs that were pre-treated with EDN1 showed significantly enhanced therapeutic ability compared with that of naïve hMSCs in in vivo regeneration of the cut sciatic nerve as well as in induction of neurite outgrowths from the spinal cord under ex vivo culture conditions. This may have been because of the paracrine effects of EDN1-primed hMSCs as well as the cellular effect, which was corroborated by the finding that a few Schwann cells were positive for human cell surface
Author contributions
Conceptualization: E.J.L. and H.K. Methodology: E.J.L., I.H., H.P., D.M., J.N.P., K.C.K., A. C., H. Y., J. L., H.P. and M.C. Data analysis: E.J.L., I.H. and H.K. Writing of the original manuscript: E.J.L. and I.H. Review and editing of the manuscript: E.J.L. and H.K. All authors read and approved the final version of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
This work was supported by the Korea Health Technology R&D Project Strategic Center of Cell and Bio Therapy [grant number HI17C2085] and Korea Research-Driven Hospital [grant number HI14C1277] through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (MHW), Republic of Korea.
The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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