The Osteogenic Action Of Erythropoietin On Human Mesenchymal Stromal Cells

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1 The Osteogenic Action Of Erythropoietin On Human Mesenchymal Stromal Cells Jan H. Rolfing 1, Anette Baatrup 1, Maik Stiehler 2, Jonas Jensen 1, Helle Lysdahl 1, Cody Bünger 1. 1 Aarhus University Hospital, Aarhus, Denmark, 2 Technische Universität Dresden, Dresden, Germany. Disclosures: J.H. Rolfing: None. A. Baatrup: None. M. Stiehler: None. J. Jensen: None. H. Lysdahl: None. C. Bünger: None. Introduction: Erythropoietin (EPO) has been demonstrated to exert an osteogenic action both in cell studies and small-animal models [1-3]. The determination of a clinically safe dose that still enhances bone formation is a necessity for clinical translation. A dose-response relationship of the osteogenic potency of EPO has not yet been described. Furthermore, the way of action has not been clearly defined. Besides other ways of action, it has been reported that the advantageous effect of EPO on bone healing can possibly be attributed to an increase in cell proliferation and mineralization [2,3]. Our hypothesis was therefore that EPO elicits a dose-dependent effect on proliferation and osteogenic differentiation of human mesenchymal stromal cells (hmscs) and that this effect is mediated through its receptor EPOR or the heterodimeric EPOR/CD131 receptor which triggers intracellular signaling pathways TOR serine-threonine kinase (mtor), Janus kinase 2 (JAK2), and phosphatidylinositol 3-kinase (PI3K). Methods: Human MSCs of two, healthy adult donors were purchased from Lonza Inc. (Allendale, NJ). The cells were characterized and classified as hmscs. The cells were seeded in 96-well plates at a density of 13,500 cells/cm2 in proliferation medium (negative control), osteogenic medium (positive control) or osteogenic medium with a wide range of EPO concentrations (5, 10, 20, 50, 100 IU/ml of epoetin alpha, Eprex, Janssen-Cilag, Birkerød, Denmark). The cells were cultured for up to 21 days. The dose-response relationship and the lowest effective dose were investigated with the primary outcome evaluation Arsenazo III assay, which quantified mineralization. Other outcome evaluations were methylene blue (MB) staining assessing proliferation and alkaline phosphatase (ALP) assay assessing early osteogenic differentiation. In receptor experiments, cells were exposed to osteogenic media with or without the lowest effective dose of 20 IU/ml EPO for 24 hours. EPOR and CD131 were stained with specific conjugated antibodies (sc-697, sc3739, sc21766 FITC, Santa Cruz Biotechnology, Santa Cruz, CA) and subsequently quantified with flow cytometry and qualitatively examined with confocal microscopy. In intracellular signaling experiments, we inhibited TOR serine-threonine kinase (mtor) with rapamycin, Janus kinase 2 (JAK2) with AG490, and phosphatidylinositol 3-kinase (PI3K) with either LY or wortmannin. Proliferation medium resembled the negative control (Neg C). Mineralization was visualized and quantified after 10 and 14 days. All experiments were conducted at least twice with six technical replicates. Normally distributed data are given as mean ± standard deviation and were analyzed with one-way ANOVA and Fisher s post hoc testing against the positive control, while skewed data are reported as median (min-max) and were analyzed with Mann-Whitney test. Statistical significance is reported when p Results: A proportional dose-response relationship between EPO-concentration and the extent of mineralization was observed after 14 and 21 days. All EPO-concentrations, but 5 and 10 IU/ml, statistically significantly increased mineralization relative to the positive control (Figure 1). Thus, the lowest effective dose was 20 IU/ml. The relative effect size of the osteogenic effect of EPO on hmscs decreased from 14 to 21 days, however it was still statistically significant at 119±8% after 21 days (p=0.0096). Notably, no statistically significant difference in cell proliferation was observed (data not shown).

2 Flow cytometry showed that 26.8% ( %) of hmscs expressed EPOR, and almost all of these cells co-expressed CD % ( %). In agreement with these data confocal imaging also documented co-expression of EPOR and CD131 by bright orange staining across the entire cell surface (Figure 2A).

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4 Blocking of any of the three signaling pathways (mtor, JAK2 or PI3K) diminished mineralization after 14 days relative to the positive control (Figure 3). This observation was not confined to a certain time point or assay. On the contrary, all assays unanimously documented the inhibition of osteogenic differentiation, e.g. ALP day 2 and 7, Alizarin red day 10 and 14, and Arsenazo III day 10 and 14 (data not shown). However, none of the inhibitors completely diminished mineralization. Hence, all three intracellular signaling pathways contributed to the osteogenic effect of EPO on hmscs. Discussion: To the best of the authors knowledge the present study is the first to describe a proportional dose-dependent effect of EPO on the osteogenic differentiation of hmscs, while cell proliferation was not altered by EPO. The osteogenic effect was mediated either through EPOR or the heterodimeric EPOR/CD131 receptor and involved the intracellular signaling pathways mtor, JAK2, and PI3K. In detail, we found that the lowest effective dose of EPO on hmscs was 20 IU/ml. This observation is in line with a previous study reporting an osteogenic effect size of 175% after stimulation of hmscs with 20 IU/ml EPO for 3 weeks [3]. In our experiment, the effect size of an identical dose and observation time was lower, namely 119±8% (Figure 1). This difference may represent biological variation or disparities in the osteogenic media utilized as positive control. We observed that EPO initially boosted osteogenic differentiation, but this stimulatory effect ceased over time. For instance the magnitude of mineralization declined from 217±21% at 14 days to 138±10% at 21 days in the 100 IU/ml EPO group relative to the positive control group (Figure 1). A time-dependent effect has also been reported in a murine fracture model [4]. Regarding the cellular mechanism of the osteogenic action of EPO, both EPOR and CD131 were present on hmscs and all three signaling pathways were involved. However, the cellular mechanisms need further elucidation. The lowest effective dose of 20 IU/ml EPO could potentially be directly applied in the clinical setting, where the risk of adverse effects needs to be minimized. However, the highest-tested dose of 100 IU/ml was most effective and could therefore be applied ex vivo in order to overcome the major limitation of large threedimensional tissue engineering, namely bony ingrowth and vascularization of scaffold constructs. Significance: The pleiotropic growth factor, EPO, could potentially be used in clinical orthopedics. Either the lowest effective dose of 20 IU/ml could be applied directly, or the highest tested dose of 100 IU/ml could be utilized in tissue engineering. The determination of the individual contribution of EPOR and EPOR/CD131 and the involved intracellular signaling pathways needs further elucidation. Acknowledgments: We thank the VELUX Foundation for financial support. References: 1.Rölfing JHD, et al. Erythropoietin augments bone formation in a rabbit posterolateral spinal fusion model. J Orthop Res Jul;30(7): Holstein JH, et al. Erythropoietin stimulates bone formation, cell proliferation, and angiogenesis in a femoral segmental defect model in mice. Bone Nov;49(5): Kim J, et al. Erythropoietin mediated bone formation is regulated by mtor signaling. J. Cell. Biochem Jan;113(1): Holstein JH, et al. Erythropoietin (EPO): EPO-receptor signaling improves early endochondral ossification and mechanical strength in fracture healing. Life Sci Feb 13;80(10): ORS 2014 Annual Meeting Poster No: 1467

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