gms | German Medical Science

Objectives: In postmenopausal or age-related osteoporosis, the osteoblastic differentiation (OD) and regenerative potential of mesenchymal stem cells (MSCs) are restricted, which may contribute to compromised bone repair. Thus, novel therapeutic strategies are needed to enhance MSC functions and promote their regenerative capacities. One promising approach is the priming of MSCs with various stimuli, such as hypoxia or different alarm signals. The aim of this study was to investigate the effect of an inflammation cocktail, which mimics the early immune response at the fracture site, on OD of MSCs.

Methods: We stimulated human bone-marrow derived MSCs with an inflammation cocktail (interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), complement component C3a and C5a, IL-1β, IL-6) for 24 h. The effect on OD was investigated on d6 by qPCR analysis and Alkaline phosphatase (ALP) staining (n=4-6/group). To identify target genes and signaling pathways, we performed RNA-seq analysis of primed MSCs and identified the immediate response 3 (IER3) gene as one of the top regulated. To validate its potential role in osteogenesis, we abrogated IER3 expression in MSCs using siRNA (siIER3), or treated MSCs with recombinant human IER3 (rhIER3). On d6 of OD, we performed ALP staining, qPCR analysis and RNA-seq analysis of both siIER3 and rhIER3-treated groups. Statistics: Student’s t-test or ANOVA/post hoc Fisher’s LSD, p<0.05.

Results and conclusion: Inflammatory priming significantly improved the OD of MSCs as shown by a significantly increased mineralization (Figure 1A, B [Fig. 1]) and osteoblastic marker gene expression, including RUNX2 (Figure 1C [Fig. 1]). Primed MSCs showed distinct changes in their transcriptome with 369 differentially regulated genes (DEGs) (Figure 1D [Fig. 1]), and modulation of various signaling pathways (Figure 1E [Fig. 1]), including the “cytokine signaling in immune system” pathway. Herein, IER3 was identified as one of the top regulated genes (Figure 1F [Fig. 1]). Notably, siRNA-mediated knockdown of IER3 reduced OD of MSCs as confirmed by a decreased ALP activity and RUNX2 expression (Figure 1G, H [Fig. 1]), while treatment with rhIER3 enhanced OD (Figure 1I, J [Fig. 1]). Transcriptomic analysis identified 499 DEGs with 42 overlapping genes between siIER3 and rhIER3-treated groups (Figure 1K [Fig. 1]). Out of these, 35 genes were differentially up- or down-regulated including the bone marrow stromal antigen 2 (BST2) (Figure 1L [Fig. 1]).

Our data showed that inflammatory priming enhanced the osteogenic differentiation of MSCs through IER3, probably dependent on BST2 expression.Thus, IER3 might offer a novel therapeutic target to enhance MSC function and bone formation, e.g., during impaired bone repair. However, further mechanistic investigations are warranted.