Abstract
ObjectivesBone morphogenetic protein 2 (BMP2) triggers hypertrophic differentiation after chondrogenic differentiation of mesenchymal stem cells (MSCs), which blocked the further application of BMP2-mediated cartilage tissue engineering. Here, we investigated the underlying mechanisms of BMP2-mediated hypertrophic differentiation of MSCs.Materials and MethodsIn vitro and in vivo chondrogenic differentiation models of MSCs were constructed. The expression of H19 in mouse limb was detected by fluorescence in situ hybridization (FISH) analysis. Transgenes BMP2, H19 silencing, and overexpression were expressed by adenoviral vectors. Gene expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), Western blot, and immunohistochemistry. Correlations between H19 expressions and other parameters were calculated with Spearman’s correlation coefficients. The combination of H19 and Runx2 was identified by RNA immunoprecipitation (RIP) analysis.ResultsWe identified that H19 expression level was highest in proliferative zone and decreased gradually from prehypertrophic zone to hypertrophic zone in mouse limbs. With the stimulation of BMP2, the highest expression level of H19 was followed after the peak expression level of Sox9; meanwhile, H19 expression levels were positively correlated with chondrogenic differentiation markers, especially in the late stage of BMP2 stimulation, and negatively correlated with hypertrophic differentiation markers. Our further experiments found that silencing H19 promoted BMP2-triggered hypertrophic differentiation through in vitro and in vivo tests, which indicated the essential role of H19 for maintaining the phenotype of BMP2-induced chondrocytes. In mechanism, we characterized that H19 regulated BMP2-mediated hypertrophic differentiation of MSCs by promoting the phosphorylation of Runx2.ConclusionThese findings suggested that H19 regulates BMP2-induced hypertrophic differentiation of MSCs by promoting the phosphorylation of Runx2.
Highlights
Stem-cell-based and gene-enhanced tissue engineered cartilage is promising in the treatment of cartilaginous pathologies, especially traumatic cartilage defects (Bishop et al, 2017; Canadas et al, 2018; Wang et al, 2019)
With the stimulation of Bone morphogenetic protein 2 (BMP2), the highest expression level of H19 was followed after the peak expression level of Sox9; H19 expression levels were positively correlated with chondrogenic differentiation markers, especially in the late stage of BMP2 stimulation, and negatively correlated with hypertrophic differentiation markers
Our further experiments found that silencing H19 promoted BMP2-triggered hypertrophic differentiation through in vitro and in vivo tests, which indicated the essential role of H19 for maintaining the phenotype of BMP2-induced chondrocytes
Summary
Stem-cell-based and gene-enhanced tissue engineered cartilage is promising in the treatment of cartilaginous pathologies, especially traumatic cartilage defects (Bishop et al, 2017; Canadas et al, 2018; Wang et al, 2019). Mesenchymal stem cells (MSCs) hold the potential for osteogenic, chondrogenic, adipogenic differentiation, etc., owing to the fact that MSCs are easy to isolate, stable in expressing exogenous genes, abundant in source, and were identified as ideal seed cells for regenerative medicine (Canadas et al, 2018; Kim et al, 2019; Mamidi et al, 2016; Wang et al, 2019). BMP2 is known to induce MSC osteogenic differentiation and stimulate hypertrophic differentiation after chondrogenic differentiation, which go against maintaining of BMP2induced cartilage phenotype (An et al, 2010; Liao et al, 2014; Zhou et al, 2016). It is important to clarify the mechanisms underlying BMP2-mediated hypertrophic differentiation of MSCs
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