The chondrogenic differentiation process of human mesenchymal stem cells (hMSCs) passes through multiple stages, which are carried out by various factors and their interactions. Recently, microRNAs that regulate chondrogenic differentiation have been reported. However, microRNA that regulates SRY-related high mobility group-box gene 9 (Sox9), a chondrogenic key factor, has not been identified in hMSC. In this study, we identified that microRNA-495 (miR-495) is an important regulator of hMSC chondrogenic differentiation. In our microarray, miR-495 was downregulated during transforming growth factor (TGF)-?3-induced chondrogenic differentiation of hMSCs in vitro. We found that there is an miR-495 binding site in the 3' untranslated region (3'UTR) of Sox9. We confirmed opposite expression between miR-495 and Sox9 by using real-time polymerase chain reaction. Further, overexpression of miR-495 inhibited Sox9 expression, and repression of miR-495 increased expression of Sox9 in SW1353 cells and hMSCs. Additionally, luciferase analysis revealed that miR-495 directly binds to the Sox9 3'UTR, and we confirmed a seed sequence of miR-495 on the Sox9 3'UTR. Subsequently, overexpression of miR-495 repressed the expression of the extracellular matrix (ECM) protein, such as type II collagen (Col2A1), aggrecan, and proteoglycan products, whereas inhibition of miR-495 increased their expression. Collectively, this study indicates that miR-495 directly targets Sox9, ultimately leading to the regulation of chondrogenic differentiation in hMSCs.
Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a heterogeneous population of cells that differ in size and morphology. BM-MSCs become committed to the osteogenic lineage as senescence approaches and lose multipotency. Nevertheless, little is known about the effects of cell-cell interaction between different populations on stemness loss and lineage commitment. The current study aimed to identify mechanisms by which cell-cell interactions between heterogeneous BM-MSCs affect stemness and lineage commitment of multipotent subpopulation. The lineage commitment of primitive multipotent cells was strongly induced in the presence of cytokines secreted by senescent-like cells in a cell culture insert system. Senescent-like cells secreted higher levels of interleukin-6 (IL-6) than primitive multipotent cells in a human cytokine array. IL-6 induced the lineage commitment and stemness loss in multipotent cells by decreasing Sox2 expression. Furthermore, we confirmed that IL-6 decreased the transcriptional activity of Sox2 through up-regulation of Runx2 and Dlx5. We suggest a mechanism by which IL-6 modulates the expression of Sox2, resulting in decreased multipotency and causing primitive multipotent cells to undergo osteogenic lineage commitment. This is the first study to identify mechanisms in which the cell-cell interactions between the different populations play important roles in the stemness loss and lineage commitment of multipotent populations.-Yoon, D. S., Kim, Y. H., Lee, S., Lee, K.-M., Park, K. H., Jang, Y., Lee, J. W. Interleukin-6 induces the lineage commitment of bone marrow-derived mesenchymal multipotent cells through down-regulation of Sox2 by osteogenic transcription factors.
microRNAs are small molecules, about 17-23 nucleotides in length, that act as translational regulators of their target gene. By binding to a target, microRNAs are known to either inhibit translation or induce degradation of the target. Despite the great interest in microRNAs, however, the exact targets of each individual microRNA in different processes remain largely unknown. In this study, we determined that the lymphoid enhancer-binding factor-1 (LEF-1) was expressed during the chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and sought to identify a novel microRNA targeting this gene. Through subsequent studies, we have identified, for the first time, one particular microRNA, miR-449a, that recognizes and regulates the expression of LEF-1 in a dose-dependent and sequence-specific manner. In addition, we observed that the inhibition of LEF-1 via miR-449a led to the subsequent repression of Sox 9, which is a well-established regulator of chondrogenesis. Collectively, this study demonstrated that miR-449a directly targets LEF-1, which in turn affects the expression of Sox 9, ultimately leading to the proper regulation of the differentiation and chondrogenesis of human MSCs (hBM-MSCs).
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.