Abstract
Recently we have demonstrated the importance of RBPjk-dependent Notch signaling in the regulation of mesenchymal stem cell (MSC) differentiation during skeletogenesis both in vivo and in vitro. Here we further performed RBPJK loss-of-function experiments to demonstrate for the first time that RBPJK deficient MSC shows enhanced differentiation and osteogenesis acts via up-regulation of the BMP signaling. In the present study, we first compared the spontaneous and osteogenic differentiation in normal and recombination signal binding protein for immunoglobulin kappa J region (RBPJK) deficient human bone marrow-derived mesenchymal stem cells (MSCs). It was found that RBPJK highly expressed in fresh isolated MSCs and its expression was progressing down-regulated during spontaneous differentiation and even greater in osteogenic media inducted differentiation. Deletion of RBPJK in MSCs not only enhances cell spontaneous differentiation, but also significantly accelerates condition media inducted osteogenic differentiation by showing enhanced alkaline phosphatase (ALP) activity, Alizarin red staining, gene expression of Runx2, Osteopontin (OPN), Type I collagen (COL1a1) in culture. Additionally, BMP signaling responsive reporter activity and phosphor-smad1/5/8 expression were also significantly increased upon removal of RBPJK in MSCs. These data proved that inhibition of Notch signaling in MSCs promotes cell osteogenic differentiation by up-regulation of BMP signaling, and RBPJK deficient MSC maybe a better cell population for cell-based bone tissue engineering.
Highlights
Mesenchymal stem cells (MSCs) hold promise for healing of injured tissue because of their capacity to differentiate into multiple cell lines and their availability from a wide variety of sources[1]
To further confirm inhibition of the Notch pathway in culture could promote the osteoblastic differentiation in human MSCs, here we aimed to investigate the following: 1) human MSC osteogenic differentiation following removal of RBPJK via short hairpin RNA (shRNA) transduction
Lymphocyte marker CD45 and hematopoietic marker CD34 were further analyzed, Fig 1D showed both CD45 and CD34 positive populations are less than 0.5% in newly isolated human MSCs confirmed that our MSCs is not contaminated with lymphocytes and hematopoietic stem cells
Summary
Mesenchymal stem cells (MSCs) hold promise for healing of injured tissue because of their capacity to differentiate into multiple cell lines and their availability from a wide variety of sources[1]. MSCs from different tissues, including dental pulp[2], adipose tissue[3], PLOS ONE | DOI:10.1371/journal.pone.0135971. MSCs from different tissues, including dental pulp[2], adipose tissue[3], PLOS ONE | DOI:10.1371/journal.pone.0135971 August 18, 2015
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