The identification and purification of murine multipotent mesenchymal stem cells (MSCs) have been difficult due to their low frequency, the presence of contaminating cell types and lack of unambiguous markers. Using a magnetic micro-beads negative selection technique to remove hematopoietic cells from mouse bone marrow stromal cells (BMSCs), our lab recently isolated a highly purified osteoprogenitor (HipOP) population that was also enriched for other mesenchymal precursors, including MSCs [Itoh and Aubin, 2009]. We now report that HipOPs are also highly enriched in vascular endothelial cells (VECs), which we hypothesized were an accessory cell type regulating osteogenesis. However, when VECs were immunodepleted from HipOPs with anti-CD31 antibodies, the resulting CD31(-) HipOP population had equal osteogenic capacity to the HipOPs in vitro and in vivo. Analysis of gene expression of Ncad, Pth1r, Ang1, Cxcl12, Jag1, Pdgfr-?, ?-sma, Desmin, and Ng2 suggested that both HipOPs and CD31(-) HipOPs are hemopoietic stem cell (HSC) niche populations. However, the data support the view that osteoblast differentiation and depletion of VECs modulate the HSC niche.
While the role of estrogen receptor-related receptor alpha (ERR?) in chondrogenesis has been investigated, the involvement of ERR gamma (ERR?) has not been determined. To assess the effect of increased ERR? activity on cartilage development in vivo, we generated two transgenic (Tg) lines overexpressing ERR?2 via a chondrocyte-specific promoter; the two lines exhibited ?3 and ?5 fold increased ERR?2 protein expression respectively in E14.5 Tg versus wild type (WT) limbs. On postnatal day seven (P7), we observed a 4-10% reduction in the size of the craniofacial, axial and appendicular skeletons in Tg versus WT mice. The reduction in bone length was already present at birth and did not appear to involve bones that are derived via intramembranous bone formation as the bones of the calvaria, clavicle, and the mandible developed normally. Histological analysis of P7 growth plates revealed a reduction in the length of the Tg versus WT growth plate, the majority of which was attributable to a reduced proliferative zone. The reduced proliferative zone paralleled a decrease in the number of Ki67-positive proliferating cells, with no significant change in apoptosis, and was accompanied by large cell-free swaths of cartilage matrix, which extended through multiple zones of the growth plate. Using a bioinformatics approach, we identified known chondrogenesis-associated genes with at least one predicted ERR binding site in their proximal promoters, as well as cell cycle regulators known to be regulated by ERR?. Of the genes identified, Col2al, Agg, Pth1r, and Cdkn1b (p27) were significantly upregulated, suggesting that ERR?2 negatively regulates chondrocyte proliferation and positively regulates matrix synthesis to coordinate growth plate height and organization.
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.