Project description:In order to assess the descendants of hypertrophic chondrocytes, we utilized Collagen10-Cre;Rosa26-tdTomato mouse bone marrow harvested at 2 months of age by centrifugation and light Collagenase II digestion. After sequencing and downstream analysis using Seurat, we observed clusters of cells with gene profiles matching classically defined skeletal stem and progenitor cells as well as CXCL12 abundant reticular (CAR) cells. These cells appear to be upstream of both osteoblasts and adipocytes. We conclude that hypertrophic chondrocytes dedifferentiate to this progentior stage before further differentiation.
Project description:In order to assess the descendants of hypertrophic chondrocytes, we utilized Collagen10-Cre;Rosa26-tdTomato mouse total bone isolated at e16.5 by Collagenase II digestion after mechanical digestion and soft tissue removal. After sequencing and downstream analysis using Seurat, we observed clusters of cells with gene profiles matching classically defined chondrocytes, skeletal stem and progenitor cells (SSPCs), and osteoblasts. Trajectory analysis reveals that the SSPCs lie intermediate to the transition of chondrocyte to osteoblast. We conclude that hypertrophic chondrocytes dedifferentiate to this progenitor stage before further differentiation.
Project description:We performed lineage tracing experiments using VE-Cadherin-Cre;LoxP-tdTomato mice. In these mice, endothelial cells (ECs) and their progeny are permanently marked by tdTomato fluorescence. We found that a substantial subset of stromal cells is derived from ECs, as indicated by their tdTomato expression. These findings support the notion that endothelial to mesenchymal transition (EndoMT) contributes to hematopoietic bone marrow niche formation in mice. Here we sought to determine the transcriptomic differences between endothelial-derived (tdTomato-positive) and non-endothelial-derived (tdTomato-negative) bone marrow stromal cells (BMSCs) and osteo/chondrolineage progenitor cells (OLCs). Murine niche populations were obtained from collagenased bone fraction of VE-Cadherin-Cre;LoxP-tdTomato mice at 3 weeks (n=2) or 11 weeks (n=2) of age. BMSCs (CD45-TER119-CD31-CD144-SCA-1+ CD51+ cells) and OLCs (CD45-TER119-CD31-CD144-Sca1-CD51+ cells) were FACS-purified and sequenced.
Project description:In order to assess the descendants of lateral plate mesoderm within the muscle interstitium, we utilized Prrx1Cre;Rosa26-tdTomato P21 tdTomato+ FACS sorted muscle interstitial cells
Project description:To assess if descendants of the hypertrophic chondrocytes behaved as skeletal stem and progenitor cells, we performed colony unit formation. We observed a near equal proportion of colonies derived from hypertrophic chondrocytes (tomato+) and derived from other sources (likely periosteum). We performed bulk RNA-sequencing on 3 colonies from each origin to compare the gene profiles and potential differentiation capacity or niche support.
Project description:The overall objective of the project is to evaluate the effects of loss of function of iron regulatory proteins (IRP1, encoded by Aco1)-1 and -2 (IRP2, encoded by Ireb2) during adult life. Mice carrying floxed Aco1 and Ireb2 alleles were crossed to a knockin strain expressing a tamoxifen-inducible CRE recombinase under the control of the Rosa26 promoter. The resulting Aco1flox/flox,Ireb2flox/flox,Rosa26+/CreERT2 mice are designated P1/2-KO; littermates lacking the CreER sequence (Aco1flox/flox,Ireb2flox/flox,Rosa26+/+) serve as reference and are designated P1/2-CTR. Adult mice were injected (i.p.) with tamoxifen on day 1 and day 3 to ablate the IRPs in the whole body, and were sacrificed on day 10 for analysis. Bone marrow cells were collected and LY6G+ cells were magnetically sorted for proteome analysis by LC-MS/MS using an Ultimate 3000 UPLC system connected to an Orbitrap Exploris 480 mass spectrometer.
Project description:We used Osx-Cre:GFP / ROSA26-loxP-stop-loxP-tdTomato mice with or without PTH1Rfl/fl. Bone marrow and bone were digested to make single cell suspension, and sorted by fluorescence. Bulk cell RNA sequencing were performed to identify specific transcripts from osterix-derived cells.