ABSTRACT: To determine lineage and differentiation relationship among skeletal cells in native environment, we performed tracking of clonal and sub-clonal mitochondrial mutations across population. Specifically, we utilized PolGD257A/D257A mice with a mitochondrial DNA “mutator” phenotype that provide a rich substrate of somatic mutations to facilitate clonal lineage tracing among skeletal cells. Here, CD24+CD29+SSCs, CD29+COPs, CD24+CAPs, CD24-CD29-cells, THY+ osteolineage cells, CD200-CD105+ adipolineage cells and CD45+ hematopoietic cells were isolated from the femurs of 3-month-old PolGD257A/D257A mice, co-labelled with both FACS and CITE-seq antibodies, and were subjected to a modified single cell ATAC-seq method for single cell sequencing of mtDNA variants. Our data establish that CD24+CD29+SSCs share a clonal lineage with adipolineage and osteolineage cells, providing the first evidence of clonal multipotency in skeletal cells and thus, establishes the direct in vivo evidence for our model of skeletal cell differentiation.
Project description:To determine lineage and differentiation relationships among skeletal cell types in their native environment, we utilized PolGD257A/D257A mice with a mitochondrial DNA “mutator” phenotype for tracking clonal and sub-clonal mitochondrial mutations across skeletal populations .
Project description:To investigate heterogeneity of CD24 osteolineage cells at the single-cell level, we performed CITE-seq analysis with the CD24 antibody on bone and marrow mesenchymal cells from mouse femurs and tibias
Project description:Recent studies have demonstrated that mitochondria can be transferred between different cell types to control metabolic homeostasis. However, whether the mitochondria transfer network occurred in the skeletal system or regulate skeletal metabolic homeostasis in vivo is not fully elucidated. Herein, we found that osteolineage cells transfer mitochondria to CD11b+ myeloid, B220+ lymphoid and hematopoietic stem and progenitor cells (HSPCs), of which monocytes/macrophages received the most transferred mitochondria. This process was inhibited by GC treatment contributing to the progression of glucocorticoid-induced osteoporosis (GIOP). Further analysis demonstrated that osteolineage cells transfer mitochondria to osteoclastic lineage cells via Miro1 mediated direct contact, and altered the glutathione metabolism, leading to the ferroptosis of osteoclastic lineage cells, thus inhibiting osteoclast activities. These findings revealed an unappreciated mechanism of mitochondrial regulation of skeletal metabolic homeostasis via mitochondria transfer and provide new insights of the mechanism of GIOP progression.
Project description:We have used SmartSeq2 to sequence single phenotypic skeletal stem cell (SSCs) populations purified via FACS from adult mouse long bones. Two putative SSC populations were isolated based on their previously reported surface marker profiles. An osteochondrogenic SSC (ocSSC, CD45-CD31-Tie2-Ter119-CD51+6C3-Thy1-CD105-) and a perivascular SSC (pvSSC, CD45-CD31-Pdgfra+Sca1+CD24+) were investigated.
Project description:Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, post-natally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.
Project description:Using a syngeneic p53 null mouse mammary gland tumor model that closely mimics human breast cancer, we have identified by limiting dilution transplantation as well as in vitro mammosphere and clonogenic assays a Lin-CD29HighCD24High subpopulation of tumor-initiating cells. Differentially expressed genes in the Lin-CD29HighCD24High mouse mammary gland tumor-initiating cell population include those involved in DNA damage response and repair, as well as genes involved in epigenetic regulation previously shown to be critical for stem cell self-renewal. Experiment Overall Design: The cells were labeled (for FACS sorting) with CD29 and CD24 antibodies, and 4 subpopulations were collected (CD29HighCD24High, CD29HighCD24Low,CD29LowCD24High, and CD29LowCD24Low) to do either in vivo transplantation or to isolate RNA from each of them for array to correlate the in vivo data. Experiment Overall Design: 12 samples (RNA of 4 subpopulations based on expression of CD29 and CD24 for each tumor of 3) were included in the identification of differentially expressed genes of tumor-initiating cells. 5 samples (RNA of subpopulations based on expression of CD29 and CD24 of normal mammary epithelial cells ) were included in the normal group analysis. Reference RNA was used to normalize all samples.
Project description:MaSC, Luminal progenitor enriched subpopulations were sorted based on CD24/CD29 expression from mammary epithelial cells of virgin female mice . (Tiede BJ et al., 2009, PLoS ONE 4(11): e8035. doi:10.1371/journal.pone.0008035), and the transcirptome profiles were determined and compared.
Project description:MaSC and luminal enriched subpopulations were sorted based on CD24/CD29 expression from mammary epithelial cells of virgin female mice . (Tiede BJ et al., 2009, PLoS ONE 4(11): e8035. doi:10.1371/journal.pone.0008035), and the microRNA profiles were determined using microRNA array and compared.
Project description:Skeletal stem cells (SSCs) reside in a 3-dimensional extracellular matrix (ECM) compartment and differentiate into multiple cell lineages, thereby controlling tissue maintenance and regeneration. Within this environment, SSCs can proteolytically remodel the surrounding ECM in response to growth factors that direct lineage commitment. In the bone tissue, type I collagen is the most abundant ECM. The dominant type I collagenolytic proteinase found in mammals is the type I transmembrane MMP, MMP14. Therefore, SSCs from Mmp14+/+ or Mmp14-/- mice were cultured in collagen hydrogels to explore the gene expression regulated by ECM remodeling. Microarrays were used to detail the gene expression underlying ECM remodeling process in SSCs.