Project description:ATAC-seq profiling of Nfat5 KO and wild type macrophages derived from bone marrow (primary cells), treated or not with Lipopolysaccharide (LPS).
Project description:The application of single-cell RNA sequencing (scRNAseq) to the bone field has led to significant advancements in our understanding of skeletal stem cell (SSC) heterogeneity, yet disparity remains in the characterization of these cells in the context of their native environment. To resolve these limitations, we combined scRNAseq and spatial transcriptomics in adult femurs to provide endogenous, in vivo context. First, predictive modeling was used to determine the spatial location of SSCs within the bone marrow. These results localized CXCL12-abundant reticular cells to the bone marrow, while PDGFRα+SCA1+ were found to enrich within the outer periosteum. Correlative analyses were next used to define the cellular niche composition, identifying smooth muscle cells, as well as endothelial cell and macrophage subtypes overrepresented within the SSC niche. Using cell-cell communication prediction and spatial transcriptomics, we defined ligand-receptor pairs specifically expressed within the niche and mapped their expression back to niche-resident cells. Finally, these SSC niches were placed in the context of bone microdomains. Signaling gradients derived from the vasculature and bone surfaces were unbiasedly assessed using SpatialTime. This data indicated a striking, spatially-restricted activation of metabolic and major morphogenetic pathways. This project demonstrates the ability of this technique to spatially localize SSC subpopulations, define the cellular components of the stem cell niche, unravel cell-cell communication and place this niche in the context of its bone microdomain to gain a comprehensive understanding of local and global SSC regulatory networks within the bone.
Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes. Mouse hematopoietic stem cells were purified from bone marrow cells using negative and positive selection with a Magnetic-Activated Cell Sorter (MACS). total RNA and mRNA were purified from the purified cells using Trizol reagent and magnetic oligo dT beads. Double strand cDNAs were synthesized using a cDNA synthesis kit and anchored oligo dT primers. After NlaIII digestion, 3’ cDNAs were isolated and amplified through 16-cycle PCR. SAGE tags were released from the 3’ cDNA after linker ligation. Ditags were formed, concatemerized and cloned into a pZERO vector. Sequencing reactions were performed with the ET sequencing terminator kit. Sequences were collected using a Megabase 1000 sequencer. SAGE tag sequences were extracted using SAGE 2000 software.
Project description:The bone tissue undergoes constant turnover, which relies on skeletal stem cells (SSCs) and/or mesenchymal stem cells (MSCs) and their niches. SSCs/MSCs and their perivascular niche within the bone marrow are well characterized in long bones. As for cranial bones, besides bone marrow, the suture mesenchyme has been identified as a unique niche for SSCs/MSCs of craniofacial bones. However, a comprehensive study of the two different cranial stem cell niches at single-cell resolution is still lacking. In addition, during the progression of aging, age-associated changes in cranial stem cell niches and resident cells remain uncovered. In this study, we investigated age-related changes in cranial stem cell niches via single-cell RNA sequencing (scRNA-seq).