Single-cell RNA sequencing of mouse periosteal cells suggests that Mitochondrial Transcription Facto

Katherine Beigel1 (M.S.), Elena Sabini2 (M.D.), Dana Mitchell1 (Ph.D.), Deanne M. Taylor1,3 (Ph.D.), Ernestina Schipani2 (M.D., Ph.D.) 1The Department of Biomedical and Health Informatics, The Children's Hospital of Philadelphia, Philadelphia PA, USA 2McKay Laboratory, Department of Orthopedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA, USA 3Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA, USA

Poster # 49

Osteoblasts play a pivotal role in the process of bone formation.Oxidative phosphorylation (OxPhos) is required for osteoblastogenesis. Mitochondrial Transcription Factor A (TFAM) is an important transcription factor that regulates expression of OxPhos genes. To examine how OxPhos affects bone mass accrual, mutant mice lacking TFAM in mesenchymal progenitor cells of the limb bud and its descendants were generated using the PRX1-Cre driver (TFAM mice). Morphologically, TFAM mice had shortened long bones with increased fragility and spontaneous fractures, and the mutant mice had an increased number of osteoclasts at the diaphyseal periosteum compared to control mice. Single cell RNA (scRNA) sequencing data from the periosteal cells of two mutant TFAM mice and two normal control mice was collected. The scRNA data was analyzed in R. Data was pre-processed using SoupX to estimate and remove cell-free mRNA contamination. The two mutant samples and two control samples were processed individually using Seurat and DoubletFinder (to remove doublets). Then, the four samples were integrated in Seurat.The following cell types were identified based on examination of marker gene expression: endothelial, hematopoietic, mesenchymal, osteoblastic, and progenitor cells. We found that thenumber of osteoblastic cells was lower in TFAM mice compared to control mice. In TFAM mice, the number of mesenchymal progenitors (identified by coexpression of Sca1, PRX1, PDGFRa, and CD90) was increased. In mutant osteoblasts, there was a decrease in expression of collagen-encoding genes, enzymes involved in post-translational collagen modification, and endoplasmic reticulum chaperones. In conclusion, we utilized single-cell expression data to demonstrate the complex role played by TFAM-regulated OxPhos in the modulation of bone balance, highlighting the intricate interplay between cellular energy generation and skeletal health.