RNA sequencing of Osteoblasts

We utilized RNA sequencing to provide the gene expression profile of mesenchymal stem cells (MSCs) derived osteoblasts in different differentiation stages as well as the gene alteration profile of H9 MSCs-derived osteoblasts following different gene regulation treatments, including SDC1 overexpression, knockout of CEBPD, knockout of IL1R1, and knockdown of CORIN. The commitment of stem cells to an osteoblastic lineage is a complex and tightly regulated process, involving coordination between extrinsic signals and intrinsic transcriptional machinery. While many rodent osteoblast studies abound, human osteoblastic signaling networks are not as well-researched due to limitations in cell sources and existing models. Here, we generated human pluripotent stem cell (hPSC)-derived osteoblasts and used this modeling platform to identify functional osteoblastic surface receptors and their downstream transcriptional networks involved in human osteogenesis. We systematically dissected osteoblastic gene expression patterns and identified critical clusters associated with osteogenesis. The osteoblast surface receptor signature study revealed enriched CORIN expression in osteoblasts and enriched SDC1 expression in MSCs. In vitro calcified staining and 3D biomimetic GelMA/microCT (μCT) studies demonstrated that depletion of CORIN as well as ectopic expression of SDC1 significantly impaired osteogenesis. Transcriptome analyses revealed that dysregulation of CORIN or SDC1 alters biological processes and pathways mainly involved in bone formation associated signaling including TGFβ regulating extracellular matrix and Wnt signaling. Genome-wide ChIP enrichment analysis further indicated that CEBPD is a downstream transcription factor involved in CORIN and SDC1-modulated osteogenesis. CEBPD ChIP-seq and RNA-seq validated its role in controlling extracellular matrix organization, bone mineralization, and TGFβ, BMP, and Wnt signaling. Depletion of CEBPD led to impairment of osteoblastic differentiation. Differential expression analysis of single-cell transcriptomes revealed enriched expression of CEBPD and its transcriptional targets during the different stages of osteoblast differentiation. In summary, our findings elucidated the vital signaling in osteoblast lineage commitment. OBs Differentiation: 44 samples. They are ad-HMSC, bm-HMSC, hFoB, H9, H1, RUES2, iPSC WT-1G, iPSC WT-1H, iPSC WT-1J, iPSC WT-F6 and iPSC WT-F37. For each cell line, we collect 4 samples at different differentiation stages: MSCs, pre-Osteoblasts (Pre-OBs), maturing OBs (Mat-OBs) and OBs. OE-SDC1: 4 samples. We construct TetO-SDC1 vector and generate lentiviruses expressing SDC1 in osteoblasts derived from H9 MSCs. For RNA-seq, we have 2 replicates for SDC1 overexpression (SDC1-1 and SDC1-2) and 2 samples as vector controls (SDC1-1 Ctrl and SDC1-2 Ctrl) which had only Tet-On empty vectors transduction. sgIL1R1: 4 samples. For RNA-seq, we have 2 replicates for sgIL1R1 (sgIL1R1-1 and sgIL1R1-2) and 2 replicates as vector controls (sgCtrl-1 and sgCtrl-2). sgCEBPD: 2 samples. For RNA-seq, we have 2 replicates for sgCEBPD (sgCEBPD-1 and sgCEBPD-2) and 2 replicates as vector controls (sgCtrl-1 and sgCtrl-2). shCORIN: 4 samples. For RNA-seq, we have 2 replicates for shCORIN (shCORIN-1 and shCORIN-2) and 2 replicates as vector controls (shCtrl-1 and shCtrl-2).