Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Temporal gene expression across osteoblastogenesis.

ABSTRACT: Purpose: Osteoblast cells mature from a mesenchymal stem cell pool to become cells capable of forming bone matrix and mineralizing this matrix. The goal of this study was to characterize temporal changes in the transcriptome across osteoblast maturation, starting with committed mesenchymal stem cell/ early pre-osteoblast stage through to mature osteoblasts capable of matrix mineralization. Methods: Enriched populations of pre-osteoblast like cells were obtained from neonatal calvaria from C57BL/6J mice expressing CFP under the control of the Col3.6 promoter. These cells were placed into culture for 4 days, removed from culture and subjected FACS sorting based on the presence/absence of CFP expression. Cells expressing CFP were returned to culture, subjected to an osteoblast differentiation cocktail and RNA was collected at 2, 4, 6, 8, 10, 12, 14, 16 and 18 days post differentiation. Methods II: mRNA profiles for each time point were generated by next generation RNA sequencing, using an Illumina HiSeq 2000. Three technical replicates per samples were sequenced. The alignments for abundance estimation of transcripts was conducted using Bowtie version 0.12.9, using the NCBIm37 reference genome. Expression level per gene was calculated using RSEM version 1.2.0 with the parameters of --fragment-length-mean 280 and --fragment-length-sd 50, and the expression level for each sample was normalized relative to the per sample upper quartile. Gene expression in calvarial osteoblasts from neonatal C57BL/6J-Col3.6 CFP mice at 9 time points post differentiation

ORGANISM(S): Mus musculus

SUBMITTER: Cheryl Ackert-Bicknell

PROVIDER: E-GEOD-54461 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description: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.

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Temporal gene expression across osteoblastogenesis.

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