Transcriptional analysis of human cranial compartments with different embryonic origins

Previous investigations suggest that the different embryonic origins of the calvarial tissues (neural crest or mesoderm) may account for the different molecular mechanisms underlying sutural development. The aim of this study was to evaluate the differences in the gene expression of human cranial tissues and assess the presence of an expression signature reflecting their embryonic origins. Using microarray technology, we investigated global gene expression of cells from the frontal and parietal bones and the metopic and sagittal intrasutural mesenchyme (ISM) of four human fetal calvaria. Tissues samples were obtained from fetal crania of 4 normal human fetuses (two females ages 94 and 103 days and two males ages 97 and 98 days). All dural and extracranial soft tissues were removed from the parietal and frontal bones. 2-3 mm tissue compartments were excised from frontal and parietal bones and metopic and sagittal ISM. To avoid contamination with osteoblasts, ISM tissue was dissected from the central portion of each suture. Similarly, bone was harvested at least 3mm from the margin of the suture to avoid contamination with ISM. Media was changed every 3-4 days. After reaching 75-80% confluence, the cells were trypsinized with TrypLEExpress (Life Technologies, Denmark) and passaged. During the fourth and final passage, 180,000 cells were plated in triplicate in 6-well plates and cultured for 5 days followed by RNA extraction.