Growing Human Dermal Fibroblasts as Spheroids Renders Them Susceptible for Early Expression of Pluripotency Genes

Suspension spheroid cultures of anchorage-dependent cell types have been widely used in cancer and stem cell research, as well as for producing organoids. It is believed that the 3-dimensional spheroid presents cells with a more physiological microenvironment to grow so that they behave more like cells in vivo, which is lacking in conventional 2-dimensional monolayer cultures. Recently, it has been reported that cancer cells grown as spheroids could express stem cell-associated genes. Hence, it is investigated whether normal mouse and human fibroblasts cultured as spheroids could also be induced to express stem cell-associated genes. The transcriptomes of human fibroblasts cultured as a monolayer and spheroids are compared and analyzed using real-time RT-qPCR, RNA-sequencing, and bioinformatics. The results reveal that the spheroid transcriptome resemble somatic cells being reprogramed into stem cells, including the induced expression of stemness-associated genes, increased expression of mesenchymal-to-epithelial transition-associated genes, and decreased expression of epithelial-to-mesenchymal transition-associated genes. In this context, it is hypothesized that during the process of spheroid formation, matrix-cell signaling is lost in favor of cell-cell contact signaling and that this subsequently increases the activity of the PI3K/Akt pathway that then upregulates Tbdx3 and stemness-associated genes.