Co-expression networks in generation of induced pluripotent stem cells

We developed an adenoviral vector, in which Yamanaka’s four reprogramming factors (RFs) were controlled by individual CMV promoters in a single cassette (Ad-GFP-SOcMK). This permitted coordinated expression of RFs (SOX2, OCT3/4, c-MYC and KLF4) in a cell for a transient period of time, synchronizing the reprogramming process with the majority of transduced cells assuming induced pluripotent stem cell (iPSC)-like characteristics as early as 3 days. These reprogrammed cells resembled human ES cells with regard to morphology, biomarker expression, and could be differentiated into cells of the germ layers in vitro and in vivo. These iPSC-like cells, however, failed to expand into larger iPSC colonies. The short and synchronized reprogramming process allowed us to study global transcription changes within short time intervals. Weighted Gene Co-Expression Network analysis (WGCNA) identified sixteen large gene co-expression modules, each including members of gene ontology categories involved in cell differentiation and development. In particular, the brown module contained a significant number of ES cell marker genes, whereas the turquoise module contained cell-cycle related genes that were downregulated in contrast to upregulation in human ESCs. Strong coordinated expression of all 4 RFs via adenoviral transduction may constrain stochastic processes and lead to silencing of genes important for cellular proliferation. Cells were dedifferentiated and gene expression assessed at 13 timepoints