Utilizing Regulatory Networks for Pluripotency Assessment in Stem Cells

Pluripotency is a term in cell biology describing a unique state present in distinct stem cell lines, which were either established from the inner cell mass of the mammalian embryo or derived from somatic cells that have been reprogrammed to induced pluripotent stem cells. Pluripotent stem cells are continuously self-renewing, and their differentiation capacity enables them to develop into all derivatives of the three germ layers of a gastrulating embryo (endoderm, ectoderm, mesoderm). Both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) are virtually indistinguishable, at least based on their global RNA expression patterns. Yet, after these in vitro cell cultures have been generated, the cell lines’ pluripotent properties may change considerably on the genetic and/or epigenetic level as a consequence of long-term propagation. Among other unphysiological changes, cell lines might acquire aneuploidies, loose physiological imprinting marks, or develop differentiation biases favoring one cell lineage over the other. As a result, stem cell researchers have to continuously monitor each stem cell line’s integrity, transcriptional profile, and functional properties. Regulatory transcription factors, protein-protein interactions, and signaling networks govern the pluripotent state. As a consequence, emerging small- and large-scale perturbations to these gene regulatory networks mediate the outlined unfavorable changes to the pluripotent phenotype. Here, we describe a reliable bioinformatic framework called PluriTest for confirmation and assessment of pluripotency as an animal-free, fast, and inexpensive way based on genome-wide transcriptional RNA profiles from microarrays. Additionally, we discuss future developments using RNA expression profiling for pluripotency assessment.