Development of a Reporter Cell Line for Visualizing PI3K Signaling During MSCs Adipogenic Differentiation

Abstract Objective: Several hormones and paracrine factors, such as insulin, adiponectin, leptin, and IGF-1,2, activate the phosphoinositide 3-kinase (PI3K) signaling pathway, thereby affecting the adipogenic differentiation of multipotent mesenchymal stromal cells (MSCs). This study aimed to develop a tool based on a genetically encoded PH-GFP biosensor for tracking the link between hormonal signaling and long-term cellular responses, such as cell differentiation. Materials and methods: Human telomerase-immortalized (hTERT) MSCs were transduced with a lentiviral vector carrying a sequence based on the pleckstrin homology (PH) domain of Bruton’s tyrosine kinase fused to GFP (PH-GFP). Subsequently, the reporter cell line was isolated using fluorescence-activated cell sorting. The resulting MSCGFP-PH cells were evaluated for PDGF- and insulin-induced fluorescent responses, multi-lineage differentiation potential using standard differentiation protocols, and proliferation rates utilizing neural networks for automated cell counting. Results: The established MSCGFP-PH cell line retains intact adipogenic and osteogenic potential and allows for the real-time detection of insulin and other PI3K-agonist signaling events in living cells, both in the undifferentiated state and throughout adipogenic differentiation. To streamline the detection of these signaling events, we applied a computational approach to improve signal visualization and used a special metric to assess signaling activation. Conclusions: The MSCGFP-PH cell line may be a useful tool for investigating PI3K signaling in real time during long-term cellular processes such as adipogenic differentiation. Furthermore, the implemented computational approach represents a convenient option for detecting PI3K signaling via the PH-GFP biosensor at the single-cell level.