Glycolysis Combined with Core Pluripotency Factors to Promote the Formation of Chicken Induced Pluripotent Stem Cells

SIMPLE SUMMARY: Chicken embryonic fibroblasts (CEF) can be induced into iPSCs by Oct4, Sox2, Nanog and Lin28 (OSNL). However, the instability of this system leads to low induction efficiency, and the mechanism of reprogramming is still unclear. Therefore, it is vital to study the mechanism of reprogramming and optimize its induction system. In this study, we first note that glycolysis was activated during the reprogramming of CEF using the OSNL induction strategy, and that the further activation of endogenous pluripotent genes promoted the reprogramming process. An optimized OSNL reprogramming cocktail was created by introducing two small-molecule inhibitors (2i-SP), TGF-β inhibitor and MEK/ERK inhibitor, which we termed the “glycolysis activator”, which improved the efficiency of iPSC formation. This study provides an enhanced theoretical basis and suggests further areas for avian cell and embryo engineering research. ABSTRACT: Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) in vitro. Previously, a lentivirus induction strategy of introducing Oct4, Sox2, Nanog and Lin28 (OSNL) into the iPSC process has been shown as a possible way to produce chicken iPSCs from chicken embryonic fibroblasts, but the induction efficiency of this method was found to be significantly limiting. In order to help resolve this efficiency obstacle, this study seeks to clarify the associated regulation mechanisms and optimizes the reprogramming strategy of chicken iPSCs. This study showed that glycolysis and the expression of glycolysis-related genes correlate with a more efficient reprogramming process. At the same time, the transcription factors Oct4, Sox2 and Nanog were found to activate the expression of glycolysis-related genes. In addition, we introduced two small-molecule inhibitors (2i-SP) as a “glycolysis activator” together with the OSNL cocktail, and found that this significantly improved the induction efficiency of the iPSC process. As such, the study identifies direct molecular connections between core pluripotency factors and glycolysis during the chicken iPSC induction process and, with its results, provides a theoretical basis and technical support for chicken somatic reprogramming.