Cost-Effective Production of Biologically Active Leukemia Inhibitory Factor for Mouse Embryonic Stem Cell Culture.

Leukemia inhibitory factor (LIF), a glycoprotein in the interleukin-6 family, is essential for maintaining the pluripotency and self-renewal of mouse embryonic stem cells (mESCs) by activating the JAK/STAT pathway and preventing spontaneous differentiation. Despite its critical role in maintaining the undifferentiated state of ES cells, the high cost of commercially available LIF poses a considerable financial challenge for research laboratories. This financial strain can limit the accessibility and feasibility of high-quality stem cell research, thereby impacting the overall progress in this field. Here, we present an efficient and cost-effective method for recombinant expression and purification of biologically active, non-glycosylated mouse LIF in Escherichia coli, which retains full functionality in mESC culture. Utilizing an N-terminal glutathione S-transferase (GST) tag enhances the solubility of LIF, facilitating rapid purification via glutathione-agarose affinity chromatography. Following purification, LIF is cleaved from the GST tag using HRV 3C protease, resulting in a product ready for direct application in mESC culture. Homemade LIF (HM-LIF) prepared with this protocol effectively maintains mESCs in an undifferentiated state, showing comparable efficacy to commercial LIF. Furthermore, mESCs cultured with HM-LIF retain pluripotency and can differentiate into glutamatergic neurons. This streamlined approach significantly reduces the cost of mESC culture while providing a reliable, high-quality alternative to commercial LIF.