Dissecting the role of LIF in human naïve pluripotent stem cells maintenance

Pluripotent stem cells have the ability to give rise to all adult cell types and to undergo self-renewal indefinitely in vitro. Particularly, two pluripotency states have been determined both in mouse and human, which differ in their differentiation potential, signaling pathways, and cytokines required for self-renewal and proliferation: naïve, resembling the pre-implantation epiblast and primed, mimicking a more developmentally advanced state. Naive mouse stem cells (mESCs) are cultured on gelatin-coated plates in an optimized and defined medium. However, given the recent derivation of naive human pluripotent stem cells (hPSCs), their culture media are still being optimized and they expand robustly only on a layer of inactivated mouse embryonic fibroblasts (MEFs). Leukemia inhibitory factor (LIF) is a common factor of naive culture conditions. mESCs pluripotency maintenance requires LIF and the downstream activation of the JAK/STAT3 pathway, which induces the nuclear transcription of naive genes and promotes genomic hypomethylation by increasing mitochondria α-Ketoglutarate production. However, it is not yet clear if LIF dependency is conserved in the human counterpart even if this cytokine is present in all the naive hPSCs media. In this project on naive hPSCs, I observed the effects on cell morphology, proliferation, and pluripotency gene expression by removing LIF from the culture medium and using different inhibitors to block the JAK/STAT3 pathway. This approach suffers from inhibitor toxicity and possible off-targets. Therefore, I set up a CRISPR interference (CRISPRi) genetic approach that allows for sequence-specific repression of gene expression. I was able to create a stable CRISPRi-expressing line for the evaluation of the effects of JAK/STAT3 pathway inhibition. In parallel, I also cultured naive hPSCs in feeder-free conditions, testing the effects of LIF removal. In sum, I found that even though LIF plays a role in human naive maintenance, it is not as fundamental as in mESCs, suggesting that hPSCs may rely on other cytokine(s) for naive pluripotency maintenance. Regarding future applications, I am going to test the effect of Interleukin-6 (IL-6), which activates the JAK/STAT3 pathway, on both MEFs and feeder-free conditions to check if it can better stimulate human naive maintenance. Moreover, I will evaluate the effects of gp130 (LIF and IL-6 co-receptor) transcriptional repression for a global JAK/STAT3 pathway inhibition.