L-proline-induced transition of mouse ES cells to a spatially distinct primitive ectoderm-like cell population primed for neural differentiation

Abstract

Naïve mouse embryonic stem cells (mESCs) derived from the preimplantation mouse blastocyst self-renew in the presence of LIF and BMP4. These cells are pluripotent, meaning they have the ability to differentiate into the ~200 cell types of the developing embryo and adult. Naïve mESCs are one discrete state along a pluripotency continuum – delimited by ground-state mESCs as the earliest cell population, followed by naïve mESCs, and with EpiSCs as the most ‘primed’ population. The amino acid L-proline has novel growth factor-like properties during development - from improving blastocyst development to driving neurogenesis. Addition of 400 μM L-proline to naïve mESCs produces a pluripotent cell population between naïve mESCs and EpiSCs. These cells, named early primitive ectoderm-like (EPL) cells, recapitulate in vivo development of the pre-implantation inner cell mass to the postimplantation primitive ectoderm. EPL cells maintain expression of the naïve marker Rex1 and upregulate expression of the primitive ectoderm genes Dnmt3b and Fgf5. This thesis identifies mechanisms underpinning L-proline-mediated differentiation to EPL cells, including a complex self-regulating signalling network involving the MAPK, Fgfr, PI3K and mTOR pathways. Statistical models were used to understand the contributions of individual signalling pathways to changes in colony morphology, cell number, gene expression, proliferation and apoptosis. Other mechanisms underpinning the naïve mESCs-to-EPL cell transition were explored, including DNA methylation, histone acetylation, proline synthesis and metabolism. In addition to expressing primed pluripotency genes, EPL cells upregulate genes associated with neurogenesis, whereas EpiSCs express genes associated with cardiovascular development. When mapped to the 7.0 dpc embryo, EPL cells and EpiSCs represent spatially distinct cell populations. This suggests that after cells transition from naïve mESCs, they diverge and are fated to become either ectoderm (from EPL cells), or mesendoderm (from EpiSCs). This thesis also explored whether L-proline plays an underappreciated role in existing neural differentiation protocols. mESCs cultured in custom N2B27 medium without L-proline had reduced selective cell death resulting in a larger yield of Sox1+ neurectoderm.