L-Proline added to mouse ES cells (mESCs) promotes their differentiation to mature neural cells via a series of embryologically relevant cell types, including pluripotent primitive ectoderm, the germ layer definitive ectoderm, and neurectoderm. Confirmation of the progression through these intermediate stages to mature neurons comes from marker analysis using flow cytometry and qPCR, and morphological analysis using microscopy and video microscopy. Our results indicate L-Proline is acting as an instructive signal, rather than by a default mechanism, by (i) acutely activating a range of signalling pathways (such as mTOR and MAPK/ERK) and (ii) stimulating anaerobic metabolism with involvement of the Pentose Phosphate Pathway. Thus, a combination of L-Proline-mediated cell signalling and metabolic changes are required to induce lineage commitment in this in vitro model of embryo development. The involvement of metabolic flux in lineage commitment is consistent with the recently emerging paradigm that changes in metabolism can be an effector of differentiation rather than just a passenger to it.
In embryos themselves, L-Proline stimulates development of pre-implantation mouse embryos from the 2-cell stage to the blastocyst stage. The effect is observed in isosmotic medium in low-density culture (1 embryo/10 μL), where autocrine support is absent, and is not observed in high-density culture, where autocrine support is available. L-Proline also stimulates hatching independent of embryo culture density.
Collectively, these data show L-Proline acts in a growth-factor-like manner by activating a range of molecular mechanisms – including signalling, metabolic, and epigenetic – which presumably integrate in complex fashion to drive development. Consistent with being a growth factor, L-Proline acts at various developmental stages including blastocyst formation, hatching, and at various points along the pre-neural to neural lineage pathway. To our knowledge, L-Proline is the first of any amino acid to be shown to act in a growth-factor-like manner in any biological system.