Signalling pathway crosstalk stimulated by L-proline drives mouse embryonic stem cells to primitive-ectoderm-like cells.

The amino acid L-proline exhibits growth factor-like properties during development - from improving blastocyst development to driving neurogenesis in vitro. Addition of 400 µM L-proline to self-renewal medium drives naïve mouse embryonic stem cells (ESCs) to early primitive ectoderm-like (EPL) cells - a transcriptionally distinct primed or partially primed pluripotent state. EPL cells retain expression of pluripotency genes, upregulate primitive ectoderm markers, undergo a morphological change and have increased cell number. These changes are facilitated by a complex signalling network hinging on the Mapk, Fgfr, Pi3k and mTor pathways. Here, we use a factorial experimental design coupled with statistical modelling to understand which signalling pathways are involved in the transition between ESCs and EPL cells, and how they underpin changes in morphology, cell number, apoptosis, proliferation and gene expression. This approach reveals pathways which work antagonistically or synergistically. Most properties were affected by more than one inhibitor, and each inhibitor blocked specific aspects of the naïve-to-primed transition. These mechanisms underpin progression of stem cells across the in vitro pluripotency continuum and serve as a model for pre-, peri- and post-implantation embryogenesis.

Application of the RBBP9 Serine Hydrolase Inhibitor, ML114, Decouples Human Pluripotent Stem Cell Proliferation and Differentiation.

Retinoblastoma binding protein 9 (RBBP9) is required for maintaining the expression of both pluripotency and cell cycle genes in human pluripotent stem cells (hPSCs). An siRNA-based study from our group showed it does so by influencing cell cycle progression through the RB/E2F pathway. In non-pluripotent cells, RBBP9 is also known to have serine hydrolase (SH) activity, acting on currently undefined target proteins. The role of RBBP9 SH activity in hPSCs, and during normal development, is currently unknown. To begin assessing whether RBBP9 SH activity might contribute to hPSC maintenance, hPSCs were treated with ML114-a selective chemical inhibitor of RBBP9 SH activity. Stem cells treated with ML114 showed significantly reduced population growth rate, colony size and progression through the cell cycle, with no observable change in cell morphology or decrease in pluripotency antigen expression-suggesting no initiation of hPSC differentiation. Consistent with this, hPSCs treated with ML114 retained the capacity for tri-lineage differentiation, as seen through teratoma formation. Subsequent microarray and Western blot analyses of ML114-treated hPSCs suggest the nuclear transcription factor Y subunit A (NFYA) may be a candidate effector of RBBP9 SH activity in hPSCs. These data support a role for RBBP9 in regulating hPSC proliferation independent of differentiation, whereby inhibition of RBBP9 SH activity de-couples decreased hPSC proliferation from initiation of differentiation.

L-Proline Supplementation Drives Self-Renewing Mouse Embryonic Stem Cells to a Partially Primed Pluripotent State: The Early Primitive Ectoderm-Like Cell.

Mouse embryonic stem cells (mESCs) can be grown under a variety of culture conditions as discrete cell states along the pluripotency continuum, ranging from the least mature "ground state" to being "primed" to differentiate. Cells along this continuum are demarcated by differences in gene expression, X chromosome inactivation, ability to form chimeras and epigenetic marks. We have developed a protocol to differentiate "naïve" mESCs to a "partially primed" state by adding the amino acid L-proline to self-renewal medium. These cells express the primitive ectoderm markers Dnmt3b and Fgf5, and are thus called early primitive ectoderm-like (EPL) cells. In addition to changes in gene expression, these cells undergo a morphological change to flattened, dispersed colonies, have an increased proliferation rate, and a predisposition to neural fate. EPL cells can be used to study the cell states along the pluripotency continuum, peri-implantation embryogenesis, and as a starting point for efficient neuronal differentiation.

Modeling Mammalian Commitment to the Neural Lineage Using Embryos and Embryonic Stem Cells.

Early mammalian embryogenesis relies on a large range of cellular and molecular mechanisms to guide cell fate. In this highly complex interacting system, molecular circuitry tightly controls emergent properties, including cell differentiation, proliferation, morphology, migration, and communication. These molecular circuits include those responsible for the control of gene and protein expression, as well as metabolism and epigenetics. Due to the complexity of this circuitry and the relative inaccessibility of the mammalian embryo in utero, mammalian neural commitment remains one of the most challenging and poorly understood areas of developmental biology. In order to generate the nervous system, the embryo first produces two pluripotent populations, the inner cell mass and then the primitive ectoderm. The latter is the cellular substrate for gastrulation from which the three multipotent germ layers form. The germ layer definitive ectoderm, in turn, is the substrate for multipotent neurectoderm (neural plate and neural tube) formation, representing the first morphological signs of nervous system development. Subsequent patterning of the neural tube is then responsible for the formation of most of the central and peripheral nervous systems. While a large number of studies have assessed how a competent neurectoderm produces mature neural cells, less is known about the molecular signatures of definitive ectoderm and neurectoderm and the key molecular mechanisms driving their formation. Using pluripotent stem cells as a model, we will discuss the current understanding of how the pluripotent inner cell mass transitions to pluripotent primitive ectoderm and sequentially to the multipotent definitive ectoderm and neurectoderm. We will focus on the integration of cell signaling, gene activation, and epigenetic control that govern these developmental steps, and provide insight into the novel growth factor-like role that specific amino acids, such as L-proline, play in this process.

Embryoid Body Differentiation of Mouse Embryonic Stem Cells into Neurectoderm and Neural Progenitors.

Mouse embryonic stem cells (mESCs) are pluripotent cells capable of differentiating in vitro to form the ~200 types of cells of the developing embryo and adult, including cells of the nervous system. This makes mESCs a useful tool for studying the molecular mechanisms of mammalian embryonic development. Many protocols involving the use of growth factors and small molecules to differentiate mESCs into neural progenitors and neurons currently exist. However, there is a paucity of protocols available that recapitulate the developmental process. Our laboratory has developed a protocol to recapitulate mammalian neural lineage development by differentiating mESCs to mature neurons via intermediate cell populations observed during in vivo embryo development. This protocol uses the amino acid L-proline to direct the differentiation of mESCs, grown as embryoid bodies, into Sox1+ neurectoderm, followed by differentiation to form Nestin+, BLBP+, and NeuN+ neural cell types.