Reversible programming of pluripotent cell differentiation

We have undertaken an in vitro differentiation analysis of two related, interconvertible, pluripotent cell populations, ES and early primitive ectoderm-like (EPL) cells, which are most similar in morphology, gene expression, cytokine responsiveness and differentiation potential in vivo to ICM and early primitive ectoderm, respectively. Pluripotent cells were differentiated in vitro as aggregates (embryoid bodies) and the appearance and abundance of cell lineages were assessed by morphology and gene expression. Differentiation in EPL cell embryoid bodies recapitulated normal developmental progression in vivo, but was advanced in comparison to ES cell embryoid bodies, with the rapid establishment of late primitive ectoderm specific gene expression, and subsequent loss of pluripotent cell markers. Nascent mesoderm was formed earlier and more extensively in EPL cell embryoid bodies, and resulted in the appearance of terminally differentiated mesodermal cell types prior to and at higher levels than in ES cell embryoid bodies. Nascent mesoderm in EPL cell embryoid bodies was not specified but could be programmed to alternative fates by the addition of exogenous factors. EPL cells remained competent to form primitive endoderm even though this is not the normal fate of primitive ectoderm in vivo. The establishment of primitive ectoderm-like gene expression and inability to participate in embryogenesis following blastocyst injection is therefore not directly associated with restriction in the ability to form extra-embryonic lineages. However, the EPL cell embryoid body environment did not support differentiation of primitive endoderm to visceral endoderm, indicating the lack of an inductive signal for visceral endoderm formation deduced to originate from the pluripotent cells. Similarly, the inability of EPL cells to form neurons when differentiated as embryoid bodies was attributable to perturbation of the differentiation environment and loss of inductive signals rather than a restricted differentiation potential. Reversion of EPL cells to ES cells was accompanied by restoration of ES cell-like differentiation potential. These results demonstrate the ability of pluripotent cells to adopt developmentally distinct, stable cell states with altered differentiation potentials.Julie-Anne Lake, Joy Rathjen, Jackie Remiszewski and Peter D. Rathje

Embryonic stem cell differentiation and the analysis of mammalian development

© UBC PressMolecular and cellular analysis of early mammalian development is compromised by the experimental inaccessibility of the embryo. Pluripotent embryonic stem (ES) cells are derived from and retain many properties of the pluripotent founder population of the embryo, the inner cell mass. Experimental manipulation of these cells and their environment in vitro provides an opportunity for the development of differentiation systems which can be used for analysis of the molecular and cellular basis of embryogenesis. In this review we discuss strengths and weaknesses of the available ES cell differentiation methodologies and their relationship to events in vivo. Exploitation of these systems is providing novel insight into embryonic processes as diverse as cell lineage establishment, cell progression during differentiation, patterning, morphogenesis and the molecular basis for cell properties in the early mammalian embryo.Stephen J. Rodda, Steven J. Kavanagh, Joy Rathjen and Peter D. Rathje

Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development.

Formation and differentiation of a pluripotent cell population is central to mammalian development, and the isolation, identification and manipulation of human pluripotent cells is predicted to be of therapeutic use. Within the early mammalian embryo, two distinct populations of pluripotent cells have been described: the inner cell mass (ICM), which differentiates to form a second pluripotent cell populations, the primitive ectoderm. Indirect evidence suggests the existence of temporally distinct intermediate pluripotent cell populations as primitive ectoderm is formed. We coupled an in vitro model of primitive ectoderm formation (the transition of embryonic stem cells to early primitive ectoderm-like (EPL) cells) with ddPCR-based techniques to identify three novel genes, Psc1, CRTR-1 and PRCE, that were expressed differently during pluripotent cell progression. Detailed mapping of these genes with Oct4, Rex1 and Fgf5 on pregastrulation embryos provided the first molecular evidence for the existence of successive, temporally distinct pluripotent cell populations in the embryo between the ICM and primitive ectoderm. No evidence was found for spatial heterogeneity within the Oct4+ pool. The transition between populations correlated with morphological or developmental alterations in pluripotent cells in vivo. Genes that are temporally expressed during pluripotent cell progression may provide an opportunity for molecular discrimination of pluripotent cells at different stages of maturation in vivo and an understanding of the cellular origins and properties of pluripotent cell lines isolated from diverse sources. Furthermore, the strong correlation of gene expression demonstrated between EPL cell formation in vitro and primitive ectoderm formation in vivo validates EPL cells as a model for primitive ectoderm, thereby providing a model system for the investigation of pluripotent differentiation and an opportunity for directed differentiation of pluripotent cells to therapeutically useful cell populations.T. A. Pelton, S. Sharma, T. C. Schulz, J. Rathjen and P. D. Rathje

Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population

During embryogenesis the central and peripheral nervous systems arise from a neural precursor population, neurectoderm, formed during gastrulation. We demonstrate the differentiation of mouse embryonic stem cells to neurectoderm in culture, in a manner which recapitulates embryogenesis, with the sequential and homogeneous formation of primitive ectoderm, neural plate and neural tube. Formation of neurectoderm occurs in the absence of extraembryonic endoderm or mesoderm and results in a stratified epithelium of cells with morphology, gene expression and differentiation potential consistent with positionally unspecified neural tube. Differentiation of this population to homogeneous populations of neural crest or glia was also achieved. Neurectoderm formation in culture allows elucidation of signals involved in neural specification and generation of implantable cell populations for therapeutic use

Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation

Manipulation of Cell:Cell Contacts and Mesoderm Suppressing Activity Direct Lineage Choice from Pluripotent Primitive Ectoderm-Like Cells in Culture

In the mammal, the pluripotent cells of embryo differentiate and commit to either the mesoderm/endoderm lineages or the ectoderm lineage during gastrulation. In culture, the ability to direct lineage choice from pluripotent cells into the mesoderm/endoderm or ectoderm lineages will enable the development of technologies for the formation of highly enriched or homogenous populations of cells. Here we show that manipulation of cell:cell contact and a mesoderm suppressing activity in culture affects the outcome of pluripotent cell differentiation and when both variables are manipulated appropriately they can direct differentiation to either the mesoderm or ectoderm lineage. The disruption of cell:cell contacts and removal of a mesoderm suppressor activity results in the differentiation of pluripotent, primitive ectoderm-like cells to the mesoderm lineage, while maintenance of cell:cell contacts and inclusion, within the culture medium, of a mesoderm suppressing activity results in the formation of near homogenous populations of ectoderm. Understanding the contribution of these variables in lineage choice provides a framework for the development of directed differentiation protocols that result in the formation of specific cell populations from pluripotent cells in culture

Endoderm complexity in the mouse gastrula is revealed through the expression of spink3.

Endoderm formation in the mammalian embryo occurs first in the blastocyst, when the primitive endoderm and pluripotent cells resolve into separate lineages, and again during gastrulation, when the definitive endoderm progenitor population emerges from the primitive streak. The formation of the definitive endoderm can be modeled using pluripotent cell differentiation in culture. The differentiation of early primitive ectoderm-like (EPL) cells, a pluripotent cell population formed from embryonic stem (ES) cells, was used to identify and characterize definitive endoderm formation. Expression of serine peptidase inhibitor, Kazal type 3 (Spink3) was detected in EPL cell-derived endoderm, and in a band of endoderm immediately distal to the embryonic-extra-embryonic boundary in pregastrula and gastrulating embryos. Later expression marked a region of endoderm separating the yolk sac from the developing gut. In the embryo, Spink3 expression marked a region of endoderm comprising the distal visceral endoderm, as determined by an endocytosis assay, and the proximal region of the definitive endoderm. This region was distinct from the more distal definitive endoderm population, marked by thyrotropin-releasing hormone (Trh). Endoderm expressing either Spink3 or Trh could be formed during EPL cell differentiation, and the prevalence of these populations could be influenced by culture medium and growth factor addition. Moreover, further differentiation suggested that the potential of these populations differed. These approaches have revealed an unexpected complexity in the definitive endoderm lineage, a complexity that will need to be accommodated in differentiation protocols to ensure the formation of the appropriate definitive endoderm progenitor in the future

Mouse ES cells: experimental exploitation of pluripotent differentiation potential

Pluripotent ES cells can be used to generate a wide variety of cell populations in vitro in a manner resembling embryonic development. Recent advances in controlling ES cell differentiation, combined with the power of genetic and biochemical manipulation, are providing insights into cell biology and the determination of cell fate.http://www.sciencedirect.com/science/journal/0959437

Lineage specific differentiation of mouse ES cells: formation and differentiation of early primitive ectoderm-like (epl) cells

http://www.sciencedirect.com/science/bookseries/0076687