A system to enrich for primitive streak-derivatives, definitive endoderm and mesoderm, from pluripotent cells in culture

Two lineages of endoderm develop during mammalian embryogenesis, the primitive endoderm in the pre-implantation blastocyst and the definitive endoderm at gastrulation. This complexity of endoderm cell populations is mirrored during pluripotent cell differentiation in vitro and has hindered the identification and purification of the definitive endoderm for use as a substrate for further differentiation. The aggregation and differentiation of early primitive ectoderm-like (EPL) cells, resulting in the formation of EPL-cell derived embryoid bodies (EPLEBs), is a model of gastrulation that progresses through the sequential formation of primitive streak-like intermediates to nascent mesoderm and more differentiated mesoderm populations. EPL cell-derived EBs have been further analysed for the formation of definitive endoderm by detailed morphological studies, gene expression and a protein uptake assay. In comparison to embryoid bodies derived from ES cells, which form primitive and definitive endoderm, the endoderm compartment of embryoid bodies formed from EPL cells was comprised almost exclusively of definitive endoderm. Definitive endoderm was defined as a population of squamous cells that expressed Sox17, CXCR4 and Trh, which formed without the prior formation of primitive endoderm and was unable to endocytose horseradish peroxidase from the medium. Definitive endoderm formed in EPLEBs provides a substrate for further differentiation into specific endoderm lineages; these lineages can be used as research tools for understanding the mechanisms controlling lineage establishment and the nature of the transient intermediates formed. The similarity between mouse EPL cells and human ES cells suggests EPLEBs can be used as a model system for the development of technologies to enrich for the formation of human ES cell-derived definitive endoderm in the future.Sveltana Vassilieva, Hweee Ngee Goh, Kevin X. Lau, James N. Hughes, Mary Familari, Peter D. Rathjen and Joy Rathje

CAVE3: A general transient heat transfer computer code utilizing eigenvectors and eigenvalues

The method of solution is a hybrid analytical numerical technique which utilizes eigenvalues and eigenvectors. The method is inherently stable, permitting large time steps even with the best of conductors with the finest of mesh sizes which can provide a factor of five reduction in machine time compared to conventional explicit finite difference methods when structures with small time constants are analyzed over long time periods. This code will find utility in analyzing hypersonic missile and aircraft structures which fall naturally into this class. The code is a completely general one in that problems involving any geometry, boundary conditions and materials can be analyzed. This is made possible by requiring the user to establish the thermal network conductances between nodes. Dynamic storage allocation is used to minimize core storage requirements. This report is primarily a user's manual for CAVE3 code. Input and output formats are presented and explained. Sample problems are included which illustrate the usage of the code as well as establish the validity and accuracy of the method

Ordinal Analysis of Intuitionistic Power and Exponentiation Kripke Platek Set Theory

Until the 1970s, proof theoretic investigations were mainly concerned with theories of inductive definitions, subsystems of analysis and finite type systems. With the pioneering work of Gerhard Jäger in the late 1970 s and early 1980s, the focus switched to set theories, furnishing ordinal-theoretic proof theory with a uniform and elegant framework. More recently it was shown that these tools can even sometimes be adapted to the context of strong axioms such as the powerset axiom, where one does not attain complete cut elimination but can nevertheless extract witnessing information and characterize the strength of the theory in terms of provable heights of the cumulative hierarchy. Here this technology is applied to intuitionistic Kripke-Platek set theories IKP(P) and IKP(E), where the operation of powerset and exponentiation, respectively, is allowed as a primitive in the separation and collection schemata. In particular, IKP(P) proves the powerset axiom whereas IKP(E) proves the exponentiation axiom. The latter expresses that given any sets A and B, the collection of all functions from A to B is a set, too. While IKP(P) can be dealt with in a similar vein as its classical cousin, the treatment of IKP(E) posed considerable obstacles. One of them was that in the infinitary system the levels of terms become a moving target as they cannot be assigned a fixed level in the formal cumulative hierarchy solely based on their syntactic structure. As adumbrated in an earlier paper, the results of this paper are an important tool in showing that several intuitionistic set theories with the collection axiom possess the existence property, i.e., if they prove an existential theorem then a witness can be provably described in the theory, one example being intuitionistic Zermelo-Fraenkel set theory with bounded separation

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

Dormancy in white-grained wheat: Mechanisms and genetic control

Dissertação de Mestrado em Ecologia, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra

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

A new foundational crisis in mathematics, is it really happening?

The article reconsiders the position of the foundations of mathematics after the discovery of HoTT. Discussion that this discovery has generated in the community of mathematicians, philosophers and computer scientists might indicate a new crisis in the foundation of mathematics. By examining the mathematical facts behind HoTT and their relation with the existing foundations, we conclude that the present crisis is not one. We reiterate a pluralist vision of the foundations of mathematics. The article contains a short survey of the mathematical and historical background needed to understand the main tenets of the foundational issues.Comment: Final versio

Transwells with Microstamped Membranes Produce Micropatterned Two-Dimensional and Three-Dimensional Co-Cultures

This article describes a simple and rapid cell patterning method to form co-culture microarrays in commercially available Transwells. A thin poly(dimethylsiloxane) (PDMS) layer is printed on the underside of a Transwell using a PDMS stamp. Arbitrary cellular patterns are generated according to the geometric features of the thin PDMS layer through hydrodynamic forces that guide cells onto the membrane only over the PDMS-uncoated regions. Micropatterns of surface-adhered cells (we refer to this as two-dimensional) or non-surface-adhered clusters of cells (we refer to this as three-dimensional) can be generated depending on the surface treatment of the filter membrane. Additionally, co-cultures can be established by introducing different types of cells on the membrane or in the bottom chamber of the Transwell. We show that this co-culture method can evaluate mouse embryonic stem (mES) cell differentiation based on heterogeneous cell-cell interactions. Co-culture of mES cells and HepG2 cells decreased SOX17 expression of mES cells, and direct cell-cell contact further decreased SOX17 expression, indicating that co-culture with HepG2 cells inhibits endoderm differentiation through soluble factors and cell-cell contact. This method is simple and user-friendly and should be broadly useful to study cell shapes and cell-cell interactions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90499/1/ten-2Etec-2E2010-2E0305.pd