A System to Enrich for Primitive Streak-Derivatives, Definitive Endoderm and Mesoderm, from Pluripotent Cells in Culture

Svetlana Vassilieva,Hwee Ngee Goh,Kevin X Lau,Peter D Rathjen,Mary Familari,James N Hughes,Joy Rathjen

doi:10.1371/journal.pone.0038645

Svetlana Vassilieva, Hwee Ngee Goh + Show 5 more

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https://doi.org/10.1371/journal.pone.0038645

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Abstract

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.

Highlights

Endoderm is first observed in the mammalian embryo as a layer of primitive endoderm that forms across the exposed surface of the inner cell mass (ICM) from the Gata6-expressing cells within the ICM [1]
Using morphological comparison with the endoderm populations of pregastrula and gastrulating mouse embryo, gene expression and a functional protein uptake assay we have shown that the outer layer of cells in EPL-cell derived embryoid bodies (EPLEBs) comprises a layer of definitive endoderm encapsulating an inner population of mesoderm
The formation of visceral and definitive endoderm in Embryoid bodies (EB) [37,43,53], a system which lacks the spatial organisation of the embryo, has hampered unequivocal identification of endoderm populations by marker analysis

Summary

Introduction

Endoderm is first observed in the mammalian embryo as a layer of primitive endoderm that forms across the exposed surface of the inner cell mass (ICM) from the Gata6-expressing cells within the ICM [1]. This population differentiates to give extraembryonic endoderm populations: visceral endoderm, which forms from cells that remain in contact with the pluripotent cells, and parietal endoderm, which forms from cells that migrate over the inner surface of the trophectoderm [2,3]. Definitive endoderm formed at gastrulation is the progenitor population of the gut tube and associated visceral organ derivatives

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