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Comparative Study
. 2012;7(6):e38645.
doi: 10.1371/journal.pone.0038645. Epub 2012 Jun 11.

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

Svetlana Vassilieva  1 , Hwee Ngee Goh, Kevin X Lau, James N Hughes, Mary Familari, Peter D Rathjen, Joy Rathjen
Affiliations
Comparative Study

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

Svetlana Vassilieva et al. PLoS One. 2012.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Posterior primitive ectoderm and primitive streak markers are expressed in differentiating EPL cells.
A. q PCR analysis of RNA isolated from ES cells, EPL cells cultured for 2 days in MEDII (EPL) and EPLEBs formed from EPL cells and cultured for 4 days for the expression of Brachyury, Mixl1, Eomesodermin, and Nanog. Gene expression has been normalised to actin and is expressed relative to EPLEB2 (Brachyury, Mixl1, Eomesodermin) or ES cells (Nanog). n = 3. Error bars represent standard error of the mean. B. RNA was extracted from EPLEBs and EBMs on day 12 and analysed by RT-PCR for the expression of a number of genes characteristic of definitive endoderm cell lineages. Reactions in which reverse transcriptase has been omitted (no RT) were included as controls.
Figure 2
Figure 2. Formation of endoderm on EBs, EPLEBs, EBMs.
A–F. Scanning electron micrographs of EBs (A, D), EBMs (B, E) and EPLEBs (C, F) on days 2.5 (A–C) and 5 (D–F). Arrowheads mark the boundary of the prospective patch of primitive endoderm on EBs on day 2.5 (A) and the prospective endoderm population forming on the surface of EPLEBs at 2.5 days of differentiation (C). The dotted line on D demarcates the boundary of two distinct surface morphologies. Size bars represent 50 μm.
Figure 3
Figure 3. Characterisation of the endoderm populations seen in EBs and EPLEBs by comparison with the endoderm populations of the 6.5 and 7.5 d.p.c. embryo.
A. (i) A 1 μm transverse section across the distal tip of a 6.5 d.p.c mouse embryo showing the distinctive morphology of the visceral endoderm (ve) surrounding the inner pluripotent cell core (ep). Size bar represents 40 μm. (ii) 1 μm transverse section across the extraembryonic region of a 7.5 d.p.c embryo showing the visceral endoderm (ve) surrounding the extraembryonic mesoderm (eem) and extraembryonic ectoderm (eee). Parietal endoderm (pa) is indicated by an arrow. (iii) Transmission electron micrographs (TEM) of visceral endoderm from the extraembryonic region of a 7.5 d.p.c. embryo, showing the typical cuboidal cell morphology with large apical vacuoles and dense microvilli on the apical surface, which can be seen at the top of the figure. Size bar represents 10 μm. B. (i) Longitudinal section of a 7.5 d.p.c. late-streak stage embryo. Size bar represents 200 μm, posterior to the right, parietal endoderm (pa) indicated by an arrow. (ii) Detail of (i), showing the trilaminar structure of the egg cylinder, with an outer layer of definitive endoderm (de), middle layer of mesoderm (m) and inner layer of ectoderm (ec) Size bar represents 40 μm. (iii, iv) TEM of definitive endoderm, showing an outer, squamous, cell layer of endoderm with a sparse decoration of microvilli on the apical surface. Inset shows the surface of the cells at a higher magnification. Size bars represent 10 μm (inset 2 μm). (v, vi) TEM of parietal endoderm, showing a dispersed, squamous, cell population in close contact with Reichart's membrane (rm), indicated by an arrow. The surface of the parietal endoderm is devoid of microvilli. Inset shows the surface of the cells at a higher magnification. Size bars represent 10 μm (inset 2 μm). C. (i, ii) TEM of the surface populations of cells observed on a day 5 EB. Cells appear reminiscent of the visceral (i) and parietal (ii) endoderm populations of the embryo. Size bars represent 10 μm. D. (i) Toluidine blue-stained 1 μm section of an EPLEB at day 5 of differentiation. Size bar represents 200 μm. (ii) Detail of (i) showing the squamous outer cell layer. (iii, iv) TEM of the outer layer of cells of EPLEB on day 5 of differentiation, showing an outer, squamous, cell layer with sparse microvilli on the apical surface, reminiscent of the morphology of the embryonic definitive endoderm. Size bars represent 10 μm (iii) and 5 μm (iv).
Figure 4
Figure 4. Localisation of definitive endoderm and mesoderm marker expression in EPLEBs.
A. RNA isolated from EBs and EPLEBs on days 1–5 of differentiation was analysed by RT-PCR for the expression of Fgf5, Brachyury, Trh, Sox17, Cdx2 and Hand1. Gapdh expression was used a loading control. Expression in undifferentiated ES cells (ES) and EPL cells (EPL) is shown for comparison. -RT (control, no reverse transcriptase) and a no template control (NTC) were included. n = 3, a representative result is shown. B. Wholemount in situ hybridisation of EPLEBs on day 5 of differentiation with a DIG-labelled probe complimentary to Sox17 (i) or Trh (ii, iii, iv). Representative aggregates are shown sectioned into 10 µm slices. Size bars represent 50 µm (i, iii, iv) or 500 µm (ii). C. EBs, EBMs, EPLEBs and EPLEBs cultured in the presence of 30 ng/mL Activin A were analysed by flow cytometry for the presence of CXCR4 positive cells. n = 3. D. Low magnification image of EPLEBs (i) and EBs (ii) on day 5 and day 7 respectively, stained for the uptake of horse radish peroxidase (HRP). Open arrowheads indicate areas of staining. Representative EPLEBs with (iii) or without (iv) a foci of HRP activity are shown sectioned and counterstained with haematoxylin to illustrate cell morphology. Open arrowheads indicate areas of staining, closed arrowheads indicate the non-staining endoderm layer on the outside of the EPLEBs. Size bars represent 500 µm (i, ii,) or 50 µm (iii, iv). E. Wholemount in situ hybridisation of EPLEBs on day 5 of differentiation using a DIG-labelled probe complimentary to Cdx2 (i) or Hand1 (ii, iii). Representative aggregates are shown sectioned into 10 µm slices. Size bars represent 50 µm.
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