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Molecular and Functional Characterization of Gastrula Organizer Cells Derived from Human Embryonic Stem Cells

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From the April 2011 Issue of Stem Cells

Paper Commentary by Stuart P. Atkinson

The gastrula organizer, a structure first described in the amphibian by Spemann and Mangold in 1923, contains a population of organizer cells which signal the allocation of anterior versus posterior structures and axis formation in the developing vertebrate embryo. It is known that the TGFb and WNT pathways are involved in the induction of amphibian organizer cells (Crease et al) which are characterised by the presence of the Goosecoid gene. However, due to obvious ethical concerns, intimate studies of organizer cells in human have not yet been established. Now, for the first time, a study from the laboratory of Nissim Benvenisty at The Hebrew University of Jerusalem, published in the April edition of Stem Cells, demonstrates that differentiating human embryonic stem cells (hESC) can be used to recapitulate the characteristics and functions of gastrula organizer cells (Sharon et al.).

Due to the high evolutionary conservation of the function and molecular basis of the organizer cells, pathways known to affect mouse organizer formation were tested in hESCs. To this end, embryoid bodies (EBs) were formed in the presence of Activin A (TGFb pathway activator), LiCl (WNT/b-catenin pathway) and DKK1 (WNT/b-catenin pathway inhibitor) and after two days, the authors assayed for genes known to be associated with organizer cells (GSC, CER1, LIM1, HHEX, NODAL, BRACHYURY, CXCR4, CHORDIN). Q-PCR analysis demonstrated that modulation of both the TGFb and WNT/b-catenin pathways in differentiating hESCs changed the expression of such genes in a way to suggest that they are important for the generation of the organizer cells in humans.

The study continued by using GSC as a marker for human gastrula organizer cells, as was done in the original amphibian model, which confirmed the expression of GSC in a fraction of cells alongside other proteins related to the organizer cells (FOXA2, CER1, NODAL and NOGGIN) and that organizer gene expression could be highly upregulated in response to Activin A. The authors then went on to create a GSC-GFP hESC reporter line to allow for better analysis and purification of cells. Microarray analysis was used to compare GFP+ and GFP- cell populations sorted from 2-3 day differentiated EBs induced with Activin A, which uncovered 75 genes that were overexpressed in the GFP+ cell fraction, allowing identification of genes involved in the organizer cell. 9 of the 11 transcription factors found were related to the gastrula organizer (GSC, FOXA2, MIXL1, LHX1, SOX17, and EOMES amongst others); while 5 of the 12 secreted molecules and 5 of the 19 receptors were also linked. Inhibitors of the WNT/b-catenin and BMP pathways and activators of the Nodal/Activin pathways were also upregulated, further suggesting the similarity of hESC-derived GSC cells to organizer cells in other mammals. To demonstrate the functionality of the Activin A induced GSC+ cells, they were purified then transplanted into late-stage blastula Xenopus embryos. Encouragingly, this led to the formation of a secondary axis in 15 of 57 embryos, while this occurred in only 5 of 51 embryos transplanted with the GSC- cell fraction. Further work went on to show the partial formation of a secondary neural tube when GSC+ cells were injected ventrally into frog blastula-stage embryos. This strongly suggests that such GCS+ cells, or at least cells within this population, are the putative human gastrula organizer cells.

Overall, this paper represents the first report of the isolation and partial characterisation of GSC-expressing cells derived from differentiating hESCs and their comparison to the gastrula organizer cells of other vertebrates. It also may provide a practical means by which to study early development and patterning in the human embryo.

 

References

Induction of embryonic primordia by implantation of organizers from a different species. 1923.
H Spemann and H Mangold
Int. J. Dev. Biol. (2001) 45: 13-38

Cooperation between the activin and Wnt pathways in the spatial control of organizer gene expression.
Crease DJ, Dyson S, Gurdon JB.
Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4398-403.

Molecular and Functional Characterization of Gastrula Organizer Cells Derived from Human Embryonic Stem Cells.
Sharon N, Mor I, Golan-Lev T, Fainsod A, Benvenisty N.
Stem Cells. 2011 Feb 15