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

PLOS ONE, May 2009

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.

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

et al. (2009) Manipulation of Cell:Cell Contacts and Mesoderm Suppressing Activity Direct Lineage Choice from Pluripotent Primitive Ectoderm-Like Cells in Culture. PLoS ONE 4(5): e5579. doi:10.1371/journal.pone.0005579 Manipulation of Cell:Cell Contacts and Mesoderm Suppressing Activity Direct Lineage Choice from Pluripotent Primitive Ectoderm-Like Cells in Culture James N. Hughes 0 Jennifer M. Washington 0 Zhiqiang Zheng 0 Xiuwen K. Lau 0 Charlotte Yap 0 Peter D. Rathjen 0 Joy Rathjen 0 Patrick Callaerts, Katholieke Universiteit Leuven, Belgium 0 1 School of Molecular and Biomedical Science, The University of Adelaide , Adelaide, South Australia , Australia , 2 The Australian Stem Cell Centre Monash University , Clayton, Victoria , Australia , 3 The Australian Research Council Special Research Centre for the Molecular Genetics of Development, The University of Adelaide , Adelaide, South Australia , Australia , 4 Department of Zoology, University of Melbourne , Parkville, Victoria , Australia 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. - Funding: This work was supported by the Australian Stem Cell Centre, Ground Floor, Building 75, STRIP, Monash University, Wellington Road, Clayton Victoria 3800 Australia. This funding body have had no role in the design and conduct of the study, in the collection, analysis and interpretation of the data or in the preparation and review of the manuscript. This manuscript has been approved for publication by the Australian Stem Cell Centre. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. At gastrulation in the mammal, pluripotent cells of the epiblast, or primitive ectoderm, lose pluripotency and commit to either the mesoderm/endoderm lineages or the ectoderm lineage. In the embryo, these events are spatially separated and occur in response to discrete signaling environments established in the anterior or posterior regions of the gastrula. The ability to recapitulate these events in vitro during pluripotent cell differentiation would enable directed differentiation technologies and the formation of highly enriched populations of normal, functional cells that can be used as research tools, as reagents in pharmacological trials and potentially as cellular adjuncts for the treatment of human disease. Moreover, recapitulation of a particular differentiation pathway in vitro would provide an accessible model to study the formation and subsequent differentiation of cellular intermediates. Embryonic stem cells were first isolated from the pluripotent cells of the inner cell mass of the mouse blastocyst [1,2] and retain many of the properties of this population in culture [3,4]. In comparison with embryonic development, these cells represent a population of pluripotent cells morphologically and genetically distinct from the primitive ectoderm. ES cells have been used widely as a model to understand the molecular regulation of lineage establishment from pluripotent cells in culture and by extrapolation in the embryo [5]. However, the use of ES cells to model molecular events at and around gastrulation is limited by the initial and spontaneous formation of extraembryonic endoderm concurrent with the establishment of a primitive ectodermlike cell [6,7]. Extraembryonic endoderm acts as a source of endogenous signaling molecules that regulate further differentiation from the pluripotent cells thereby confounding the interpretation of the actions of exogenously added molecules. Considerable success has been achieved with the purification of differentiating cells from ES cell-based differentiation models and subsequent manipulation in culture to define immediate postgastrulati (...truncated)


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James N. Hughes, Jennifer M. Washington, Zhiqiang Zheng, Xiuwen K. Lau, Charlotte Yap, Peter D. Rathjen, Joy Rathjen. Manipulation of Cell:Cell Contacts and Mesoderm Suppressing Activity Direct Lineage Choice from Pluripotent Primitive Ectoderm-Like Cells in Culture, PLOS ONE, 2009, 5, DOI: 10.1371/journal.pone.0005579