Spatially Organized Differentiation of Mouse Pluripotent Stem Cells on Micropatterned Surfaces
Pluripotent stem cells (PSCs) are the in vitro counterpart of the pluripotent epiblast of the mammalian embryo with the capacity to generate all cell types of the adult organism. During development, the three definitive germ layers are specified and simul
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Introduction Gastrulation is the developmental process whereby pluripotent cells within the epiblast (Epi) of the embryo differentiate and spatially organize into three embryonic (or definitive) germ layers. Studying this complex process in vivo in mammalian models presents several challenges. At the onset of gastrulation, embryos get implanted into the uterus of the mother, rendering them somewhat inaccessible. In order to study this process, embryos must be extracted and cultured ex vivo under precisely optimized conditions. At early gastrulation stages, the embryo comprises of only a few thousand cells [1], hence experimental design is constrained by the availability of embryonic material. Throughout gastrulation the embryo is vastly increasing in size while undergoing morphogenetic processes. These rapid changes in size and shape mean that imaging
Katia Ancelin and Maud Borensztein (eds.), Epigenetic Reprogramming During Mouse Embryogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 2214, https://doi.org/10.1007/978-1-0716-0958-3_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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Sophie M. Morgani and Anna-Katerina Hadjantonakis
gastrulating embryos in their entirety, at a frequency and resolution to allow for single cell tracking, while maintaining viability is difficult and requires substantial computational capacity [2]. Furthermore, it is not possible to easily and accurately manipulate multiple factors simultaneously in vivo with temporal control. As a result of these compounding factors, fundamental questions remain regarding the dynamic regulation of mammalian gastrulation. Pluripotent stem cells (PSCs) are the in vitro counterparts of the pluripotent Epi. PSCs have the potential to give rise to all cell types of the adult organism and are used as a model of in vivo development. The majority of PSC differentiation protocols generate cell fates without obvious spatial organization, but the advent of organoid systems [3] revealed that, under appropriate conditions, PSCs can self-organize in vitro as they would during embryonic development. While most organoid systems mimic (post-gastrulation) organogenesis and comprise derivatives of a single germ layer, pluripotent human embryonic stem cells (ESCs) differentiated on circular micropatterns generate organized multi-germ layer colonies [4–7] reminiscent of gastrulating embryos [8]. However, ethical restrictions [9] prevent comparisons with human embryos. Based on the human ESC micropattern differentiation, we established a mouse PSC micropattern system. Comparisons to gastrulating mouse embryos revealed that micropatterned colonies can spatially organize both posterior and anterior cell fates (Fig. 1) in a manner that temporally recapitulates aspects of in vivo development [10]. First, mouse ESCs are converted to epiblast-like cells (EpiLCs) [11, 12] (Fig. 2), akin to the early post-implantation Epi. EpiLCs are seeded onto Laminin-coated micropatterns generating uniform circular colonies that are then exposed
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