Generating Human Gastruloids from Human Embryonic Stem Cells

Abstract

Gastruloids are aggregates of Pluripotent Stem Cells (PSCs) which, when exposed to differentiation medium and plated within defined conditions, undergo trilineage differentiation to all three germ layers (mesoderm, ectoderm and endoderm) with constitutive cell types organised spatiotemporally along 3 axes (Becarri et. al. 2018). They also undergo morphological shape changes including axial elongation through convergent extension cell movements. The gastruloid method has been well-established using mouse Embryonic Stem Cells (mESCs) and mouse induced PSCs (iPSCs) (van den Brink et. al., 2014; Turner et. al. 2016a; Turner et. al. 2016b; Bailie-Johnson et. al. 2015; Turner et. al. 2017a; Turner et. al. 2017b, Beccari et. al., 2018, See method in Girgin et. al., 2018). Here, we describe a new method to generate equivalent gastruloids from human pluripotent stem cells (hPSCs). hPSCs are able to generate axially elongated human gastruloids with evidence of spatially organised germ layers and comparable features to their mouse counterparts (Moris et. al., 2020).

An in vitro model of early anteroposterior organization during human development

“…Medium was exchanged for fresh E6 medium daily following this timepoint. Detailed instructions for generating human gastruloids can be found in Protocol Exchange 28 .…”

In vitro teratogenicity testing using a 3D, embryo-like gastruloid system

“…The full protocol for generating human gastruloids can be found on Protocol Exchange (DOI: 10.21203/rs.3.pex-812/v1 ) and a full characterisation of the structures has been previously described [ 15 ]. Before human gastruloid culture, cells were passaged to single cell suspension and plated to achieve 65,000 cells per well of a 6-well plate in Nutristem supplemented with 1:2,000 Y-27632 (ROCK inhibitor; Sigma Aldrich, Y0503) [ 23 ]. Adherent cultures were pre-treated in Nutristem supplemented with 3.25 μM CHIR99021 (Chiron; Tocris Biosciences, 4423) and either 0.2 % DMSO (vehicle), water (vehicle for penicillin G) or the different compound concentrations on the fourth day of adherent culture.…”

“…Images of the mouse gastruloids were then cropped and the bit-depth of the images of both human and mouse gastruloids was reduced with custom ImageJ macros (details available at https://github.com/naomi-moris/humanGastruloids_shapeDescriptors ). The 8-bit TIFF files were passed through a custom analysis pipeline, implemented in the Project Jupyter Python 3.6 distribution [ 23 ]. Input files of the mouse gastruloids were cropped to a size that favored optimal performance of the computer vision methods used by the pipeline.…”

3D gastruloids: a novel frontier in stem cell-based in vitro modeling of mammalian gastrulation

“…Similar to mouse 3D gastruloids, human 3D gastruloids also generate derivatives of all three germ layers in the correct spatial location [24] and comparisons with human embryo samples from the Carnegie collection revealed that they recapitulate events associated with the post-occipital region of~18-21 days post fertilization (dpf) human embryos. However, both human and mouse 3D gastruloids generated with the first protocols [16, 17,24,42] do not generate anterior neural (brain) or extraembryonic tissues and lack embryonic architecture (i.e., they are not able to form somite or neural tube-like structures) (Figure 1).…”