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  • In vitro generation of mouse polarized embryo-like structures from embryonic and trophoblast stem cells.

In vitro generation of mouse polarized embryo-like structures from embryonic and trophoblast stem cells.

Nature protocols (2018-07-11)
Sarah Ellys Harrison, Berna Sozen, Magdalena Zernicka-Goetz
ABSTRACT

Mammalian embryogenesis requires the coordination of embryonic and extra-embryonic tissues to enable implantation into the uterus and post-implantation development to establish the body plan. Mouse embryonic stem cells (ESCs) are a useful tool for studying pluripotent embryonic tissue in vitro. However, they cannot undertake correct embryogenesis alone. Many attempts to model the early embryo in vitro involve the aggregation of ESCs into spheroids of variable size and cell number that undertake germ-layer specification but fail to recapitulate the characteristic architecture and arrangement of tissues of the early embryo. Here, we describe a protocol to generate the first embryo-like structures by directing the assembly of mouse ESCs and extra-embryonic trophoblast stem cells (TSCs) in a 3D extracellular matrix (ECM) into structures we call 'polarized embryo-like structures'. By establishing the medium and culture conditions needed to support the growth of both stem cell types simultaneously, embryonic architecture is generated within 4 d of co-culture. This protocol can be performed by those proficient in standard ESC culture techniques and can be used in developmental studies to investigate the interactions between embryonic and extra-embryonic tissues during mammalian development.

MATERIALS
Product Number
Brand
Product Description

SAFC
MegaCell RPMI-1640 Medium, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Mitomycin C from Streptomyces caespitosus, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Glycine, ReagentPlus®, ≥99% (HPLC)
Sigma-Aldrich
Gelatin from cold water fish skin, 40-50% in H2O
Sigma-Aldrich
apo-Transferrin human, powder, BioReagent, suitable for cell culture, ≥98% (agarose gel electrophoresis)
Sigma-Aldrich
Sodium selenite, BioReagent, suitable for cell culture, ≥98%
Sigma-Aldrich
Insulin solution human, sterile-filtered, BioXtra, suitable for cell culture