Stem Cell

The blastocysts consist of dozens of cells of three distinct lineages: epiblast (Epi), trophoblast (TB), and primitive endoderm (PrE). All embryonic and extraembryonic tissues are derived from Epi, TB, and PrE. Stem cell lines representing preimplantation Epi and TB have been established and are known as embryonic stem cells (ESCs) and trophoblast stem cells (TSCs). Extraembryonic endoderm cells (XENCs) constitute a cell line that has been established from PrE. Although in vivo, PrE gives rise to visceral endoderm (VE), parietal endoderm (PE), and marginal zone endoderm (MZE); XENCs, on blastocyst injection into chimeras, primarily contribute to the distal region of PE. Here, we provide a comprehensive protocol for the establishment of fully potent primitive endoderm stem cell (PrESC) lines. PrESCs are established and maintained on mouse embryonic fibroblast (MEF) feeder cells in a serum-free medium supplemented with fibroblast growth factor 4 (FGF4), heparin, CHIR99021, and platelet-derived growth factor-AA (PDGF-AA). PrESCs co-express markers indicative of pluripotency and endoderm lineage commitment, exhibiting characteristics akin to those of PrE. On transplantation of PrESCs into blastocysts, they demonstrate a high efficiency in contributing to VE, PE, and MZE. PrESCs serve as a valuable model for studying PrE, sharing similarities in gene expression profiles and differentiation potential. PrESCs constitute a pivotal cornerstone for in vitro analysis of early developmental mechanisms and for studies of embryo reconstitution in vitro, particularly in conjunction with ESCs and TSCs.

Key features

• Establishment and maintenance of primitive endoderm stem cell (PrESCs) capable of recapitulating the developmental prowess inherent to PrE.

• Offering a source of PrE lineage for embryo-like organoid reconstitution studies.

Graphical overview

Model organisms offer the opportunity to decipher the dynamic and complex behavior of stem cells in their native environment; however, imaging stem cells in situ remains technically challenging. C. elegans germline stem cells (GSCs) are distinctly accessible for in situ live imaging but relatively few studies have taken advantage of this potential. Here we provide our protocol for mounting and live imaging dividing C. elegans GSCs, as well as analysis tools to facilitate the processing of large datasets. While the present protocol was optimized for imaging and analyzing mitotic GSCs, it can easily be adapted to visualize dividing cells or other subcellular processes in C. elegans at multiple developmental stages. Our image analysis pipeline can also be used to analyze mitosis in other cell types and model organisms.

The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016.

Allogeneic organ transplantation is a powerful tool for clinical and basic research studies. However, the graft is often rejected by the host organism. Here, we describe a protocol that uses immunodeficient rag1 mutant zebrafish. These zebrafish escaped rejection, which made it possible to successfully transplant fragments of an allogeneic testis and testicular hyperplasia. This protocol can be used to amplify and maintain testicular hyperplasia grafts for several years (Kawasaki et al., 2016). The amplified hyperplasias are likely to be a good source of somatic and germ cells such as Sertoli cells and spermatogonial stem cells.