Faithful embryogenesis requires precise coordination between embryonic and extraembryonic tissues. Although stem cells from embryonic and extraembryonic origins have been generated for several mammalian species(Bogliotti et al., 2018; Choi et al., 2019; Cui et al., 2019; Evans and Kaufman, 1981; Kunath et al., 2005; Li et al., 2008; Martin, 1981; Okae et al., 2018; Tanaka et al., 1998; Thomson et al., 1998; Vandevoort et al., 2007; Vilarino et al., 2020; Yu et al., 2021b; Zhong et al., 2018), they are grown in different culture conditions with diverse media composition, which makes it difficult to study cross-lineage communication. Here, by using the same culture condition that activates FGF, TGF-β and WNT signaling pathways, we derived stable embryonic stem cells (ESCs), extraembryonic endoderm stem cells (XENs) and trophoblast stem cells (TSCs) from all three founding tissues of mouse and cynomolgus monkey blastocysts. This allowed us to establish embryonic and extraembryonic stem cell co-cultures to dissect lineage crosstalk during early mammalian development. Co-cultures of ESCs and XENs uncovered a conserved and previously unrecognized growth inhibition of pluripotent cells by extraembryonic endoderm cells, which is in part mediated through extracellular matrix signaling. Our study unveils a more universal state of stem cell self-renewal stabilized by activation, as opposed to inhibition, of developmental signaling pathways. The embryonic and extraembryonic stem cell co-culture strategy developed here will open new avenues for creating more faithful embryo models and developing more developmentally relevant differentiation protocols.

Serum-containing medium is widely used to support cell attachment, stable growth and serial passaging of various cancer cell lines. However, the presence of cholesterols and lipids in serum greatly hinders the analysis of the effects of cholesterol depletion on cells in culture. In this study, we developed a defined serum-free culture condition accessible to a variety of different types of adherent cancer cells. We tested different factors that are considered essential for cell culture and various extracellular matrix for plate coating, and found cells cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) basal media supplemented with Albumin (BSA) and insulin-transferrin-selenium-ethanolamine (ITS-X) on fibronectin-precoated plate (called as “DA-X condition”) showed comparable proliferation and survival to those in a serum-containing medium. Interestingly, we observed that DA-X condition could be adapted to a wide variety of adherent cancer cell lines, which enabled the analysis of how cholesterol depletion affected cancer cells in culture. Mechanistically, we found the beneficial effects of the DA-X condition in part can be attributed to the appropriate level of membrane cholesterol, and fibronectin-mediated signaling plays an important role in the suppression of cholesterol production.

Serum-containing medium is widely used to support cell attachment, stable growth and serial passaging of various cancer cell lines.   However, the presence of cholesterols and lipids in serum greatly hinders the analysis of the effects of cholesterol depletion on cells in culture.   In this study, we develop a defined serum-free culture condition accessible to a variety of different types of adherent cancer cells. We tested different factors that are considered essential for cell culture and various extracellular matrix for plate coating, and found cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) basal media supplemented with Albumin (BSA) and insulin-transferrin-selenium-ethanolamine (ITS-X) on fibronectin-precoated well (called as “DA-X condition”) showed comparable proliferation and survival to those in a serum-containing medium. Interestingly, we observed that DA-X condition could be adapted to a wide variety of adherent cancer cell lines, which enabled the analysis of how cholesterol depletion affected cancer cells in culture. Mechanistically, we found the beneficial effects of the DA-X condition in part can be attributed to the appropriate level of membrane cholesterol, and fibronectin-mediated signaling plays an important role in the suppression of cholesterol production.