A key challenge of analyzing data from high-resolution spatial profiling technologies is to suitably represent the features of cellular neighborhoods or niches. Here we introduce the covariance environment (COVET), a representation that leverages the gene–gene covariate structure across cells in the niche to capture the multivariate nature of cellular interactions within it. We define a principled optimal transport-based distance metric between COVET niches that scales to millions of cells. Using COVET to encode spatial context, we developed environmental variational inference (ENVI), a conditional variational autoencoder that jointly embeds spatial and single-cell RNA sequencing data into a latent space. ENVI includes two decoders: one to impute gene expression across the spatial modality and a second to project spatial information onto single-cell data. ENVI can confer spatial context to genomics data from single dissociated cells and outperforms alternatives for imputing gene expression on diverse spatial datasets.

Just over ten years have passed since the seminal Takahashi-Yamanaka paper, and while most attention nowadays is on induced, embryonic, and cancer stem cells, much of the pioneering work arose from studies with embryonal carcinoma cells (ECCs) derived from teratocarcinomas. This original work was broad in scope, but eventually led the way for us to focus on the components involved in the gene regulation of stemness and differentiation. As the name implies, ECCs are malignant in nature, yet maintain the ability to differentiate into the 3 germ layers and extraembryonic tissues, as well as behave normally when reintroduced into a healthy blastocyst. Retinoic acid signaling has been thoroughly interrogated in ECCs, especially in the F9 and P19 murine cell models, and while we have touched on this aspect, this review purposely highlights how some key transcription factors regulate pluripotency and cell stemness prior to this signaling. Another major focus is on the epigenetic regulation of ECCs and stem cells, and, towards that end, this review closes on what we see as a new frontier in combating aging and human disease, namely, how cellular metabolism shapes the epigenetic landscape and hence the pluripotency of all stem cells.

Glucose metabolism has a crucial role for providing substrates required to generate ATP and regulate the epigenetic landscape. We reported that F9 embryonal carcinoma stem-like cells require cytosolic reactive oxygen species to differentiate into extraembryonic endoderm; however, mitochondrial sources were not examined. To extend these studies, we examined the metabolic profile of early and late-passage F9 cells, and show that their ability to differentiate is similar, even though each population has dramatically different metabolic profiles. Differentiated early-passage cells relied on glycolysis, while differentiated late-passage cells transitioned towards oxidative phosphorylation (OXPHOS). Unexpectedly, electron transport chain protein stoichiometry was disrupted in differentiated late-passage cells, whereas genes encoding mitofusion 1 and 2, which promote mitochondrial fusion and favor OXPHOS, were upregulated in differentiated early-passage cells. Despite this, early-passage cells cultured under conditions to promote glycolysis showed enhanced differentiation, whereas promoting OXPHOS in late-passage cells showed a similar trend. Further analysis revealed that the distinct metabolic profiles seen between the two populations is largely associated with changes in genomic integrity, linking metabolism to passage number. Together, these results indicate that passaging has no effect on the potential for F9 cells to differentiate into extraembryonic endoderm; however, it does impact their metabolic profile. Thus, it is imperative to determine the molecular and metabolic status of a stem cell population before considering its utility as a therapeutic tool for regenerative medicine.

Metabolism plays a crucial role for cell survival and function; however, recent evidence has implicated it in regulating embryonic development. In the embryo, the inner cell mass undergoes orchestrated cellular divisions resulting in the formation of pluripotent epiblast stem cells and primitive endoderm cells. However, both lineages can be captured in vitro as embryonic stem (ES) cells and extraembryonic endoderm (XEN) cells. Concomitantly, changes in the metabolic profile occurs during development, and are well documented in the embryonic lineages. However, a comprehensive multi-omic analysis of these features in XEN cells remains lacking. We observed that mouse XEN cells exhibited high sensitivity to glycolytic inhibition in addition to maintaining elevated intra- and extracellular lactate levels in vitro. Extraembryonic endoderm cells maintain high lactate levels by increased LDHA activity, and re-routing pyruvate away from the mitochondria resulting in reduced mitochondrial activity due to disruptions in electron transport chain stoichiometry. Importantly, exogenous lactate supplementation or promoting intracellular lactate accumulation enhances XEN differentiation in vitro. These results highlight how lactate contributes to XEN differentiation in vitro and may serve to enhance reprogramming efficiency of cells used for regenerative medicine.

Mouse F9 cells differentiate to primitive endoderm (PrE) when treated with retinoic acid (RA). Differentiation is accompanied by increased reactive oxygen species (ROS) levels, and while treating F9 cells with antioxidants attenuates differentiation, H₂O₂ treatment alone is sufficient to induce PrE. We identified the NADPH oxidase (NOX) complexes as candidates for the source of this endogenous ROS, and within this gene family, and over the course of differentiation, Nox1 and Nox 4 show the greatest upregulation induced by RA. Gata6, encoding a master regulator of extraembryonic endoderm is also up-regulated by RA and we provide evidence that NOX1 and NOX4 protein levels increase in F9 cells overexpressing Gata6. Pan-NOX and NOX1-specific inhibitors significantly reduced the ability of RA to induce PrE, and this was recapitulated using a genetic approach to knockdown Nox1 and/or Nox4 transcripts. Interestingly, overexpressing either gene in untreated F9 cells did not induce differentiation, even though each elevated ROS levels. Thus, the data suggests that ROS produced during PrE differentiation is dependent in part on increased NOX1 and NOX4 levels, which is under the control of GATA6. Furthermore, these results suggest that the combined activity of multiple NOX proteins is necessary for the differentiation of F9 cells to primitive endoderm.

The regulation of proteins through the addition and removal of O-linked β-N-acetylglucosamine (O-GlcNAc) plays a role in many signaling events, specifically in stem cell pluripotency and the regulation of differentiation. However, these post-translational modifications have not been explored in extraembryonic endoderm (XEN) differentiation. Of the plethora of proteins regulated through O-GlcNAc, we explored galectin-3 as a candidate protein known to have various intracellular and extracellular functions. Based on other studies, we predicted a reduction in global O-GlcNAcylation levels and a distinct galectin expression profile in XEN cells relative to embryonic stem (ES) cells. By conducting dot blot analysis, XEN cells had decreased levels of global O-GlcNAc than ES cells, which reflected a disbalance in the expression of genes encoding O-GlcNAc cycle enzymes. Immunoassays (Western blot and ELISA) revealed that although XEN cells (low O-GlcNAc) had lower concentrations of both intracellular and extracellular galectin-3 than ES cells (high O-GlcNAc), the relative secretion of galectin-3 was significantly increased by XEN cells. Inducing ES cells toward XEN in the presence of an O-GlcNAcase inhibitor was not sufficient to inhibit XEN differentiation. However, global O-GlcNAcylation was found to decrease in differentiated cells and the extracellular localization of galectin-3 accompanies these changes. Inhibiting global O-GlcNAcylation status does not, however, impact pluripotency and the ability of ES cells to differentiate to the XEN lineage.