The advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias.

The Chromosome-centric Human Proteome Project (C-HPP) aims at identifying the proteins as gene products encoded by the human genome, characterizing their isoforms and functions. The existence of products has now been confirmed for 93.2% of the genes at the protein level. The remaining mostly correspond to proteins of low abundance or difficult to access. Over the past years, we have significantly contributed to the identification of missing proteins in the human spermatozoa. We pursue our search in the reproductive sphere with a focus on early human embryonic development. Pluripotent cells, developing into the fetus, and trophoblast cells, giving rise to the placenta, emerge during the first weeks. This emergence is a focus of scientists working in the field of reproduction, placentation and regenerative medicine. Most knowledge has been harnessed by transcriptomic analysis. Interestingly, some genes are uniquely expressed in those cells, giving the opportunity to uncover new proteins that might play a crucial role in setting up the molecular events underlying early embryonic development. Here, we analyzed naive pluripotent and trophoblastic stem cells and discovered 4 new missing proteins, thus contributing to the C-HPP. The mass spectrometry proteomics data was deposited on ProteomeXchange under the data set identifier PXD035768.

Development of new 2D and 3D models of human development such as trophoblast stem cells, gastruloids or blastoids widened possibilities to study early timepoints of development and brightened up ever so slightly the black box of human development. While opening new horizons, the cell sources of those models need proper benchmarking to clarify which hallmark is associated with which lineage and developmental stage. Here, we propose a thorough characterization of pluripotent and trophoblastic stem cell models by transcriptomic, proteomic, epigenetic and metabolic approaches. Extended pluripotent stem cells are similar to primed pluripotent stem cells for most criteria, except metabolic activity, which might explain their ability to convert directly into trophoblast stem cells. We show that trophoblast stem cells are hypo-methylated and that they have a high metabolic activity. Our results clarify the fact that hallmarks of pluripotency are not predictive of each other and have to be used in combination. Multiplying hallmarks alleviate stage matching bias.

Generating patient-specific stem cells representing the onset of development has become possible since the discovery of somatic cell reprogramming into induced pluripotent stem cells. However, human pluripotent stem cells are generally cultured in a primed pluripotent state: they are poised for differentiation and represent a stage of development corresponding to post-implantation epiblast. Here, we describe a protocol to reprogram human fibroblasts into naive pluripotent stem cells by overexpressing the transcription factors OCT4, SOX2, KLF4, and c-MYC using Sendai viruses. The resulting cells represent an earlier stage of development that corresponds to pre-implantation epiblast. We also discuss validation methods for human naive pluripotent stem cells.