Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids.

In vitro stem cell models that replicate human gastrulation have been generated, but they lack the essential extraembryonic cells needed for embryonic development, morphogenesis, and patterning. Here, we describe a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, termed peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. Although peri-gastruloids are not viable due to the exclusion of trophoblasts, they recapitulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and undergoing early neurulation and organogenesis. Single-cell RNA-sequencing unveiled transcriptomic similarities between advanced human peri-gastruloids and primary peri-gastrulation cell types found in humans and non-human primates. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.

Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo.

Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages inside monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These results may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.

Global molecular features in transcription and chromatin accessibility in human extended pluripotent stem cells.

Human extended pluripotent stem (hEPS) cell is a newly established human embryonic stem cell (hESC) line with the capacity of chimerizing both embryonic and extraembryonic tissues compared with primed hESCs which are inefficient to contribute to the inner cell mass (ICM). The molecular mechanism underlying the pluripotency of hEPS cells is still not clear. We conducted RNA-seq and ATAC-seq analysis to investigate the differential expression profiling and genomic chromatin accessibility features. According to our data, more than 2000 genes were specially up-regulated in hEPS cells. Furthermore, the open chromatin regions in these two human embryonic stem cell lines were quite different. In hEPS cells, transcriptional factors binding motifs associated with pluripotency maintenance were enriched in chromatin accessible regions. Integrating the results from ATAC-seq and RNA-seq, we identified new regulatory features which were important for pluripotency maintenance and cell development in hEPS cells. Together, these results provided a new perspective on the understanding of molecular features of hESCs in different pluripotent states and a novel resource for further studies on regenerative medicine by using hEPS cells.

Comparative cytotoxicity studies of halophenolic disinfection byproducts using human extended pluripotent stem cells.

2,4,6-trichlorophenol (TCP), 2,4,6-tribromophenol (TBP) and 2,4,6-triiodophenol (TIP) are a new class of halophenolic disinfection byproducts (DBPs) which have been widely detected in drinking water. In recent years, their developmental toxicity has got increasing public attention due to their potential toxic effects on embryo development towards lower organisms. Nonetheless, the application of human embryos for embryonic toxicologic studies is rendered by ethical and moral considerations, as well as the technical barrier to sustaining normal development beyond a few days. Human extended pluripotent stem (EPS) cells (novel totipotent-like stem cells) represent a much more appropriate cellular model for studying human embryo development. In this study, we utilized human EPS cells to study the developmental toxicity of TCP, TBP and TIP, respectively. All three halophenolic DBPs showed cytotoxicity against human EPS cells in an obvious dose-dependent manner, among which TIP was the most cytotoxic one. Notably, the expression of pluripotent genes in human EPS cells significantly declined after 2,4,6-trihalophenol exposure. Meanwhile, 2,4,6-trihalophenol exposure promoted ectodermal differentiation of human EPS cells in an embryoid bodies (EBs) differentiation assay, while both endodermal and mesodermal differentiation were impaired. These results implied that phenolic halogenated DBPs have specific effects on human embryo development even in the early stage of pregnancy. In summary, we applied human EPS cells as a novel research model for human embryo developmental toxicity study of environmental pollutants, and demonstrated the toxicity of phenolic halogenated DBPs on early embryo development of human beings.