Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency.

Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research. VIDEO ABSTRACT.

Generation of a human extended pluripotent stem cell line (SKLRMe002-A) carrying a doxycycline-inducible Cas9 expression cassette.

Human extended pluripotent stem cell (hEPS) is a novel type of pluripotent stem cell, which possesses bi-potency towards both embryonic and extraembryonic lineages. Here, we generated a hEPS cell line (hEPS1-iCas9-B) from the cell line named hEPS1, carrying a doxycycline-inducible Cas9 expression cassette along with a constitutive reverse tetracycline transactivator (M2rtTA) expression cassette at the AAVS1 locus, thus we could efficiently generate genetically modified hEPS for studies. This cell lined remained self-renewal, differentiation potential and normal karyotype. Meanwhile, it showed robust transcriptional expression of Cas9 with doxycycline induction and could target the site where the sgRNA guided.

Embryotoxic effects of tribromophenol on early post-implantation development of mouse embryos in vitro.

2,4,6-Tribromophenol (TBP, CAS No. 118-79-6), the most widely produced brominated phenol, is frequently detected in environmental components. The detection of TBP in human bodies has earned great concerns about its adverse effects on human beings, especially for early embryonic development. Here, we optimized the mouse embryo in vitro culture (IVC) system for early post-implantation embryos and employed it to determine the embryotoxicity of TBP. With this new research model, we revealed the dose-dependent toxic effects of TBP on mouse embryos from peri-implantation to egg cylinder stages. Furthermore, TBP exposure inhibited the differentiation and survival of epiblast (EPI) cells and extraembryonic endoderm (ExEn) cells, while those of extraembryonic ectoderm (ExEc) cells were not influenced. These results implied that TBP might inhibit embryonic development by influencing the generation of three primary germ layers and fetal membranes (the amnion, chorionic disk, umbilical cord, and yolk sac). In summary, we showed a proof of concept for applying mouse embryo IVC system as a novel research model for studying mammalian embryonic toxicology of environmental pollutants. This study also demonstrated the toxicity of TBP on early embryonic development of mammals.

Effect of SOCS3 on apoptosis of human trophoblasts via adjustment of the JAK2/STAT3 signaling pathway in preterm birth.

BACKGROUND: The expression of suppressor of cytokine signaling 3 (SOCS3) was induced by interleukin-6 (IL-6) in preterm placental tissues. However, its role in IL-6 induced apoptosis of trophoblast cells derived from preterm placental tissues remains to be elucidated. METHODS: Primary cytotrophoblasts from human preterm placental tissues were used to stably knock down and overexpress the level of SOCS3 by corresponding lentiviral vectors and the expression of SOCS3 was validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. The effect of SOCS3 overexpression or knockdown on the proliferation and apoptosis of IL-6 treated human cytotrophoblasts were determined by Cell Counting Kit-8 (CCK8) assay and Annexin-V/Propidium Iodide (PI) double-staining assay, respectively. Based on it, we detected the proteins associated with the Janus Tyrosine Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway and apoptosis, such as JAK2, p-JAK2, STAT3, p-STAT3, B-cell lymphoma-2 (Bcl-2) and BCL2-associated X (Bax) by Western blot. RESULTS: IL-6-treatment resulted in significant apoptosis of human cytotrophoblasts. Overexpressing SOCS3 in the cytotrophoblasts reduced cell apoptosis, while the knockdown of SCOS3 had the opposite effects. Further analyses showed that SOCS3 overexpression inhibited JAK2 and STAT3 phosphorylation, which was induced by IL-6 stimulation. CONCLUSIONS: SOCS3 plays a protective role in human preterm placental tissue-derived cytotrophoblasts from IL-6 induced apoptosis by feedback inhibition of JAK2/STAT3 signaling.

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.

Generation of naive induced pluripotent stem cells from rhesus monkey fibroblasts.

Conventional embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) derived from primates resemble mouse epiblast stem cells, raising an intriguing question regarding whether the naive pluripotent state resembling mouse embryonic stem cells (mESCs) exists in primates and how to capture it in vitro. Here we identified several specific signaling modulators that are sufficient to generate rhesus monkey fibroblast-derived iPSCs with the features of naive pluripotency in terms of growth properties, gene expression profiles, self-renewal signaling, X-reactivation, and the potential to generate cross-species chimeric embryos. Interestingly, together with recent reports of naive human pluripotent stem cells, our findings suggest several conserved signaling pathways shared with rodents and specific to primates, providing significant insights for acquiring naive pluripotency from other species. In addition, the derivation of rhesus monkey naive iPSCs also provides a valuable cell source for use in preclinical research and disease modeling.

Systematically labeling developmental stage-specific genes for the study of pancreatic ß-cell differentiation from human embryonic stem cells.

The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic ß cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP(+) cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature ß cells in vitro.