Complete human day 14 post-implantation embryo models from naive ES cells.

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.

Human primed and naïve PSCs are both able to differentiate into trophoblast stem cells.

The recent derivation of human trophoblast stem cells (TSCs) from placental cytotrophoblasts and blastocysts opened opportunities for studying the development and function of the human placenta. Recent reports have suggested that human naïve, but not primed, pluripotent stem cells (PSCs) retain an exclusive potential to generate TSCs. Here we report that, in the absence of WNT stimulation, transforming growth factor ß (TGF-ß) pathway inhibition leads to direct and robust conversion of primed human PSCs into TSCs. The resulting primed PSC-derived TSC lines exhibit self-renewal, can differentiate into the main trophoblast lineages, and present RNA and epigenetic profiles that are indistinguishable from recently established TSC lines derived from human placenta, blastocysts, or isogenic human naïve PSCs expanded under human enhanced naïve stem cell medium (HENSM) conditions. Activation of nuclear Yes-associated protein (YAP) signaling is sufficient for this conversion and necessary for human TSC maintenance. Our findings underscore a residual plasticity in primed human PSCs that allows their in vitro conversion into extra-embryonic trophoblast lineages.

Recent insights into mammalian natural and synthetic ex utero embryogenesis.

Research on early postimplantation mammalian development has been limited by the small size and intrauterine confinement of the developing embryos. Owing to the inability to observe and manipulate living embryos at these stages in utero, the establishment of robust ex utero embryo-culture systems that capture prolonged periods of mouse development has been an important research goal. In the last few years, these methods have been significantly improved by the optimization and enhancement of in vitro culture systems sustaining embryo development during peri-implantation stages for several species, and more recently, proper growth of natural mouse embryos from pregastrulation to late organogenesis stages and of embryonic stem cell (ES)-derived synthetic embryo models until early organogenesis stages. Here, we discuss the most recent ex utero embryo-culture systems established to date for rodents, nonhuman primates, and humans. We emphasize their technical aspects and developmental timeframe and provide insights into the new opportunities that these methods will contribute to the study of natural and synthetic mammalian embryogenesis and the stem-cell field.

Post-gastrulation synthetic embryos generated ex utero from mouse naive ESCs.

In vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation.

SUMOylation of linker histone H1 drives chromatin condensation and restriction of embryonic cell fate identity.

The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.

Principles of signaling pathway modulation for enhancing human naive pluripotency induction.

Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/ß-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.

Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis.

The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro1,2, approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesis in mammals.

In Vitro Employment of Recombinant Taenia solium Calreticulin as a Novel Strategy Against Breast and Ovarian Cancer Stem-like Cells.

BACKGROUND AND AIMS: Calreticulin is a chaperone and master regulator of intracellular calcium homeostasis. Several additional functions have been discovered. Human and parasite calreticulin have been shown to suppress mammary tumor growth in vivo. Here, we explored the capacity of recombinant Taenia solium calreticulin (rTsCRT) to modulate cancer cell growth in vitro. METHODS: We used different concentrations of rTsCRT to treat cancer cell lines and analyzed viability and colony formation capacity. We also tested the combination of the IC20 or IC50 doses of rTsCRT and of the chemotherapeutic drug 5-fluorouracil on MCF7 and SKOV3 cell lines. As a control, the non-tumorigenic cell line MCF10-A was employed. The effect of the drug combinations was also assessed in cancer stem-like cells. Additionally, scavenger receptor ligands were employed to identify the role of this receptor in the rTsCRT anti-tumoral effect. RESULTS: rTsCRT has a dose-dependent in vitro anti-tumoral effect, being SKOV3 the most sensitive cell line followed by MCF7. When rTsCRT/5-fluorouracil were used, MCF7 and SKOV3 showed a 60% reduction in cell viability; colony formation capacity was also diminished. Treatment of cancer stem-like cells from MCF7 showed a higher reduction in cell viability, while those from SKOV3 were more sensitive to colony disaggregation. Finally, pharmacological inhibition of the scavenger receptor, abrogated the reduction in viability induced by rTsCRT in both the parental and stem-like cells. CONCLUSION: Our data suggest that rTsCRT alone or in combination with 5-fluorouracil inhibits the growth of breast and ovarian cancer cell lines through its interaction with scavenger receptors.

Calreticulin in phagocytosis and cancer: opposite roles in immune response outcomes.

Calreticulin (CRT) is a pleiotropic and highly conserved molecule that is mainly localized in the endoplasmic reticulum. Recently, CRT has gained special interest for its functions outside the endoplasmic reticulum where it has immunomodulatory properties. CRT translocation to the cell membrane serves as an "eat me" signal and promotes efferocytosis of apoptotic cells and cancer cell removal with completely opposite outcomes. Efferocytosis results in a silenced immune response and homeostasis, while removal of dying cancer cells brought about by anthracycline treatment, ionizing-irradiation or photodynamic therapy results in immunogenic cell death with activation of the innate and adaptive immune responses. In addition, CRT impacts phagocyte activation and cytokine production. The effects of CRT on cytokine production depend on its conformation, species specificity, degree of oligomerization and/or glycosylation, as well as its cellular localization and the molecular partners involved. The controversial roles of CRT in cancer progression and the possible role of the CALR gene mutations in myeloproliferative neoplasms are also addressed. The release of CRT and its influence on the different cells involved during efferocytosis and immunogenic cell death points to additional roles of CRT besides merely acting as an "eat me" signal during apoptosis. Understanding the contribution of CRT in physiological and pathological processes could give us some insight into the potential of CRT as a therapeutic target.

Reproducibility of BiliCare™ Transcutaneus Bilirrubin Meter in Mexican Newborns.

BACKGROUND: Newborn hyperbilirubinemia is considered a worldwide health problem that demands medical evaluation. Noninvasive transcutaneous bilirubin (TcB) has been used as a screening method with different devices but there has not been any evaluation of reproducibility of the same brand devices. The BiliCare™ system is evaluated to demonstrate consistency between measurements with four different devices. METHODS: 336 TcB measurements were obtained with four BiliCare™ devices in 21 Mexican icteric newborns with a mean postnatal age of 44.1 hours of life and 38 weeks of gestation (33-41). Two measurements were taken in the same ear alternatively at the scaphoid fossa with each device. TcB values were compared between devices. Validity was compared with total serum bilirubin (TB). RESULTS: intraclass correlation coefficient demonstrates a minimum limit in the study of 0.945 and maximum of 0.988 with the same device. Correlations with serum and between devices gave results above 0.932. CONCLUSIONS: BiliCare™ transcutaneous bilirubin measurement instrument has very good intra- and interdevice reproducibility; also correlation of TcS with serum bilirubin gave statistically the same results.

RNA Purity, Real-Time PCR Sensitivity, and Colon Segment Influence mRNA Relative Expression in Murine Dextran Sodium Sulfate Experimental Colitis.

The dextran sodium sulfate (DSS) model of colitis is widely used as a result of its simplicity and reproducibility and because it mimics clinicopathological disease features. Its effectiveness depends on the mouse strain, DSS MW, and brand. Quantitative RT-PCR (qRT-PCR) is highly sensitive for analyzing cytokine mRNA expression. We analyzed an acute model of DSS treatment in Balb/c mice for the onset of colitis using qRT-PCR for the quantification of a mouse cytokine transcript. We compared differences among 1--and 2-step qRT-PCR for transcript quantification, the effect of multiple concentrations of DSS, and the use of 2 reference genes in 3 portions of the colon. A reliable and sensitive 1-step protocol for qRT-PCR was established with a modified double LiCl precipitation for RNA isolation. The variability of 2 reference genes, ß-actin and eukaryotic elongation factor 2, was compared, and expression of IL-6 was analyzed in 3 segments of the colon. The RNA cleaning protocol prevented inhibition of qRT-PCR by DSS, and RNA loss was minimized. No clinical differences among the different DSS concentrations were seen on d 7, but higher concentrations resulted in the appearance of earlier symptoms. Higher efficiency and sensitivity of the 1-step qRT-PCR reaction using eukaryotic elongation factor 2 were obtained and also less variability. Although expression levels of IL-6 were high in the middle and distal colon, the middle section had consistently less variability in values. Thus, this segment is recommended for future studies. These factors influence the statistical significance of data and need to be considered to get accurate and reliable results and to improve comparisons of the published colitis experiments.