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The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids—three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development. Human gastruloids—three-dimensional aggregates derived from human embryonic stem cells—show features of human embryos at around 19–21 days, and provide a model for the study of early human development. [ABSTRACT FROM AUTHOR]

Summary Variation in the level of mtDNA heteroplasmy in adult tissues is commonly seen in patients with a mixture of wild-type and mutant mtDNA. A mixture of different mtDNA variants may influence such variation and cause mtDNA segregation bias. We analyzed cellular heterogeneity in embryonic stem cells (ESCs) derived from a polymorphic mouse model containing NZB and BALB mtDNA genotypes. In ESCs, inter-colony heterogeneity varied up to 61%, whereas intra-colony heterogeneity varied up to 100%. Three out of five cell lines displayed nearly homoplasmic BALB and NZB mtDNA haplotypes in differentiated single cells. The proportion of NZB mtDNA genotype increased with progressive passaging (0.39%; p = 0.002). These results demonstrate the bimodal segregation of mtDNA haplotypes, indicating the occurrence of tissues with variable levels of heteroplasmies in individuals with mtDNA mutations. Furthermore, proliferation of one mtDNA genotype over another may pose the risk of accumulating mutant mtDNAs during subsequent cell divisions. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation. S tem C ells 2015;33:2686-2698 [ABSTRACT FROM AUTHOR]

In mice, inhibition of both the fibroblast growth factor (FGF) mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/Erk) and the Wnt signaling inhibitor glycogen synthase-3β (GSK3β) enables the derivation of mouse embryonic stem cells (mESCs) from nonpermissive strains in the presence of leukemia inhibitory factor (LIF). Whereas mESCs are in an uncommitted naïve state, human embryonic stem cells (hESCs) represent a more advanced state, denoted as primed pluripotency. This burdens hESCs with a series of characteristics, which, in contrast to naïve ESCs, makes them not ideal for key applications such as cell-based clinical therapies and human disease modeling. In this study, different small molecule combinations were applied during human ESC derivation. Hereby, we aimed to sustain the naïve pluripotent state, by interfering with various key signaling pathways. First, we tested several combinations on existing, 2i (PD0325901 and CHIR99021)-derived mESCs. All combinations were shown to be equally adequate to sustain the expression of naïve pluripotency markers. Second, these conditions were tested during hESC derivation. Overall, the best results were observed in the presence of medium supplemented with 2i, LIF, and the noncanonical Wnt signaling agonist Wnt5A, alone and combined with epinephrine. In these conditions, outgrowths repeatedly showed an ESC progenitor-like morphology, starting from day 3. Culturing these 'progenitor cells' did not result in stable, naïve hESC lines in the current conditions. Although Wnt5A could not promote naïve hESC derivation, we found that it was sustaining the conversion of established hESCs toward a more naïve state. Future work should aim to distinct the effects of the various culture formulations, including our Wnt5A-supplemented medium, reported to promote stable naïve pluripotency in hESCs. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the reliability of preimplantation genetic diagnosis (PGD) based on polar body (PB), blastomere or trophectoderm (TE) analysis in a heteroplasmic mitochondrial mouse model? SUMMARY ANSWER The reliability of PGD to determine the level of mitochondrial DNA (mtDNA) heteroplasmy is questionable based on either the first or second PB analysis; however, PGD based on blastomere or TE analysis seems more reliable. WHAT IS KNOWN ALREADY PGD has been suggested as a technique to determine the level of mtDNA heteroplasmy in oocytes and embryos to avoid the transmission of heritable mtDNA disorders. A strong correlation between first PBs and oocytes and between second PBs and zygotes was reported in mice but is controversial in humans. So far, the levels of mtDNA heteroplasmy in first PBs, second PBs and their corresponding oocytes, zygotes and blastomeres, TE and blastocysts have not been analysed within the same embryo. STUDY DESIGN, SIZE AND DURATION We explored the suitability of PGD by comparing the level of mtDNA heteroplasmy between first PBs and metaphase II (MII) oocytes (n = 33), between first PBs, second PBs and zygotes (n = 30), and between first PBs, second PBs and their corresponding blastomeres of 2- (n = 10), 4- (n = 10) and 8-cell embryos (n = 11). Levels of mtDNA heteroplasmy in second PBs (n = 20), single blastomeres from 8-cell embryos (n = 20), TE (n = 20) and blastocysts (n = 20) were also compared. PARTICIPANTS/MATERIALS, SETTING, METHODS Heteroplasmic mice (BALB/cOlaHsd), containing mtDNA mixtures of BALB/cByJ and NZB/OlaHsd, were used in this study. The first PBs were biopsied from in vivo matured MII oocytes. The ooplasm was then subjected to ICSI. After fertilization, second PBs were biopsied and zygotes were cultured to recover individual blastomeres from 2-, 4- and 8-cell embryos. Similarly, second PBs were biopsied from in vivo fertilized zygotes and single blastomeres were biopsied from 8-cell stage embryos. The remaining embryo was cultured until the blastocyst stage to isolate TE cells. Polymerase chain reaction followed by restriction fragment length polymorphism was performed to measure the level of mtDNA heteroplasmy in individual samples. MAIN RESULTS AND THE ROLE OF CHANCE Modest correlations and wide prediction interval [PI at 95% confidence interval (CI)] were observed in the level of mtDNA heteroplasmy between first PBs and their corresponding MII oocytes (r2 = 0.56; PI = 45.96%) and zygotes (r2 = 0.69; PI = 37.07%). The modest correlations and wide PI were observed between second PBs and their corresponding zygotes (r2 = 0.65; PI = 39.69%), single blastomeres (r2 = 0.42; PI = 48.04%), TE (r2 = 0.26; PI = 54.79%) and whole blastocysts (r2 = 0.40; PI = 57.48%). A strong correlation with a narrow PI was observed among individual blastomeres of 2-, 4- and 8-cell stage embryos (r2 = 0.92; PI = 11.73%, r2 = 0.86; PI = 18.85% and r2 = 0.85; PI = 21.42%, respectively), and also between TE and whole blastocysts (r2 = 0.90; PI = 23.58%). Moreover, single blastomeres from 8-cell stage embryos showed a close correlation and an intermediate PI with corresponding TE cells (r2 = 0.81; PI = 28.15%) and blastocysts (r2 = 0.76; PI = 36.43%). LIMITATIONS, REASONS FOR CAUTION These results in a heteroplasmic mitochondrial mouse model should be further verified in patients with mtDNA disorders to explore the reliability of PGD. WIDER IMPLICATIONS OF THE FINDINGS To avoid the transmission of heritable mtDNA disorders, PGD techniques should accurately determine the level of heteroplasmy in biopsied cells faithfully representing the heteroplasmic load in oocytes and preimplantation embryos. Unlike previous PGD studies in mice, our results accord with PGD results for mitochondrial disorders in humans, ... [ABSTRACT FROM AUTHOR]

STUDY QUESTION Is there an effect of the TGFβ inhibitor SB431542 (SB) on the epiblast compartment of human blastocysts, and does it affect subsequent human embryonic stem cell (hESC) derivation? SUMMARY ANSWER SB increases the mean number of NANOG-positive cells in the inner cell mass (ICM), and allows for subsequent hESC derivation. WHAT IS KNOWN ALREADY It is known that inhibition of TGFβ by SB has a positive effect on mouse ESC self-renewal, while active TGFβ signalling is needed for self-renewal of primed ESC. STUDY DESIGN, SIZE, DURATION From December 2011 until March 2012, 263 donated spare embryos were used from patients who had undergone IVF/ICSI in our centre. PARTICIPANTS/MATERIALS, SETTING, METHODS Donated human embryos were cultured in the presence of SB or Activin A, and immunocytochemistry was performed on Day 6 blastocysts for NANOG and GATA6. Moreover, blastocysts were used for the derivation of hESC, with or without exposure to SB. MAIN RESULTS AND THE ROLE OF CHANCE Immunocytochemistry revealed a significantly higher number of NANOG-positive ICM cells in the SB group compared with the control (12.0 ± 5.9 versus 6.1 ± 4.7), while no difference was observed in the Activin A group compared with other groups (6.7 ± 3.7). The number of GATA6-positive ICM cells did not differ between the SB, Activin A and control group (8.8 ± 4.3, 8.0 ± 4.6 and 7.2 ± 4.0, respectively). Blocking TGFβ signalling did not prevent subsequent hESC line derivation. LIMITATIONS, REASONS FOR CAUTION The number of human blastocysts available for this study was too low to reveal if the observed increase in NANOG-positive epiblast cells after exposure to SB affected the efficiency of hESC derivation (12.5% compared with 16.7%). WIDER IMPLICATIONS OF THE FINDINGS This work can contribute to the derivation of naive hESC lines in the future. STUDY FUNDING/COMPETING INTEREST(S) M.V.d.J. is holder of a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT, grant number SB093128), Belgium. G.D. and this research are supported by the Research Foundation Flanders (FWO), grant number FWO-3G062910) and a Concerted Research Actions funding from BOF (Bijzonder Onderzoeksfonds University Ghent, grant number BOF GOA 01G01112). S.M.C.d. S.L. is supported by the Netherlands Organization of Scientific Research (NWO) (ASPASIA 015.007.037) and the Interuniversity Attraction Poles (PAI) (no. P7/07). P.D.S. is holder of a fundamental clinical research mandate by the FWO. We would like to thank Ferring Company (Aalst, Belgium) for financial support of this study. The authors do not have any competing interests to declare. TRIAL REGISTRATION NUMBER Not applicable. [ABSTRACT FROM PUBLISHER]