Human embryos cultured in vitro to 14 days

We know a great deal about the development of the mammalian embryo until the time that the blastocyst implants into the uterus. With model organisms such as the mouse, we have also developed a considerable understanding of development immediately around gastrulation as embryos can be recovered at this stage for short-term in vitro culture. However, the intervening period of development remained a ‘black box’ because it takes place as the blastocyst is implanting into the uterus. Over the past 6 years, techniques pioneered and developed in Magdalena Zernicka-Goetz's laboratory for the in vitro culture of embryos through these implantation stages have opened up this box, affording the first glimpse of embryonic development through these previously hidden stages. Remarkably, the techniques developed with mouse embryos are equally applicable to human embryos, ushering the very first opportunities for studying our own development throughout this time. Here, I outline how the culture methods were developed, paving the way to culture of the human embryo to the point of gastrulation, an accomplishment recognized as the People's Choice for the Scientific Breakthrough of 2016 in Science magazine. I also discuss the new ethical challenges raised by the possibility of extending the time limits for human embryo culture

Nitric oxide production from nitrite by a series of zeolites produced via the ADOR route

We would like to thank the Engineering and Physical Sciences Research Council (EPSRC), University of St Andrews, and CRITICAT Centre for Doctoral Training for financial support [Ph.D. studentship to SR; Grant code: EP/L016419/1]. We also thank the EPSRC for funding part of this work through grant EP/K025112/1. R.E.M. would like to acknowledge OP VVV "Excellent Research Teams", project No. CZ.02.1.01/0.0/0.0/15_003/0000417 – CUCAM.The nitric oxide (NO) production for a series of aluminosilicate zeolites, prepared using the ADOR method, was investigated. Al-UTL and Al-IPC-2,-4 and −6 were prepared and characterised to determine their elemental composition. Positive trends were found to exist between zeolite pore size and the rate and total amount of NO produced. A trend between the number of acid sites and the initial rate of NO produced by the zeolite was also discovered.PostprintPeer reviewe

Gene expression dynamics underlying cell fate emergence in 2D micropatterned human embryonic stem cell gastruloids

Human embryonic stem cells cultured in 2D micropatterns with BMP4 differentiate into a radial arrangement of germ layers and extraembryonic cells. Single-cell transcriptomes demonstrate generation of cell types transcriptionally similar to their in vivo counterparts in Carnegie stage 7 human gastrula. Time-course analyses indicate sequential differentiation, where the epiblast arises by 12 h between the prospective ectoderm in the center and the cells initiating differentiation toward extraembryonic fates at the edge. Extraembryonic and mesendoderm precursors arise from the epiblast by 24 h, while nascent mesoderm, endoderm, and primordial germ cell-like cells form by 44 h. Dynamic changes in transcripts encoding signaling components support a BMP, WNT, and Nodal hierarchy underlying germ-layer specification conserved across mammals, and FGF and HIPPO pathways being active throughout differentiation. This work also provides a resource for mining genes and pathways expressed in a stereotyped 2D gastruloid model, common with other species or unique to human gastrulation

Dissecting cell identity via network inference and in silico gene perturbation

Cell identity is governed by the complex regulation of gene expression, represented as gene-regulatory network

Gene regulatory network reconfiguration in direct lineage reprogramming

In direct lineage conversion, transcription factor (TF) overexpression reconfigures gene regulatory networks (GRNs) to reprogram cell identity. We previously developed CellOracle, a computational method to infer GRNs from single-cell transcriptome and epigenome data. Using inferred GRNs, CellOracle simulates gene expression changes in response to TF perturbation, enabling in silico interrogation of network reconfiguration. Here, we combine CellOracle analysis with lineage tracing of fibroblast to induced endoderm progenitor (iEP) conversion, a prototypical direct reprogramming paradigm. By linking early network state to reprogramming outcome, we reveal distinct network configurations underlying successful and failed fate conversion. Via in silico simulation of TF perturbation, we identify new factors to coax cells into successfully converting their identity, uncovering a central role for the AP-1 subunit Fos with the Hippo signaling effector, Yap1. Together, these results demonstrate the efficacy of CellOracle to infer and interpret cell-type-specific GRN configurations, providing new mechanistic insights into lineage reprogramming

CellTag Indexing: Genetic barcode-based sample multiplexing for single-cell genomics

High-throughput single-cell assays increasingly require special consideration in experimental design, sample multiplexing, batch effect removal, and data interpretation. Here, we describe a lentiviral barcode-based multiplexing approach, CellTag Indexing, which uses predefined genetic barcodes that are heritable, enabling cell populations to be tagged, pooled, and tracked over time in the same experimental replicate. We demonstrate the utility of CellTag Indexing by sequencing transcriptomes using a variety of cell types, including long-term tracking of cell engraftment and differentiation in vivo. Together, this presents CellTag Indexing as a broadly applicable genetic multiplexing tool that is complementary with existing single-cell technologies

High-resolution transcriptional and morphogenetic profiling of cells from micropatterned human ESC gastruloid cultures

During mammalian gastrulation, germ layers arise and are shaped into the body plan while extraembryonic layers sustain the embryo. Human embryonic stem cells, cultured with BMP4 on extracellular matrix micro-discs, reproducibly differentiate into gastruloids, expressing markers of germ layers and extraembryonic cells in radial arrangement. Using single-cell RNA sequencing and cross-species comparisons with mouse, cynomolgus monkey gastrulae, and post-implantation human embryos, we reveal that gastruloids contain cells transcriptionally similar to epiblast, ectoderm, mesoderm, endoderm, primordial germ cells, trophectoderm, and amnion. Upon gastruloid dissociation, single cells reseeded onto micro-discs were motile and aggregated with the same but segregated from distinct cell types. Ectodermal cells segregated from endodermal and extraembryonic but mixed with mesodermal cells. Our work demonstrates that the gastruloid system models primate-specific features of embryogenesis, and that gastruloid cells exhibit evolutionarily conserved sorting behaviors. This work generates a resource for transcriptomes of human extraembryonic and embryonic germ layers differentiated in a stereotyped arrangement

The differential response to Fgf signalling in cells internalized at different times influences lineage segregation in preimplantation mouse embryos

Lineage specification in the preimplantation mouse embryo is a regulative process. Thus, it has been difficult to ascertain whether segregation of the inner-cell-mass (ICM) into precursors of the pluripotent epiblast (EPI) and the differentiating primitive endoderm (PE) is random or influenced by developmental history. Here, our results lead to a unifying model for cell fate specification in which the time of internalization and the relative contribution of ICM cells generated by two waves of asymmetric divisions influence cell fate. We show that cells generated in the second wave express higher levels of Fgfr2 than those generated in the first, leading to ICM cells with varying Fgfr2 expression. To test whether such heterogeneity is enough to bias cell fate, we upregulate Fgfr2 and show it directs cells towards PE. Our results suggest that the strength of this bias is influenced by the number of cells generated in the first wave and, mostly likely, by the level of Fgf signalling in the ICM. Differences in the developmental potential of eight-cell- and 16-cell-stage outside blastomeres placed in the inside of chimaeric embryos further support this conclusion. These results unite previous findings demonstrating the importance of developmental history and Fgf signalling in determining cell fate