Inducible stem cell-derived embryos capture mouse morphogenetic events in vitro

The development of mouse embryos can be partially recapitulated by combining embryonic (ES), trophoblast (TS) and extra-embryonic endoderm (XEN) stem cells to generate ETX-embryos. Although ETX-embryos transcriptionally capture the mouse gastrula, their ability to recapitulate complex morphogenic events such as gastrulation is limited, possibly due to the limited potential of XEN cells. To address this, we generated ES cells transiently expressing transcription factor Gata4 that drives the extra-embryonic endoderm fate and combined them together with ES cells and TS cells to generate induced ETX-embryos (iETX-embryos). We show that iETX-embryos establish a robust anterior signalling centre that migrates unilaterally to break embryo symmetry. Furthermore, iETX-embryos gastrulate generating embryonic and extra-embryonic mesoderm, and definitive endoderm. Our findings reveal that replacement of XEN cells with ES cells transiently expressing Gata4 endows iETX-embryos with greater developmental potential, thus enabling the study of the establishment of anterior-posterior patterning and gastrulation in an in vitro system.This work was supported by a European Research Council Grant (RG77946)Wellcome Trust (207415/Z/17/Z), Open Philanthropy, Shurl and Kay Curci, and Weston Havens Foundations grants awarded to M.Z.G.; K.Y.C.L. is supported by the Croucher Foundation and Cambridge Trust. F.H. is supported by a European Research Council Grant (695669) and Wellcome Trust (WT108438/C/15/Z). J.D.J. is supported by the Biotechnology and Biological Sciences Research Council

Inducible stem cell-derived embryos capture mouse morphogenetic events in vitro

The development of mouse embryos can be partially recapitulated by combining embryonic (ES), trophoblast (TS) and extra-embryonic endoderm (XEN) stem cells to generate ETX-embryos. Although ETX-embryos transcriptionally capture the mouse gastrula, their ability to recapitulate complex morphogenic events such as gastrulation is limited, possibly due to the limited potential of XEN cells. To address this, we generated ES cells transiently expressing transcription factor Gata4 that drives the extra-embryonic endoderm fate and combined them together with ES cells and TS cells to generate induced ETX-embryos (iETX-embryos). We show that iETX-embryos establish a robust anterior signalling centre that migrates unilaterally to break embryo symmetry. Furthermore, iETX-embryos gastrulate generating embryonic and extra-embryonic mesoderm, and definitive endoderm. Our findings reveal that replacement of XEN cells with ES cells transiently expressing Gata4 endows iETX-embryos with greater developmental potential, thus enabling the study of the establishment of anterior-posterior patterning and gastrulation in an in vitro system.This work was supported by a European Research Council Grant (RG77946)Wellcome Trust (207415/Z/17/Z), Open Philanthropy, Shurl and Kay Curci, and Weston Havens Foundations grants awarded to M.Z.G.; K.Y.C.L. is supported by the Croucher Foundation and Cambridge Trust. F.H. is supported by a European Research Council Grant (695669) and Wellcome Trust (WT108438/C/15/Z). J.D.J. is supported by the Biotechnology and Biological Sciences Research Council

Thermal stress induces glycolytic beige fat formation via a myogenic state.

Environmental cues profoundly affect cellular plasticity in multicellular organisms. For instance, exercise promotes a glycolytic-to-oxidative fibre-type switch in skeletal muscle, and cold acclimation induces beige adipocyte biogenesis in adipose tissue. However, the molecular mechanisms by which physiological or pathological cues evoke developmental plasticity remain incompletely understood. Here we report a type of beige adipocyte that has a critical role in chronic cold adaptation in the absence of β-adrenergic receptor signalling. This beige fat is distinct from conventional beige fat with respect to developmental origin and regulation, and displays enhanced glucose oxidation. We therefore refer to it as glycolytic beige fat. Mechanistically, we identify GA-binding protein α as a regulator of glycolytic beige adipocyte differentiation through a myogenic intermediate. Our study reveals a non-canonical adaptive mechanism by which thermal stress induces progenitor cell plasticity and recruits a distinct form of thermogenic cell that is required for energy homeostasis and survival

A single cell characterisation of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast centre.

Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development

A single cell characterisation of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast centre

Abstract: Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development

Garbage in, garbage out: how reliable training data improved a virtual screening approach against SARS-CoV-2 MPro

Introduction: The identification of chemical compounds that interfere with SARS-CoV-2 replication continues to be a priority in several academic and pharmaceutical laboratories. Computational tools and approaches have the power to integrate, process and analyze multiple data in a short time. However, these initiatives may yield unrealistic results if the applied models are not inferred from reliable data and the resulting predictions are not confirmed by experimental evidence.Methods: We undertook a drug discovery campaign against the essential major protease (MPro) from SARS-CoV-2, which relied on an in silico search strategy –performed in a large and diverse chemolibrary– complemented by experimental validation. The computational method comprises a recently reported ligand-based approach developed upon refinement/learning cycles, and structure-based approximations. Search models were applied to both retrospective (in silico) and prospective (experimentally confirmed) screening.Results: The first generation of ligand-based models were fed by data, which to a great extent, had not been published in peer-reviewed articles. The first screening campaign performed with 188 compounds (46 in silico hits and 100 analogues, and 40 unrelated compounds: flavonols and pyrazoles) yielded three hits against MPro (IC50 ≤ 25 μM): two analogues of in silico hits (one glycoside and one benzo-thiazol) and one flavonol. A second generation of ligand-based models was developed based on this negative information and newly published peer-reviewed data for MPro inhibitors. This led to 43 new hit candidates belonging to different chemical families. From 45 compounds (28 in silico hits and 17 related analogues) tested in the second screening campaign, eight inhibited MPro with IC50 = 0.12–20 μM and five of them also impaired the proliferation of SARS-CoV-2 in Vero cells (EC50 7–45 μM).Discussion: Our study provides an example of a virtuous loop between computational and experimental approaches applied to target-focused drug discovery against a major and global pathogen, reaffirming the well-known “garbage in, garbage out” machine learning principle

Investigating Brain Repair and Development Using Stem Cells

© 2019 Carlos William Baevski GantnerThe directed differentiation of human pluripotent stem cells into neuronal subtypes has generated immense enthusiasm that they could be used therapeutically or to study development in vitro. Two areas of particular interest are the replacement of midbrain dopaminergic neurons that degenerate in Parkinson’s disease and the ability to study the early development of the human cortex. The loss of midbrain dopamine neurons in Parkinson’s disease leads to a breakdown in basal ganglia circuitry and motor dysfunction. Previous clinical trials utilizing fetal dopamine tissue have provided proof-of-principle that transplantation of new dopamine neurons can relieve motor symptoms for up to two decades. However, the widespread use of fetal tissue in the clinic presents multiple ethical and logistical hurdles. As a result, the generation of human pluripotent stem cell-derived dopamine neurons has been an area of intense focus in recent years. Current protocols are not amenable to clinical translation and generate dopamine neurons that poorly reinnervate the striatum following transplantation. Here we develop a fully-defined protocol for midbrain dopamine generation and improve transplantation survival, plasticity and function via overexpression of glial cell line-derived neurotrophic factor. In contrast to dopaminergic differentiation strategies, protocols for deriving neurons of the neocortex are relatively limited, generating heterogenous populations of progenitors, neurons and glia. The human neocortex is arguably the most altered structure during mammalian evolution and underlies the cognitive abilities that define human intelligence. However, the majority of research into cortical development has been carried out in frog, chick or mouse models. Therefore, there is an unmet need to investigate how the relative complexity of the human cortex is developed. In particular, the intrinsic and extrinsic cues that control temporal development and drive the progressive generation of neuronal subtypes that form the six-layered mammalian neocortex remain unknown. Here we investigated the role of FGF-ERK signaling in the development of early-born, deep layer neurons of the neocortex in a reductionist pluripotent stem cell model. We find that FGF-ERK signaling, in part, modulates the timing and temporal progression of neocortical progenitors. Together, these studies advance the use of pluripotent stem cell tools to model neural development and to aid in neuroregeneration

Tissue programmed hydrogels functionalized with GDNF improve human neural grafts in Parkinson's disease

The survival and synaptic integration of transplanted dopaminergic (DA) progenitors are essential for ameliorating motor symptoms in Parkinson's disease (PD). Human pluripotent stem cell (hPSC)-derived DA progenitors are, however, exposed to numerous stressors prior to, and during, implantation that result in poor survival. Additionally, hPSC-derived grafts show inferior plasticity compared to fetal tissue grafts. These observations suggest that a more conducive host environment may improve graft outcomes. Here, tissue-specific support to DA progenitor grafts is provided with a fully characterized self-assembling peptide hydrogel. This biomimetic hydrogel matrix is programmed to support DA progenitors by i) including a laminin epitope within the matrix; and ii) shear encapsulating glial cell line-derived neurotrophic factor (GDNF) to ensure its sustained delivery. The biocompatible hydrogel biased a 51% increase in A9 neuron specification—a subpopulation of DA neurons critical for motor function. The sustained delivery of GDNF induced a 2.7-fold increase in DA neurons and enhanced graft plasticity, resulting in significant improvements in motor deficits at 6 months. These findings highlight the therapeutic benefit of stepwise customization of tissue-specific hydrogels to improve the physical and trophic support of human PSC-derived neural transplants, resulting in improved standardization, predictability and functional efficacy of grafts for PD. © 2021 Wiley-VCH GmbH. **Please note that there are multiple authors for this article therefore only the name of the first 5 including Federation University Australia affiliate “Benjamin Long” is provided in this record*

Mouse embryo model derived exclusively from embryonic stem cells undergoes neurulation and heart development.

Several in vitro models have been developed to recapitulate mouse embryogenesis solely from embryonic stem cells (ESCs). Despite mimicking many aspects of early development, they fail to capture the interactions between embryonic and extraembryonic tissues. To overcome this difficulty, we have developed a mouse ESC-based in vitro model that reconstitutes the pluripotent ESC lineage and the two extraembryonic lineages of the post-implantation embryo by transcription-factor-mediated induction. This unified model recapitulates developmental events from embryonic day 5.5 to 8.5, including gastrulation; formation of the anterior-posterior axis, brain, and a beating heart structure; and the development of extraembryonic tissues, including yolk sac and chorion. Comparing single-cell RNA sequencing from individual structures with time-matched natural embryos identified remarkably similar transcriptional programs across lineages but also showed when and where the model diverges from the natural program. Our findings demonstrate an extraordinary plasticity of ESCs to self-organize and generate a whole-embryo-like structure