Modelling post-implantation human development to yolk sac blood emergence.

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system1,2. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons3-5. Stem cell models of human embryo have emerged to help unlock the mysteries of this stage6-16. Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling.

Biliary atresia is associated with polygenic susceptibility in ciliogenesis and planar polarity effector genes.

BACKGROUND & AIMS: Biliary atresia (BA) is poorly understood and leads to liver transplantation (LT), with the requirement for and associated risks of lifelong immunosuppression, in most children. We performed a genome-wide association study (GWAS) to determine the genetic basis of BA. METHODS: We performed a GWAS in 811 European BA cases treated with LT in US, Canadian and UK centers, and 4,654 genetically matched controls. Whole-genome sequencing of 100 cases evaluated synthetic association with rare variants. Functional studies included whole liver transcriptome analysis of 64 BA cases and perturbations in experimental models. RESULTS: A GWAS of common single nucleotide polymorphisms (SNPs), i.e. allele frequencies >1%, identified intronic SNPs rs6446628 in AFAP1 with genome-wide significance (p = 3.93E-8) and rs34599046 in TUSC3 at sub-threshold genome-wide significance (p = 1.34E-7), both supported by credible peaks of neighboring SNPs. Like other previously reported BA-associated genes, AFAP1 and TUSC3 are ciliogenesis and planar polarity effectors (CPLANE). In gene-set-based GWAS, BA was associated with 6,005 SNPs in 102 CPLANE genes (p = 5.84E-15). Compared with non-CPLANE genes, more CPLANE genes harbored rare variants (allele frequency <1%) that were assigned Human Phenotype Ontology terms related to hepatobiliary anomalies by predictive algorithms, 87% vs. 40%, p <0.0001. Rare variants were present in multiple genes distinct from those with BA-associated common variants in most BA cases. AFAP1 and TUSC3 knockdown blocked ciliogenesis in mouse tracheal cells. Inhibition of ciliogenesis caused biliary dysgenesis in zebrafish. AFAP1 and TUSC3 were expressed in fetal liver organoids, as well as fetal and BA livers, but not in normal or disease-control livers. Integrative analysis of BA-associated variants and liver transcripts revealed abnormal vasculogenesis and epithelial tube formation, explaining portal vein anomalies that co-exist with BA. CONCLUSIONS: BA is associated with polygenic susceptibility in CPLANE genes. Rare variants contribute to polygenic risk in vulnerable pathways via unique genes. IMPACT AND IMPLICATIONS: Liver transplantation is needed to cure most children born with biliary atresia, a poorly understood rare disease. Transplant immunosuppression increases the likelihood of life-threatening infections and cancers. To improve care by preventing this disease and its progression to transplantation, we examined its genetic basis. We find that this disease is associated with both common and rare mutations in highly specialized genes which maintain normal communication and movement of cells, and their organization into bile ducts and blood vessels during early development of the human embryo. Because defects in these genes also cause other birth defects, our findings could lead to preventive strategies to lower the incidence of biliary atresia and potentially other birth defects.

Modelling Human Post-Implantation Development via Extra-Embryonic Niche Engineering.

Implantation of the human embryo commences a critical developmental stage that comprises profound morphogenetic alteration of embryonic and extra-embryonic tissues, axis formation, and gastrulation events. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons. Additionally, human stem cell models of early post-implantation development with both embryonic and extra-embryonic tissue morphogenesis are lacking. Here, we present iDiscoid, produced from human induced pluripotent stem cells via an engineered a synthetic gene circuit. iDiscoids exhibit reciprocal co-development of human embryonic tissue and engineered extra-embryonic niche in a model of human post-implantation. They exhibit unanticipated self-organization and tissue boundary formation that recapitulates yolk sac-like tissue specification with extra-embryonic mesoderm and hematopoietic characteristics, the formation of bilaminar disc-like embryonic morphology, the development of an amniotic-like cavity, and acquisition of an anterior-like hypoblast pole and posterior-like axis. iDiscoids offer an easy-to-use, high-throughput, reproducible, and scalable platform to probe multifaceted aspects of human early post-implantation development. Thus, they have the potential to provide a tractable human model for drug testing, developmental toxicology, and disease modeling.

Platform-agnostic CellNet enables cross-study analysis of cell fate engineering protocols.

Optimization of cell engineering protocols requires standard, comprehensive quality metrics. We previously developed CellNet, a computational tool to quantitatively assess the transcriptional fidelity of engineered cells compared with their natural counterparts, based on bulk-derived expression profiles. However, this platform and others were limited in their ability to compare data from different sources, and no current tool makes it easy to compare new protocols with existing state-of-the-art protocols in a standardized manner. Here, we utilized our prior application of the top-scoring pair transformation to build a computational platform, platform-agnostic CellNet (PACNet), to address both shortcomings. To demonstrate the utility of PACNet, we applied it to thousands of samples from over 100 studies that describe dozens of protocols designed to produce seven distinct cell types. We performed an in-depth examination of hepatocyte and cardiomyocyte protocols to identify the best-performing methods, characterize the extent of intra-protocol and inter-lab variation, and identify common off-target signatures, including a surprising neural/neuroendocrine signature in primary liver-derived organoids. We have made PACNet available as an easy-to-use web application, allowing users to assess their protocols relative to our database of reference engineered samples, and as open-source, extensible code.

Genetically engineering endothelial niche in human kidney organoids enables multilineage maturation, vascularization and de novo cell types.

Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation in a clinical setting thereby hinges on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcome this hurdle by combining an inducible ETS translocation variant 2 (ETV2) human induced pluripotent stem cell (iPSC) line, which directs endothelial fate, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive vascularization by endothelial cells with an identity most closely related to endogenous kidney endothelia. Vascularized organoids also show increased maturation of nephron structures including more mature podocytes with improved marker expression, foot process interdigitation, an associated fenestrated endothelium, and the presence of renin+ cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Furthermore, this approach is orthogonal to native tissue differentiation paths, hence readily adaptable to other organoid systems and thus has the potential for a broad impact on basic and translational organoid studies.

Therapeutic Cell Repopulation of the Liver: From Fetal Rat Cells to Synthetic Human Tissues.

Progenitor cells isolated from the fetal liver can provide a unique cell source to generate new healthy tissue mass. Almost 20 years ago, it was demonstrated that rat fetal liver cells repopulate the normal host liver environment via a mechanism akin to cell competition. Activin A, which is produced by hepatocytes, was identified as an important player during cell competition. Because of reduced activin receptor expression, highly proliferative fetal liver stem/progenitor cells are resistant to activin A and therefore exhibit a growth advantage compared to hepatocytes. As a result, transplanted fetal liver cells are capable of repopulating normal livers. Important for cell-based therapies, hepatic stem/progenitor cells containing repopulation potential can be separated from fetal hematopoietic cells using the cell surface marker δ-like 1 (Dlk-1). In livers with advanced fibrosis, fetal epithelial stem/progenitor cells differentiate into functional hepatic cells and out-compete injured endogenous hepatocytes, which cause anti-fibrotic effects. Although fetal liver cells efficiently repopulate the liver, they will likely not be used for human cell transplantation. Thus, utilizing the underlying mechanism of repopulation and developed methods to produce similar growth-advantaged cells in vitro, e.g., human induced pluripotent stem cells (iPSCs), this approach has great potential for developing novel cell-based therapies in patients with liver disease. The present review gives a brief overview of the classic cell transplantation models and various cell sources studied as donor cell candidates. The advantages of fetal liver-derived stem/progenitor cells are discussed, as well as the mechanism of liver repopulation. Moreover, this article reviews the potential of in vitro developed synthetic human fetal livers from iPSCs and their therapeutic benefits.

TraSig: inferring cell-cell interactions from pseudotime ordering of scRNA-Seq data.

A major advantage of single cell RNA-sequencing (scRNA-Seq) data is the ability to reconstruct continuous ordering and trajectories for cells. Here we present TraSig, a computational method for improving the inference of cell-cell interactions in scRNA-Seq studies that utilizes the dynamic information to identify significant ligand-receptor pairs with similar trajectories, which in turn are used to score interacting cell clusters. We applied TraSig to several scRNA-Seq datasets and obtained unique predictions that improve upon those identified by prior methods. Functional experiments validate the ability of TraSig to identify novel signaling interactions that impact vascular development in liver organoids.Software https://github.com/doraadong/TraSig .

Synthetic living machines: A new window on life.

Increased control of biological growth and form is an essential gateway to transformative medical advances. Repairing of birth defects, restoring lost or damaged organs, normalizing tumors, all depend on understanding how cells cooperate to make specific, functional large-scale structures. Despite advances in molecular genetics, significant gaps remain in our understanding of the meso-scale rules of morphogenesis. An engineering approach to this problem is the creation of novel synthetic living forms, greatly extending available model systems beyond evolved plant and animal lineages. Here, we review recent advances in the emerging field of synthetic morphogenesis, the bioengineering of novel multicellular living bodies. Emphasizing emergent self-organization, tissue-level guided self-assembly, and active functionality, this work is the essential next generation of synthetic biology. Aside from useful living machines for specific functions, the rational design and analysis of new, coherent anatomies will greatly increase our understanding of foundational questions in evolutionary developmental and cell biology.

Computational tools for analyzing single-cell data in pluripotent cell differentiation studies.

Single-cell technologies are revolutionizing the ability of researchers to infer the causes and results of biological processes. Although several studies of pluripotent cell differentiation have recently utilized single-cell sequencing data, other aspects related to the optimization of differentiation protocols, their validation, robustness, and usage are still not taking full advantage of single-cell technologies. In this review, we focus on computational approaches for the analysis of single-cell omics and imaging data and discuss their use to address many of the major challenges involved in the development, validation, and use of cells obtained from pluripotent cell differentiation.

Human biomimetic liver microphysiology systems in drug development and precision medicine.

Microphysiology systems (MPS), also called organs-on-chips and tissue chips, are miniaturized functional units of organs constructed with multiple cell types under a variety of physical and biochemical environmental cues that complement animal models as part of a new paradigm of drug discovery and development. Biomimetic human liver MPS have evolved from simpler 2D cell models, spheroids and organoids to address the increasing need to understand patient-specific mechanisms of complex and rare diseases, the response to therapeutic treatments, and the absorption, distribution, metabolism, excretion and toxicity of potential therapeutics. The parallel development and application of transdisciplinary technologies, including microfluidic devices, bioprinting, engineered matrix materials, defined physiological and pathophysiological media, patient-derived primary cells, and pluripotent stem cells as well as synthetic biology to engineer cell genes and functions, have created the potential to produce patient-specific, biomimetic MPS for detailed mechanistic studies. It is projected that success in the development and maturation of patient-derived MPS with known genotypes and fully matured adult phenotypes will lead to advanced applications in precision medicine. In this Review, we examine human biomimetic liver MPS that are designed to recapitulate the liver acinus structure and functions to enhance our knowledge of the mechanisms of disease progression and of the absorption, distribution, metabolism, excretion and toxicity of therapeutic candidates and drugs as well as to evaluate their mechanisms of action and their application in precision medicine and preclinical trials.

Neighborhood Impact Factor to Study Cell-Fate Decision-Making in Cellular Communities.

Cell-fate determination is a function of cell-intrinsic and -extrinsic signaling cues. Understanding the design principles governing fate control in multicellular systems remains difficult to understand and analyze. To address the current challenges of spatial analysis of potential signaling events, we have developed a pipeline for assessment of the neighboring cells at defined areas in the vicinity of target cells using a newly defined concept of Neighborhood Impact Factor. We have used our pipeline to interrogate cellular decision-making in a genetically derived multi-lineage liver organoid from induced pluripotent stem cells. We examined endothelial versus hepatocyte fate determination for cells with similar expression level of an engineered driver gene circuit. Our analysis suggests that the relative level of gene expression to the neighbor population can control the final fate choice in our engineered liver multicellular system.

Gene Regulatory Network Analysis and Engineering Directs Development and Vascularization of Multilineage Human Liver Organoids.

Pluripotent stem cell (PSC)-derived organoids have emerged as novel multicellular models of human tissue development but display immature phenotypes, aberrant tissue fates, and a limited subset of cells. Here, we demonstrate that integrated analysis and engineering of gene regulatory networks (GRNs) in PSC-derived multilineage human liver organoids direct maturation and vascular morphogenesis in vitro. Overexpression of PROX1 and ATF5, combined with targeted CRISPR-based transcriptional activation of endogenous CYP3A4, reprograms tissue GRNs and improves native liver functions, such as FXR signaling, CYP3A4 enzymatic activity, and stromal cell reactivity. The engineered tissues possess superior liver identity when compared with other PSC-derived liver organoids and show the presence of hepatocyte, biliary, endothelial, and stellate-like cell populations in single-cell RNA-seq analysis. Finally, they show hepatic functions when studied in vivo. Collectively, our approach provides an experimental framework to direct organogenesis in vitro by systematically probing molecular pathways and transcriptional networks that promote tissue development.

Synthetic immunomodulation with a CRISPR super-repressor in vivo.

Transient modulation of the genes involved in immunity, without exerting a permanent change in the DNA code, can be an effective strategy to modulate the course of many inflammatory conditions. CRISPR-Cas9 technology represents a promising platform for achieving this goal. Truncation of guide RNA (gRNA) from the 5' end enables the application of a nuclease competent Cas9 protein for transcriptional modulation of genes, allowing multifunctionality of CRISPR. Here, we introduce an enhanced CRISPR-based transcriptional repressor to reprogram immune homeostasis in vivo. In this repressor system, two transcriptional repressors-heterochromatin protein 1 (HP1a) and Krüppel-associated box (KRAB)-are fused to the MS2 coat protein and subsequently recruited by gRNA aptamer binding to a nuclease competent CRISPR complex containing truncated gRNAs. With the enhanced repressor, we demonstrate transcriptional repression of the Myeloid differentiation primary response 88 (Myd88) gene in vitro and in vivo. We demonstrate that this strategy can efficiently downregulate Myd88 expression in lung, blood and bone marrow of Cas9 transgenic mice that receive systemic injection of adeno-associated virus (AAV)2/1-carrying truncated gRNAs targeting Myd88 and the MS2-HP1a-KRAB cassette. This downregulation is accompanied by changes in downstream signalling elements such as TNF-α and ICAM-1. Myd88 repression leads to a decrease in immunoglobulin G (IgG) production against AAV2/1 and AAV2/9 and this strategy modulates the IgG response against AAV cargos. It improves the efficiency of a subsequent AAV9/CRISPR treatment for repression of proprotein convertase subtilisin/kexin type 9 (PCSK9), a gene that, when repressed, can lower blood cholesterol levels. We also demonstrate that CRISPR-mediated Myd88 repression can act as a prophylactic measure against septicaemia in both Cas9 transgenic and C57BL/6J mice. When delivered by nanoparticles, this repressor can serve as a therapeutic modality to influence the course of septicaemia. Collectively, we report that CRISPR-mediated repression of endogenous Myd88 can effectively modulate the host immune response against AAV-mediated gene therapy and influence the course of septicaemia. The ability to control Myd88 transcript levels using a CRISPR-based synthetic repressor can be an effective strategy for AAV-based CRISPR therapies, as this pathway serves as a key node in the induction of humoral immunity against AAV serotypes.

CRISPR-Based Synthetic Transcription Factors In Vivo: The Future of Therapeutic Cellular Programming.

Pinpoint control over endogenous gene expression in vivo has long been a fevered dream for clinicians and researchers alike. With the recent repurposing of programmable, RNA-guided DNA endonucleases from the CRISPR bacterial immune system, this dream is becoming a powerful reality. Engineered CRISPR/Cas9-based transcriptional regulators and epigenome editors have enabled researchers to perturb endogenous gene expression in vivo, allowing for the therapeutic reprogramming of cell and tissue behavior. For this technology to be of maximal use, a variety of technological hurdles still need to be addressed. Better understanding of the design principle controlling gene expression together with technologies that enable spatiotemporal control of transcriptional engineering are fundamental for rational design, improved efficacy, and ultimately safe translation to humans. In this review, we will discuss recent advances and integrative strategies that can help pave the path toward a new class of transcriptional therapeutics.

Multicellular Systems to Translate Somatic Cell Genome Editors to Humans.

As genome editors move into clinical trials, there is a need to establish ex vivo multicellular systems to rapidly assess and predict toxic effects of genome editors in physiologically relevant human models. Advancements in organoid and organs-on-chip technologies offer the possibility to create multicellular systems that replicate the cellular composition and metabolic function of native tissues. Some multicellular systems have been validated in multiple applications for drug discovery and could be easily adapted to test genome editors; other models, especially those of the adaptive immune system, will require validation before being used as benchmarks for testing genome editors. Likewise, protocols to assess immunogenicity, to detect off-target effects, and to predict ex vivo to in vivo translation will need to be established and validated. This review will discuss key aspects to consider when designing, building, and/or adopting in vitro human multicellular systems for testing genome editors.

Synthetic developmental biology: build and control multicellular systems.

Synthetic biology offers a bottom-up engineering approach that intends to understand complex systems via design-build-test cycles. Embryonic development comprises complex processes that originate at the level of gene regulatory networks in a cell and emerge into collective cellular behaviors with multicellular forms and functions. Here, we review synthetic biology approaches to development that involve building de novo developmental trajectories or engineering control in stem cell-derived multicellular systems. The field of synthetic developmental biology is rapidly growing with the help of recent advances in artificial gene circuits, self-organizing organoids, and controllable tissue microenvironments. The outcome will be a blueprint to decode principles of morphogenesis and to create programmable organoids with novel designs or improved functions.

Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes.

The CRISPR-Cas9 system has raised hopes for developing personalized gene therapies for complex diseases. Its application for genetic and epigenetic therapies in humans raises concerns over immunogenicity of the bacterially derived Cas9 protein. Here we detect antibodies to Streptococcus pyogenes Cas9 (SpCas9) in at least 5% of 143 healthy individuals. We also report pre-existing human CD8+T cell immunity in the majority of healthy individuals screened. We identify two immunodominant SpCas9 T cell epitopes for HLA-A*02:01 using an enhanced prediction algorithm that incorporates T cell receptor contact residue hydrophobicity and HLA binding and evaluated them by T cell assays using healthy donor PBMCs. In a proof-of-principle study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity. Our study highlights the problem of pre-existing immunity against CRISPR-associated nucleases and offers a potential solution to mitigate the T cell immune response.