Toward developing human organs via embryo models and chimeras.

Developing functional organs from stem cells remains a challenging goal in regenerative medicine. Existing methodologies, such as tissue engineering, bioprinting, and organoids, only offer partial solutions. This perspective focuses on two promising approaches emerging for engineering human organs from stem cells: stem cell-based embryo models and interspecies organogenesis. Both approaches exploit the premise of guiding stem cells to mimic natural development. We begin by summarizing what is known about early human development as a blueprint for recapitulating organogenesis in both embryo models and interspecies chimeras. The latest advances in both fields are discussed before highlighting the technological and knowledge gaps to be addressed before the goal of developing human organs could be achieved using the two approaches. We conclude by discussing challenges facing embryo modeling and interspecies organogenesis and outlining future prospects for advancing both fields toward the generation of human tissues and organs for basic research and translational applications.

Advancing the ethical dialogue about monkey/human chimeric embryos.

In this issue of Cell, Tan et al. report the first injection of human stem cells into in vitro non-human primate blastocysts with significant survival of the human cells, raising new scientific possibilities but also important ethical issues.

Niche-Selective Inhibition of Pathogenic Th17 Cells by Targeting Metabolic Redundancy.

Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

Adenovirus vector vaccination reprograms pulmonary fibroblastic niches to support protective inflating memory CD8+ T cells.

Pathogens and vaccines that produce persisting antigens can generate expanded pools of effector memory CD8+ T cells, described as memory inflation. While properties of inflating memory CD8+ T cells have been characterized, the specific cell types and tissue factors responsible for their maintenance remain elusive. Here, we show that clinically applied adenovirus vectors preferentially target fibroblastic stromal cells in cultured human tissues. Moreover, we used cell-type-specific antigen targeting to define critical cells and molecules that sustain long-term antigen presentation and T cell activity after adenovirus vector immunization in mice. While antigen targeting to myeloid cells was insufficient to activate antigen-specific CD8+ T cells, genetic activation of antigen expression in Ccl19-cre-expressing fibroblastic stromal cells induced inflating CD8+ T cells. Local ablation of vector-targeted cells revealed that lung fibroblasts support the protective function and metabolic fitness of inflating memory CD8+ T cells in an interleukin (IL)-33-dependent manner. Collectively, these data define a critical fibroblastic niche that underpins robust protective immunity operating in a clinically important vaccine platform.

Identification of piRNA Binding Sites Reveals the Argonaute Regulatory Landscape of the C. elegans Germline.

piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs tolerate mismatched pairing and in principle could target every transcript. Here we employ in vivo cross-linking to identify transcriptome-wide interactions between piRNAs and target RNAs. We show that piRNAs engage all germline mRNAs and that piRNA binding follows microRNA-like pairing rules. Targeting correlates better with binding energy than with piRNA abundance, suggesting that piRNA concentration does not limit targeting. In mRNAs silenced by piRNAs, secondary small RNAs accumulate at the center and ends of piRNA binding sites. In germline-expressed mRNAs, however, targeting by the CSR-1 Argonaute correlates with reduced piRNA binding density and suppression of piRNA-associated secondary small RNAs. Our findings reveal physiologically important and nuanced regulation of individual piRNA targets and provide evidence for a comprehensive post-transcriptional regulatory step in germline gene expression.

Clonal Evolution of Autoreactive Germinal Centers.

Germinal centers (GCs) are the primary sites of clonal B cell expansion and affinity maturation, directing the production of high-affinity antibodies. This response is a central driver of pathogenesis in autoimmune diseases, such as systemic lupus erythematosus (SLE), but the natural history of autoreactive GCs remains unclear. Here, we present a novel mouse model where the presence of a single autoreactive B cell clone drives the TLR7-dependent activation, expansion, and differentiation of other autoreactive B cells in spontaneous GCs. Once tolerance was broken for one self-antigen, autoreactive GCs generated B cells targeting other self-antigens. GCs became independent of the initial clone and evolved toward dominance of individual clonal lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease.

Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency.

Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research. VIDEO ABSTRACT.

Generation and Application of Mouse-Rat Allodiploid Embryonic Stem Cells.

Mammalian interspecific hybrids provide unique advantages for mechanistic studies of speciation, gene expression regulation, and X chromosome inactivation (XCI) but are constrained by their limited natural resources. Previous artificially generated mammalian interspecific hybrid cells are usually tetraploids with unstable genomes and limited developmental abilities. Here, we report the generation of mouse-rat allodiploid embryonic stem cells (AdESCs) by fusing haploid ESCs of the two species. The AdESCs have a stable allodiploid genome and are capable of differentiating into all three germ layers and early-stage germ cells. Both the mouse and rat alleles have comparable contributions to the expression of most genes. We have proven AdESCs as a powerful tool to study the mechanisms regulating X chromosome inactivation and to identify X inactivation-escaping genes, as well as to efficiently identify genes regulating phenotypic differences between species. A similar method could be used to create hybrid AdESCs of other distantly related species.

Lessons from Interspecies Mammalian Chimeras.

As chimeras transform from beasts of Greek mythology into tools of contemporary bioscience, secrets of developmental biology and evolutionary divergence are being revealed. Recent advances in stem cell biology and interspecies chimerism have generated new models with extensive basic and translational applications, including generation of transplantable, patient-specific organs.

The transcription factor Rfx7 limits metabolism of NK cells and promotes their maintenance and immunity.

Regulatory factor X 7 (Rfx7) is an uncharacterized transcription factor belonging to a family involved in ciliogenesis and immunity. Here, we found that deletion of Rfx7 leads to a decrease in natural killer (NK) cell maintenance and immunity in vivo. Genomic approaches showed that Rfx7 coordinated a transcriptional network controlling cell metabolism. Rfx7-/- NK lymphocytes presented increased size, granularity, proliferation, and energetic state, whereas genetic reduction of mTOR activity mitigated those defects. Notably, Rfx7-deficient NK lymphocytes were rescued by interleukin 15 through engagement of the Janus kinase (Jak) pathway, thus revealing the importance of this signaling for maintenance of such spontaneously activated NK cells. Rfx7 therefore emerges as a novel transcriptional regulator of NK cell homeostasis and metabolic quiescence.

Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.

Haploid mouse embryonic stem cells: rapid genetic screening and germline transmission.

Most animal genomes are diploid, and mammalian development depends on specific adaptations that have evolved secondary to diploidy. Genomic imprinting and dosage compensation restrict haploid development to early embryos. Recently, haploid mammalian development has been reinvestigated since the establishment of haploid embryonic stem cells (ESCs) from mouse embryos. Haploid cells possess one copy of each gene, facilitating the generation of loss-of-function mutations in a single step. Recessive mutations can then be assessed in forward genetic screens. Applications of haploid mammalian cell systems in screens have been illustrated in several recent publications. Haploid ESCs are characterized by a wide developmental potential and can contribute to chimeric embryos and mice. Different strategies for introducing genetic modifications from haploid ESCs into the mouse germline have been further developed. Haploid ESCs therefore introduce new possibilities in mammalian genetics and could offer an unprecedented tool for genome exploration in the future.

Standard Deviations: The Biological Bases of Transmission Ratio Distortion.

The rule of Mendelian inheritance is remarkably robust, but deviations from the equal transmission of alternative alleles at a locus [a.k.a. transmission ratio distortion (TRD)] are also commonly observed in genetic mapping populations. Such TRD reveals locus-specific selection acting at some point between the diploid heterozygous parents and progeny genotyping and therefore can provide novel insight into otherwise-hidden genetic and evolutionary processes. Most of the classic selfish genetic elements were discovered through their biasing of transmission, but many unselfish evolutionary and developmental processes can also generate TRD. In this review, we describe methodologies for detecting TRD in mapping populations, detail the arenas and genetic interactions that shape TRD during plant and animal reproduction, and summarize patterns of TRD from across the genetic mapping literature. Finally, we point to new experimental approaches that can accelerate both detection of TRD and characterization of the underlying genetic mechanisms.

Living with Two Genomes: Grafting and Its Implications for Plant Genome-to-Genome Interactions, Phenotypic Variation, and Evolution.

Plant genomes interact when genetically distinct individuals join, or are joined, together. Individuals can fuse in three contexts: artificial grafts, natural grafts, and host-parasite interactions. Artificial grafts have been studied for decades and are important platforms for studying the movement of RNA, DNA, and protein. Yet several mysteries about artificial grafts remain, including the factors that contribute to graft incompatibility, the prevalence of genetic and epigenetic modifications caused by exchanges between graft partners, and the long-term effects of these modifications on phenotype. Host-parasite interactions also lead to the exchange of materials, and RNA exchange actively contributes to an ongoing arms race between parasite virulence and host resistance. Little is known about natural grafts except that they can be frequent and may provide opportunities for evolutionary innovation through genome exchange. In this review, we survey our current understanding about these three mechanisms of contact, the genomic interactions that result, and the potential evolutionary implications.

Generation of chimeric rhesus monkeys.

Totipotent cells in early embryos are progenitors of all stem cells and are capable of developing into a whole organism, including extraembryonic tissues such as placenta. Pluripotent cells in the inner cell mass (ICM) are the descendants of totipotent cells and can differentiate into any cell type of a body except extraembryonic tissues. The ability to contribute to chimeric animals upon reintroduction into host embryos is the key feature of murine totipotent and pluripotent cells. Here, we demonstrate that rhesus monkey embryonic stem cells (ESCs) and isolated ICMs fail to incorporate into host embryos and develop into chimeras. However, chimeric offspring were produced following aggregation of totipotent cells of the four-cell embryos. These results provide insights into the species-specific nature of primate embryos and suggest that a chimera assay using pluripotent cells may not be feasible.

Induced pluripotent stem cells in disease modelling and drug discovery.

The derivation of induced pluripotent stem cells (iPSCs) over a decade ago sparked widespread enthusiasm for the development of new models of human disease, enhanced platforms for drug discovery and more widespread use of autologous cell-based therapy. Early studies using directed differentiation of iPSCs frequently uncovered cell-level phenotypes in monogenic diseases, but translation to tissue-level and organ-level diseases has required development of more complex, 3D, multicellular systems. Organoids and human-rodent chimaeras more accurately mirror the diverse cellular ecosystems of complex tissues and are being applied to iPSC disease models to recapitulate the pathobiology of a broad spectrum of human maladies, including infectious diseases, genetic disorders and cancer.

Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells.

The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment.

A single-cell molecular map of mouse gastrulation and early organogenesis.

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.

A human liver cell atlas reveals heterogeneity and epithelial progenitors.

The human liver is an essential multifunctional organ. The incidence of liver diseases is rising and there are limited treatment options. However, the cellular composition of the liver remains poorly understood. Here we performed single-cell RNA sequencing of about 10,000 cells from normal liver tissue from nine human donors to construct a human liver cell atlas. Our analysis identified previously unknown subtypes of endothelial cells, Kupffer cells, and hepatocytes, with transcriptome-wide zonation of some of these populations. We show that the EPCAM+ population is heterogeneous, comprising hepatocyte-biased and cholangiocyte populations as well as a TROP2int progenitor population with strong potential to form bipotent liver organoids. As a proof-of-principle, we used our atlas to unravel the phenotypic changes that occur in hepatocellular carcinoma cells and in human hepatocytes and liver endothelial cells engrafted into a mouse liver. Our human liver cell atlas provides a powerful resource to enable the discovery of previously unknown cell types in normal and diseased livers.

Uncovering the enigmatic evolution of bears in greater depth: The hybrid origin of the Asiatic black bear.

Bears are fascinating mammals because of their complex pattern of speciation and rapid evolution of distinct phenotypes. Interspecific hybridization has been common and has shaped the complex evolutionary history of bears. In this study, based on the largest population-level genomic dataset to date involving all Ursinae species and recently developed methods for detecting hybrid speciation, we provide explicit evidence for the hybrid origin of Asiatic black bears, which arose through historical hybridization between the ancestor of polar bear/brown bear/American black bears and the ancestor of sun bear/sloth bears. This was inferred to have occurred soon after the divergence of the two parental lineages in Eurasia due to climate-driven population expansion and dispersal. In addition, we found that the intermediate body size of this hybrid species arose from its combination of relevant genes derived from two parental lineages of contrasting sizes. This and alternate fixation of numerous other loci that had diverged between parental lineages may have initiated the reproductive isolation of the Asiatic black bear from its two parents. Our study sheds further light on the evolutionary history of bears and documents the importance of hybridization in new species formation and phenotypic evolution in mammals.