RESUMEN
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.
Asunto(s)
Quimera , Organogénesis , Animales , Humanos , Quimera/embriología , Implantación del Embrión , Embrión de Mamíferos/citología , Desarrollo Embrionario , Células Madre Embrionarias , Modelos Biológicos , Organoides , Medicina Regenerativa , Ingeniería de Tejidos/métodosRESUMEN
Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.
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Prosencéfalo , Animales , Prosencéfalo/metabolismo , Prosencéfalo/embriología , Ratones , Ratas , Blastocisto/metabolismo , Femenino , Sistemas CRISPR-Cas/genética , Transcriptoma , Organogénesis , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Masculino , Ratones Endogámicos C57BLRESUMEN
A central question for regenerative neuroscience is whether synthetic neural circuits, such as those built from two species, can function in an intact brain. Here, we apply blastocyst complementation to selectively build and test interspecies neural circuits. Despite approximately 10-20 million years of evolution, and prominent species differences in brain size, rat pluripotent stem cells injected into mouse blastocysts develop and persist throughout the mouse brain. Unexpectedly, the mouse niche reprograms the birth dates of rat neurons in the cortex and hippocampus, supporting rat-mouse synaptic activity. When mouse olfactory neurons are genetically silenced or killed, rat neurons restore information flow to odor processing circuits. Moreover, they rescue the primal behavior of food seeking, although less well than mouse neurons. By revealing that a mouse can sense the world using neurons from another species, we establish neural blastocyst complementation as a powerful tool to identify conserved mechanisms of brain development, plasticity, and repair.
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Neuronas , Animales , Ratones , Ratas , Neuronas/metabolismo , Neuronas/citología , Neuronas/fisiología , Blastocisto/metabolismo , Blastocisto/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Encéfalo/citología , Encéfalo/fisiología , Femenino , Hipocampo/citología , Hipocampo/fisiología , Especificidad de la Especie , Ratones Endogámicos C57BL , MasculinoRESUMEN
In vitro stem cell models that replicate human gastrulation have been generated, but they lack the essential extraembryonic cells needed for embryonic development, morphogenesis, and patterning. Here, we describe a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, termed peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. Although peri-gastruloids are not viable due to the exclusion of trophoblasts, they recapitulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and undergoing early neurulation and organogenesis. Single-cell RNA-sequencing unveiled transcriptomic similarities between advanced human peri-gastruloids and primary peri-gastrulation cell types found in humans and non-human primates. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.
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Implantación del Embrión , Gastrulación , Células Madre Pluripotentes , Animales , Femenino , Humanos , Embarazo , Diferenciación Celular , Embrión de Mamíferos , Desarrollo Embrionario , Organogénesis , Células Madre Pluripotentes/metabolismo , PrimatesRESUMEN
Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-ß, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.
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Embrión de Mamíferos , Células Madre Embrionarias , Animales , Técnicas de Cocultivo , Macaca fascicularis , Células Madre Embrionarias/metabolismo , Diferenciación Celular , Endodermo/metabolismo , Linaje de la CélulaRESUMEN
The third and fourth weeks of gestation in primates are marked by several developmental milestones, including gastrulation and the formation of organ primordia. However, our understanding of this period is limited due to restricted access to in vivo embryos. To address this gap, we developed an embedded 3D culture system that allows for the extended ex utero culture of cynomolgus monkey embryos for up to 25 days post-fertilization. Morphological, histological, and single-cell RNA-sequencing analyses demonstrate that ex utero cultured monkey embryos largely recapitulated key events of in vivo development. With this platform, we were able to delineate lineage trajectories and genetic programs involved in neural induction, lateral plate mesoderm differentiation, yolk sac hematopoiesis, primitive gut, and primordial germ-cell-like cell development in monkeys. Our embedded 3D culture system provides a robust and reproducible platform for growing monkey embryos from blastocysts to early organogenesis and studying primate embryogenesis ex utero.
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Embrión de Mamíferos , Desarrollo Embrionario , Animales , Macaca fascicularis , Blastocisto , Organogénesis , PrimatesRESUMEN
Tumor angiogenesis and immunity show an inverse correlation in cancer progression and outcome1. Here, we report that ZBTB46, a repressive transcription factor and a widely accepted marker for classical dendritic cells (DCs)2,3, controls both tumor angiogenesis and immunity. Zbtb46 was downregulated in both DCs and endothelial cells by tumor-derived factors to facilitate robust tumor growth. Zbtb46 downregulation led to a hallmark pro-tumor microenvironment (TME), including dysfunctional vasculature and immunosuppressive conditions. Analysis of human cancer data revealed a similar association of low ZBTB46 expression with an immunosuppressive TME and a worse prognosis. In contrast, enforced Zbtb46 expression led to TME changes to restrict tumor growth. Mechanistically, Zbtb46-deficient endothelial cells were highly angiogenic, and Zbtb46-deficient bone marrow progenitors upregulated Cebpb and diverted the DC program to immunosuppressive myeloid lineage output, potentially explaining the myeloid lineage skewing phenomenon in cancer4. Conversely, enforced Zbtb46 expression normalized tumor vessels and, by suppressing Cebpb, skewed bone marrow precursors toward immunostimulatory myeloid lineage output, leading to an immune-hot TME. Remarkably, Zbtb46 mRNA treatment synergized with anti-PD1 immunotherapy to improve tumor management in preclinical models. These findings identify ZBTB46 as a critical factor for angiogenesis and for myeloid lineage skewing in cancer and suggest that maintaining its expression could have therapeutic benefits.
RESUMEN
Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages inside monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These results may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.
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Quimerismo , Embrión de Mamíferos/citología , Células Madre Pluripotentes/citología , Animales , Blastocisto/citología , Blastocisto/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Embrión de Mamíferos/metabolismo , Femenino , Humanos , Macaca fascicularis , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/trasplante , RNA-Seq , Análisis de la Célula Individual , TranscriptomaRESUMEN
A single mouse blastomere from an embryo until the 8-cell stage can generate an entire blastocyst. Whether laboratory-cultured cells retain a similar generative capacity remains unknown. Starting from a single stem cell type, extended pluripotent stem (EPS) cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization. EPS-blastoids resembled blastocysts in morphology and cell-lineage allocation and recapitulated key morphogenetic events during preimplantation and early postimplantation development in vitro. Upon transfer, some EPS-blastoids underwent implantation, induced decidualization, and generated live, albeit disorganized, tissues in utero. Single-cell and bulk RNA-sequencing analysis revealed that EPS-blastoids contained all three blastocyst cell lineages and shared transcriptional similarity with natural blastocysts. We also provide proof of concept that EPS-blastoids can be generated from adult cells via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis and pave the way to creating viable synthetic embryos by using cultured cells.
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Blastocisto/citología , Linaje de la Célula , Implantación del Embrión , Células Madre Pluripotentes Inducidas/citología , Células Madre Embrionarias de Ratones/citología , Creación de Embriones para Investigación/métodos , Animales , Blastocisto/metabolismo , Diferenciación Celular , Línea Celular , Células Cultivadas , Técnicas de Reprogramación Celular/métodos , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Células Madre Embrionarias de Ratones/metabolismo , TranscriptomaRESUMEN
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.
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Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes/citología , Animales , Blastocisto/citología , Línea Celular , Quimera/metabolismo , Dimetindeno/farmacología , Humanos , Indicadores y Reactivos/química , Ratones , Minociclina/química , Minociclina/farmacología , Células Madre Pluripotentes/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1/metabolismoRESUMEN
Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.
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Quimerismo , Edición Génica , Mamíferos/embriología , Animales , Blastocisto , Sistemas CRISPR-Cas , Bovinos , Embrión de Mamíferos/citología , Femenino , Humanos , Masculino , Mamíferos/clasificación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Células Madre Pluripotentes , Ratas , Ratas Sprague-Dawley , Sus scrofaRESUMEN
Random or not, the mechanisms at play during the first cell-fate determination in mammalian embryos have been debated for years. Studies by Goolam et al. and White et al. shed new light on the molecular mechanisms underlying the intrinsic biases that lead to non-random lineage segregation in early mammalian development.
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Diferenciación Celular , Embrión de Mamíferos , Animales , Tipificación del Cuerpo , Linaje de la CélulaRESUMEN
The understanding of human biology and how it relates to that of other species represents an ancient quest. Limited access to human material, particularly during early development, has restricted researchers to only scratching the surface of this inherently challenging subject. Recent technological innovations, such as single cell "omics" and human stem cell derivation, have now greatly accelerated our ability to gain insights into uniquely human biology. The opportunities afforded to delve molecularly into scarce material and to model human embryogenesis and pathophysiological processes are leading to new insights of human development and are changing our understanding of disease and choice of therapy options.
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Técnicas Citológicas , Desarrollo Embrionario , Investigación con Células Madre , Animales , Implantación del Embrión , Embrión de Mamíferos/metabolismo , Humanos , Células Madre Pluripotentes/metabolismoRESUMEN
The discovery of induced pluripotent stem cells (iPSCs) a decade ago, which we are celebrating in this issue of Cell, represents a landmark discovery in biomedical research. Together with somatic cell nuclear transfer, iPSC generation reveals the remarkable plasticity associated with differentiated cells and provides an unprecedented means for modeling diseases using patient samples. In addition to transcriptional and epigenetic remodeling, cellular reprogramming to pluripotency is also accompanied by a rewiring of metabolic pathways, which ultimately leads to changes in cell identities.
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Células Madre Pluripotentes Inducidas/metabolismo , Redes y Vías Metabólicas , Animales , Reprogramación Celular , Humanos , Células Madre Pluripotentes Inducidas/citología , Enfermedades Metabólicas/metabolismo , Modelos BiológicosRESUMEN
Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber's hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.
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Marcación de Gen , Enfermedades Mitocondriales/genética , Animales , Fusión Celular , ADN Mitocondrial , Embrión de Mamíferos/metabolismo , Endonucleasas/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NZB , Enfermedades Mitocondriales/prevención & control , Mutación , Oocitos/metabolismoRESUMEN
A clear relationship exists between visceral obesity and type 2 diabetes, whereas subcutaneous obesity is comparatively benign. Here, we show that adipocyte-specific deletion of the coregulatory protein PRDM16 caused minimal effects on classical brown fat but markedly inhibited beige adipocyte function in subcutaneous fat following cold exposure or ß3-agonist treatment. These animals developed obesity on a high-fat diet, with severe insulin resistance and hepatic steatosis. They also showed altered fat distribution with markedly increased subcutaneous adiposity. Subcutaneous adipose tissue in mutant mice acquired many key properties of visceral fat, including decreased thermogenic and increased inflammatory gene expression and increased macrophage accumulation. Transplantation of subcutaneous fat into mice with diet-induced obesity showed a loss of metabolic benefit when tissues were derived from PRDM16 mutant animals. These findings indicate that PRDM16 and beige adipocytes are required for the "browning" of white fat and the healthful effects of subcutaneous adipose tissue.
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Tejido Adiposo Pardo/metabolismo , Tejido Adiposo/metabolismo , Proteínas de Unión al ADN/metabolismo , Obesidad/metabolismo , Factores de Transcripción/metabolismo , Adipocitos/metabolismo , Animales , Proteínas de Unión al ADN/genética , Dieta Alta en Grasa , Resistencia a la Insulina , Ratones , Ratones Noqueados , Factores de Transcripción/genéticaRESUMEN
Our understanding of human early development is severely hampered by limited access to embryonic tissues. Due to their close evolutionary relationship with humans, nonhuman primates are often used as surrogates to understand human development but currently suffer from a lack of in vivo datasets, especially from gastrulation to early organogenesis during which the major embryonic cell types are dynamically specified. To fill this gap, we collected six Carnegie stage 8-11 cynomolgus monkey (Macaca fascicularis) embryos and performed in-depth transcriptomic analyses of 56,636 single cells. Our analyses show transcriptomic features of major perigastrulation cell types, which help shed light on morphogenetic events including primitive streak development, somitogenesis, gut tube formation, neural tube patterning and neural crest differentiation in primates. In addition, comparative analyses with mouse embryos and human embryoids uncovered conserved and divergent features of perigastrulation development across species-for example, species-specific dependency on Hippo signalling during presomitic mesoderm differentiation-and provide an initial assessment of relevant stem cell models of human early organogenesis. This comprehensive single-cell transcriptome atlas not only fills the knowledge gap in the nonhuman primate research field but also serves as an invaluable resource for understanding human embryogenesis and developmental disorders.
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Gastrulación , Macaca fascicularis , Organogénesis , Análisis de la Célula Individual , Animales , Humanos , Ratones , Gastrulación/genética , Macaca fascicularis/embriología , Macaca fascicularis/genética , Organogénesis/genética , Cuerpos Embrioides , Perfilación de la Expresión Génica , Línea Primitiva/citología , Línea Primitiva/embriología , Tubo Neural/citología , Tubo Neural/embriología , Cresta Neural/citología , Cresta Neural/embriología , Vía de Señalización Hippo , Mesodermo/citología , Mesodermo/embriología , Células MadreRESUMEN
Metabolic dysfunction-associated steatohepatitis (MASH) is the progressive form of liver steatosis, the most common liver disease, and substantially increases the mortality rate. However, limited therapies are currently available to prevent MASH development. Identifying potential pharmacological treatments for the condition has been hampered by its heterogeneous and complex nature. Here, we identified a hepatic nonneuronal cholinergic signaling pathway required for metabolic adaptation to caloric overload. We found that cholinergic receptor nicotinic alpha 2 subunit (CHRNA2) is highly expressed in hepatocytes of mice and humans. Further, CHRNA2 is activated by a subpopulation of local acetylcholine-producing macrophages during MASH development. The activation of CHRNA2 coordinates defensive programs against a broad spectrum of MASH-related pathogenesis, including steatosis, inflammation, and fibrosis. Hepatocyte-specific loss of CHRNA2 signaling accelerates the disease onset in different MASH mouse models. Activation of this pathway via pharmacological inhibition of acetylcholine degradation protects against MASH development. Our study uncovers a hepatic nicotinic cholinergic receptor pathway that constitutes a cell-autonomous self-defense route against prolonged metabolic stress and holds therapeutic potential for combatting human MASH.
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Hígado Graso , Hepatocitos , Hígado , Receptores Nicotínicos , Transducción de Señal , Animales , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Humanos , Hígado/metabolismo , Hígado/patología , Ratones , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Ratones Endogámicos C57BL , Masculino , Macrófagos/metabolismo , Acetilcolina/metabolismo , Ratones Noqueados , Modelos Animales de EnfermedadRESUMEN
PGC-1α is a transcriptional coactivator induced by exercise that gives muscle many of the best known adaptations to endurance-type exercise but has no effects on muscle strength or hypertrophy. We have identified a form of PGC-1α (PGC-1α4) that results from alternative promoter usage and splicing of the primary transcript. PGC-1α4 is highly expressed in exercised muscle but does not regulate most known PGC-1α targets such as the mitochondrial OXPHOS genes. Rather, it specifically induces IGF1 and represses myostatin, and expression of PGC-1α4 in vitro and in vivo induces robust skeletal muscle hypertrophy. Importantly, mice with skeletal muscle-specific transgenic expression of PGC-1α4 show increased muscle mass and strength and dramatic resistance to the muscle wasting of cancer cachexia. Expression of PGC-1α4 is preferentially induced in mouse and human muscle during resistance exercise. These studies identify a PGC-1α protein that regulates and coordinates factors involved in skeletal muscle hypertrophy.