RESUMO
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.
Assuntos
Quimerismo , Embrião de Mamíferos/citologia , Células-Tronco Pluripotentes/citologia , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Macaca fascicularis , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/transplante , RNA-Seq , Análise de Célula Única , TranscriptomaRESUMO
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.
Assuntos
Blastocisto/citologia , Linhagem da Célula , Implantação do Embrião , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Embrionárias Murinas/citologia , Criação de Embriões para Pesquisa/métodos , Animais , Blastocisto/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Técnicas de Reprogramação Celular/métodos , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Células-Tronco Embrionárias Murinas/metabolismo , TranscriptomaRESUMO
Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.
Assuntos
Envelhecimento/genética , Reprogramação Celular , Epigênese Genética , Doenças Metabólicas/genética , Fatores de Transcrição/metabolismo , Senilidade Prematura/genética , Senilidade Prematura/metabolismo , Animais , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Lamina Tipo A/genética , Doenças Metabólicas/metabolismo , Doenças Metabólicas/prevenção & controle , Camundongos , Modelos Animais , Pâncreas/metabolismo , Sarcopenia/metabolismoRESUMO
Nodal, a member of the TGF-ß superfamily, plays an important role in vertebrate and invertebrate early development. The biochemical study of Nodal and its signaling pathway has been a challenge, mainly because of difficulties in producing the protein in sufficient quantities. We have developed a library of stable, chemically refoldable Nodal/BMP2 chimeric ligands (NB2 library). Three chimeras, named NB250, NB260, and NB264, show Nodal-like signaling properties including dependence on the co-receptor Cripto and activation of the Smad2 pathway. NB250, like Nodal, alters heart looping during the establishment of embryonic left-right asymmetry, and both NB250 and NB260, as well as Nodal, induce chondrogenic differentiation of human adipose-derived stem cells. This Nodal-induced differentiation is shown to be more efficient than BPM2-induced differentiation. Interestingly, the crystal structure of NB250 shows a backbone scaffold similar to that of BMP2. Our results show that these chimeric ligands may have therapeutic implications in cartilage injuries.
Assuntos
Tecido Adiposo/metabolismo , Proteína Morfogenética Óssea 2 , Condrogênese/efeitos dos fármacos , Proteína Nodal , Proteínas Recombinantes de Fusão , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , Tecido Adiposo/patologia , Adulto , Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/farmacologia , Cartilagem/lesões , Cartilagem/metabolismo , Cartilagem/patologia , Linhagem Celular , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Nodal/química , Proteína Nodal/genética , Proteína Nodal/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Células-Tronco/patologiaRESUMO
BACKGROUND: Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. Multiple identified mutations in nexilin (NEXN) have been suggested to be linked with severe DCM. However, the exact association between multiple mutations of Nexn and DCM remains unclear. Moreover, it is critical for the development of precise and effective therapeutics in treatments of DCM. RESULTS: In our study, Nexn global knockout mice and mice carrying human equivalent G645del mutation are studied using functional gene rescue assays. AAV-mediated gene delivery is conducted through systemic intravenous injections at the neonatal stage. Heart tissues are analyzed by immunoblots, and functions are assessed by echocardiography. Here, we identify functional components of Nexilin and demonstrate that exogenous introduction could rescue the cardiac function and extend the lifespan of Nexn knockout mouse models. Similar therapeutic effects are also obtained in G645del mice, providing a promising intervention for future clinical therapeutics. CONCLUSIONS: In summary, we demonstrated that a single injection of AAV-Nexn was capable to restore the functions of cardiomyocytes and extended the lifespan of Nexn knockout and G645del mice. Our study represented a long-term gene replacement therapy for DCM that potentially covers all forms of loss-of-function mutations in NEXN.
Assuntos
Cardiomiopatia Dilatada , Terapia Genética , Camundongos Knockout , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/terapia , Camundongos , Humanos , Dependovirus/genética , Miócitos Cardíacos/metabolismo , Modelos Animais de Doenças , Mutação , Vetores Genéticos/administração & dosagem , Técnicas de Transferência de GenesRESUMO
Defect in the SMN1 gene causes spinal muscular atrophy (SMA), which shows loss of motor neurons, muscle weakness and atrophy. While current treatment strategies, including small molecules or viral vectors, have shown promise in improving motor function and survival, achieving a definitive and long-term correction of SMA's endogenous mutations and phenotypes remains highly challenging. We have previously developed a CRISPR-Cas9 based homology-independent targeted integration (HITI) strategy, enabling unidirectional DNA knock-in in both dividing and non-dividing cells in vivo. In this study, we demonstrated its utility by correcting an SMA mutation in mice. When combined with Smn1 cDNA supplementation, it exhibited long-term therapeutic benefits in SMA mice. Our observations may provide new avenues for the long-term and efficient treatment of inherited diseases.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Terapia Genética , Atrofia Muscular Espinal , Proteína 1 de Sobrevivência do Neurônio Motor , Atrofia Muscular Espinal/terapia , Atrofia Muscular Espinal/genética , Animais , Edição de Genes/métodos , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Camundongos , Terapia Genética/métodos , Modelos Animais de Doenças , Humanos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação , Masculino , FemininoRESUMO
Tissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue homeostasis and function. Here, we present a biochemical intervention that induces an intermediate cell state mirroring a transition identified during normal differentiation of myoblasts and other multipotent and pluripotent cells to mature cells. When applied in somatic differentiated cells, the intervention, composed of one-carbon metabolites, reduces some dedifferentiation markers without losing the lineage identity, thus inducing limited reprogramming into a more flexible cell state. Moreover, the intervention enabled accelerated repair after muscle injury in young and aged mice. Overall, our study uncovers a conserved biochemical transitional phase that enhances cellular plasticity in vivo and hints at potential and scalable biochemical interventions of use in regenerative medicine and rejuvenation interventions that may be more tractable than genetic ones.
Assuntos
Músculos , Mioblastos , Camundongos , Animais , Diferenciação Celular , Mioblastos/metabolismoRESUMO
Human pluripotent stem cell-derived kidney organoids offer unprecedented opportunities for studying polycystic kidney disease (PKD), which still has no effective cure. Here, we developed both in vitro and in vivo organoid models of PKD that manifested tubular injury and aberrant upregulation of renin-angiotensin aldosterone system. Single-cell analysis revealed that a myriad of metabolic changes occurred during cystogenesis, including defective autophagy. Experimental activation of autophagy via ATG5 overexpression or primary cilia ablation significantly inhibited cystogenesis in PKD kidney organoids. Employing the organoid xenograft model of PKD, which spontaneously developed tubular cysts, we demonstrate that minoxidil, a potent autophagy activator and an FDA-approved drug, effectively attenuated cyst formation in vivo. This in vivo organoid model of PKD will enhance our capability to discover novel disease mechanisms and validate candidate drugs for clinical translation.
Assuntos
Cílios , Doenças Renais Policísticas , Humanos , Rim , Doenças Renais Policísticas/tratamento farmacológico , Autofagia , OrganoidesRESUMO
Constitutive heterochromatin is responsible for genome repression of DNA enriched in repetitive sequences, telomeres, and centromeres. During physiological and pathological premature aging, heterochromatin homeostasis is profoundly compromised. Here, we showed that LINE-1 (Long Interspersed Nuclear Element-1; L1) RNA accumulation was an early event in both typical and atypical human progeroid syndromes. L1 RNA negatively regulated the enzymatic activity of the histone-lysine N-methyltransferase SUV39H1 (suppression of variegation 3-9 homolog 1), resulting in heterochromatin loss and onset of senescent phenotypes in vitro. Depletion of L1 RNA in dermal fibroblast cells from patients with different progeroid syndromes using specific antisense oligonucleotides (ASOs) restored heterochromatin histone 3 lysine 9 and histone 3 lysine 27 trimethylation marks, reversed DNA methylation age, and counteracted the expression of senescence-associated secretory phenotype genes such as p16, p21, activating transcription factor 3 (ATF3), matrix metallopeptidase 13 (MMP13), interleukin 1a (IL1a), BTG anti-proliferation factor 2 (BTG2), and growth arrest and DNA damage inducible beta (GADD45b). Moreover, systemic delivery of ASOs rescued the histophysiology of tissues and increased the life span of a Hutchinson-Gilford progeria syndrome mouse model. Transcriptional profiling of human and mouse samples after L1 RNA depletion demonstrated that pathways associated with nuclear chromatin organization, cell proliferation, and transcription regulation were enriched. Similarly, pathways associated with aging, inflammatory response, innate immune response, and DNA damage were down-regulated. Our results highlight the role of L1 RNA in heterochromatin homeostasis in progeroid syndromes and identify a possible therapeutic approach to treat premature aging and related syndromes.
Assuntos
Senilidade Prematura , Síndrome de Cockayne , Proteínas Imediatamente Precoces , Progéria , Senilidade Prematura/genética , Animais , Antígenos de Diferenciação , Heterocromatina , Histonas/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Lisina/metabolismo , Camundongos , Fenótipo , Progéria/genética , RNA , Telômero/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
It is widely believed that cellular senescence plays a critical role in both aging and cancer, and that senescence is a fundamental, permanent growth arrest that somatic cells cannot avoid. Here we show that Myc plays an important role in self-renewal of esophageal epithelial cells, contributing to their resistance to cellular senescence. Myc is homogeneously expressed in basal cells of the esophageal epithelium and Myc positively regulates their self-renewal by maintaining their undifferentiated state. Indeed, Myc knockout induced a loss of the undifferentiated state of esophageal epithelial cells resulting in cellular senescence while forced MYC expression promoted oncogenic cell proliferation. A superoxide scavenger counteracted Myc knockout-induced senescence, therefore suggesting that a mitochondrial superoxide takes part in inducing senescence. Taken together, these analyses reveal extremely low levels of cellular senescence and senescence-associated phenotypes in the esophageal epithelium, as well as a critical role for Myc in self-renewal of basal cells in this organ. This provides new avenues for studying and understanding the links between stemness and resistance to cellular senescence.
RESUMO
During embryogenesis, cells are spatially patterned as a result of highly coordinated and stereotyped morphogenetic events. In the vertebrate embryo, information on laterality is conveyed to the node, and subsequently to the lateral plate mesoderm, by a complex cascade of epigenetic and genetic events, eventually leading to a left-right asymmetric body plan. At the same time, the paraxial mesoderm is patterned along the anterior-posterior axis in metameric units, or somites, in a bilaterally symmetric fashion. Here we characterize a cascade of laterality information in the zebrafish embryo and show that blocking the early steps of this cascade (before it reaches the lateral plate mesoderm) results in random left-right asymmetric somitogenesis. We also uncover a mechanism mediated by retinoic acid signalling that is crucial in buffering the influence of the flow of laterality information on the left-right progression of somite formation, and thus in ensuring bilaterally symmetric somitogenesis.
Assuntos
Padronização Corporal/fisiologia , Embrião não Mamífero/metabolismo , Transdução de Sinais , Somitos/metabolismo , Tretinoína/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Cílios/fisiologia , Embrião não Mamífero/embriologia , Modelos Biológicos , Dados de Sequência Molecular , Somitos/citologia , Peixe-Zebra/genéticaRESUMO
Organ shape and size, and, ultimately, organ function, relate in part to the cell and tissue spatial arrangement that takes place during embryonic development. Despite great advances in the genetic regulatory networks responsible for tissue and organ development, it is not yet clearly understood how specific gene functions are linked to the specific morphogenetic processes underlying the internal organ asymmetries found in vertebrate animals. During female chick embryogenesis, and in contrast to males where both testes develop symmetrically, asymmetrical gonad morphogenesis results in only one functional ovary. The disposition of paired organs along the left-right body axis has been shown to be regulated by the activity of the homeobox containing gene pitx2. We have found that pitx2 regulates cell adhesion, affinity, and cell recognition events in the developing gonad primordium epithelia. This in turn not only allows for proper somatic development of the gonad cortex but also permits the proliferation and differentiation of primordial germ cells. We illustrate how Pitx2 activity directs asymmetrical gonad morphogenesis by controlling mitotic spindle orientation of the developing gonad cortex and how, by modulating cyclinD1 expression during asymmetric ovarian development, Pitx2 appears to control gonad organ size. All together our observations indicate that the effects elicited by Pitx2 during the development of the female chick ovary are critical for cell topology, growth, fate, and ultimately organ morphogenesis and function.
Assuntos
Diferenciação Celular/fisiologia , Galinhas/fisiologia , Células Germinativas/fisiologia , Ovário/embriologia , Animais , Adesão Celular/fisiologia , Proliferação de Células , Embrião de Galinha , Ciclina D1/metabolismo , Epitélio/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Germinativas/citologia , Proteínas de Homeodomínio , Masculino , Tamanho do Órgão , Ovário/citologia , Fuso Acromático/metabolismo , Testículo/citologia , Testículo/embriologia , Fatores de Transcrição , Proteína Homeobox PITX2RESUMO
Short-term, systemic expression of the Yamanaka reprogramming factors (Oct-3/4, Sox2, Klf4 and c-Myc [OSKM]) has been shown to rejuvenate aging cells and promote tissue regeneration in vivo. However, the mechanisms by which OSKM promotes tissue regeneration are unknown. In this work, we focus on a specific tissue and demonstrate that local expression of OSKM, specifically in myofibers, induces the activation of muscle stem cells or satellite cells (SCs), which accelerates muscle regeneration in young mice. In contrast, expressing OSKM directly in SCs does not improve muscle regeneration. Mechanistically, expressing OSKM in myofibers regulates the expression of genes important for the SC microenvironment, including upregulation of p21, which in turn downregulates Wnt4. This is critical because Wnt4 is secreted by myofibers to maintain SC quiescence. Thus, short-term induction of the Yamanaka factors in myofibers may promote tissue regeneration by modifying the stem cell niche.
Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Miofibrilas/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/metabolismo , Nicho de Células-Tronco , Animais , Células Cultivadas , Feminino , Expressão Gênica , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos Transgênicos , Miofibrilas/fisiologia , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Células Satélites de Músculo Esquelético/citologia , Proteína Wnt4/genéticaRESUMO
Epimorphic regeneration is a unique and complex instance of postembryonic growth observed in certain metazoans that is usually triggered by severe injury [Akimenko et al., 2003; Alvarado and Tsonis, 2006; Brockes, 1997; Endo et al., 2004]. Cell division and migration are two fundamental biological processes required for supplying replacement cells during regeneration [Endo et al., 2004; Slack, 2007]. However, the connection between the early stimuli generated after injury and the signals regulating proliferation and migration during regeneration remain largely unknown. Here we show that the oncogenes ErbB2 and ErbB3, two members of the EGFR family, are essential for mounting a successful regeneration response in vertebrates. Importantly, amputation-induced progenitor proliferation and migration are significantly reduced upon genetic and/or chemical modulation of ErbB function. Moreover, we also found that NRG1 and PI3K functionally interact with ErbB2 and ErbB3 during regeneration and interfering with their function also abrogates the capacity of progenitor cells to regenerate lost structures upon amputation. Our findings suggest that ErbB, PI3K and NRG1 are components of a permissive switch for migration and proliferation continuously acting across the amputated fin from early stages of vertebrate regeneration onwards that regulate the expression of the transcription factors lef1 and msxB.
Assuntos
Amputação Cirúrgica , Receptor ErbB-2/fisiologia , Receptor ErbB-3/fisiologia , Regeneração , Células-Tronco/fisiologia , Animais , Movimento Celular , Proliferação de Células , Proteínas de Homeodomínio/genética , Neuregulina-1/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/genética , Vertebrados , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
During vertebrate embryo development, the breaking of the initial bilateral symmetry is translated into asymmetric gene expression around the node and/or in the lateral plate mesoderm. The earliest conserved feature of this asymmetric gene expression cascade is the left-sided expression of Nodal, which depends on the activity of the Notch signalling pathway. Here we present a mathematical model describing the dynamics of the Notch signalling pathway during chick embryo gastrulation, which reveals a complex and highly robust genetic network that locally activates Notch on the left side of Hensen's node. We identify the source of the asymmetric activation of Notch as a transient accumulation of extracellular calcium, which in turn depends on left-right differences in H+/K+-ATPase activity. Our results uncover a mechanism by which the Notch signalling pathway translates asymmetry in epigenetic factors into asymmetric gene expression around the node.
Assuntos
Padronização Corporal , Sinalização do Cálcio , Ácido Egtázico/análogos & derivados , Proteínas de Membrana/metabolismo , Fatores de Transcrição , Animais , Proteínas Aviárias , Padronização Corporal/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio , Linhagem Celular Tumoral , Embrião de Galinha , Ácido Egtázico/farmacologia , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Proteínas de Membrana/genética , Modelos Biológicos , Proteína Nodal , Omeprazol/farmacologia , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch1 , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Notch , Proteínas Serrate-Jagged , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Identification of the precise molecular pathways involved in oncogene-induced transformation may help us gain a better understanding of tumor initiation and promotion. Here, we demonstrate that SOX2+ foregut epithelial cells are prone to oncogenic transformation upon mutagenic insults, such as KrasG12D and p53 deletion. GFP-based lineage-tracing experiments indicate that SOX2+ cells are the cells-of-origin of esophagus and stomach hyperplasia. Our observations indicate distinct roles for oncogenic KRAS mutation and P53 deletion. p53 homozygous deletion is required for the acquisition of an invasive potential, and KrasG12D expression, but not p53 deletion, suffices for tumor formation. Global gene expression analysis reveals secreting factors upregulated in the hyperplasia induced by oncogenic KRAS and highlights a crucial role for the CXCR2 pathway in driving hyperplasia. Collectively, the array of genetic models presented here demonstrate that stratified epithelial cells are susceptible to oncogenic insults, which may lead to a better understanding of tumor initiation and aid in the design of new cancer therapeutics.
Assuntos
Neoplasias Esofágicas/metabolismo , Mutação , Receptores de Interleucina-8B/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Proliferação de Células , Neoplasias Esofágicas/patologia , Feminino , Masculino , Camundongos , Camundongos Mutantes , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Human pluripotent stem cell-derived kidney organoids recapitulate developmental processes and tissue architecture, but intrinsic limitations, such as lack of vasculature and functionality, have greatly hampered their application. Here we establish a versatile protocol for generating vascularized three-dimensional (3D) kidney organoids. We employ dynamic modulation of WNT signaling to control the relative proportion of proximal versus distal nephron segments, producing a correlative level of vascular endothelial growth factor A (VEGFA) to define a resident vascular network. Single-cell RNA sequencing identifies a subset of nephron progenitor cells as a potential source of renal vasculature. These kidney organoids undergo further structural and functional maturation upon implantation. Using this kidney organoid platform, we establish an in vitro model of autosomal recessive polycystic kidney disease (ARPKD), the cystic phenotype of which can be effectively prevented by gene correction or drug treatment. Our studies provide new avenues for studying human kidney development, modeling disease pathogenesis, and performing patient-specific drug validation.
Assuntos
Rim/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Rim Policístico Autossômico Recessivo/patologia , Diferenciação Celular , Células Cultivadas , Descoberta de Drogas , Terapia Genética , Humanos , Rim/irrigação sanguínea , Neovascularização Fisiológica , Técnicas de Cultura de Órgãos , Organogênese , Organoides/irrigação sanguínea , Rim Policístico Autossômico Recessivo/metabolismo , Rim Policístico Autossômico Recessivo/terapia , Medicina de Precisão , Fator A de Crescimento do Endotélio Vascular/metabolismo , Via de Sinalização WntRESUMO
In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.
Assuntos
Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas/genética , Reparo do DNA , Dependovirus/genética , Fator de Transcrição GATA3/genética , Técnicas de Introdução de Genes , Terapia Genética/métodos , Vetores Genéticos/metabolismo , Células-Tronco Embrionárias Humanas , Humanos , Íntrons , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Neurônios/citologia , Neurônios/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Ratos , Tubulina (Proteína)/genéticaRESUMO
The IkappaB kinase (IKK) activity is critical for processing IkappaB inhibitory proteins and activating the NF-kappaB signaling, which is involved in a series of physiological and developmental steps in vertebrates. The IKK activity resides in two catalytic subunits, IKK1 and IKK2, and two regulatory subunits, NEMO and ELKS. IKK2 is the major cytokine-responsive IkappaB kinase because depletion of IKK1 does not interfere with the IKK activity. In fact, IKK1-/- mice display morphological abnormalities that are independent of its kinase activity and NF-kappaB activation. Hence, using zebrafish (Danio rerio) as a model, we examined the evolutionary role of IKK1 in modulating NF-kappaB. Ikk1-/- zebrafish embryos present head and tail malformations and, surprisingly, show upregulation of NF-kappaB-responsive genes and increased NF-kappaB-dependent apoptosis. Overexpression of ikk1 leads to midline structure defects that resemble NF-kappaB blockage in vivo. Zebrafish Ikk1 forms complexes with NEMO that represses NF-kappaB in vertebrate cells. Indeed, truncation of its NEMO binding domain (NBD) restores NF-kappaB-dependent transcriptional activity and, consequently, the ikk1-overexpressing phenotype. Here, we report that Ikk1 negatively regulates NF-kappaB by sequestering NEMO from active IKK complexes, indicating that IKK1 can function as a repressor of NF-kappaB.
Assuntos
Regulação para Baixo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Peixe-Zebra/metabolismo , Animais , Western Blotting , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Biologia Computacional , Primers do DNA , Componentes do Gene , Humanos , Quinase I-kappa B , Imuno-Histoquímica , Hibridização In Situ , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Embryonic stem cells (ESCs) can be propagated indefinitely in culture, while retaining the ability to differentiate into any cell type in the organism. The molecular and cellular mechanisms underlying ESC pluripotency are, however, poorly understood. We characterize a population of early mesoderm-specified (EM) progenitors that is generated from mouse ESCs by bone morphogenetic protein stimulation. We further show that pluripotent ESCs are actively regenerated from EM progenitors by the action of the divergent homeodomain-containing protein Nanog, which, in turn, is upregulated in EM progenitors by the combined action of leukemia inhibitory factor and the early mesoderm transcription factor T/Brachyury. These findings uncover specific roles of leukemia inhibitory factor, Nanog, and bone morphogenetic protein in the self-renewal of ESCs and provide novel insights into the cellular bases of ESC pluripotency.