RESUMO
After fertilization, the quiescent zygote experiences a burst of genome activation that initiates a short-lived totipotent state. Understanding the process of totipotency in human cells would have broad applications. However, in contrast to in mice1,2, demonstration of the time of zygotic genome activation or the eight-cell (8C) stage in in vitro cultured human cells has not yet been reported, and the study of embryos is limited by ethical and practical considerations. Here we describe a transgene-free, rapid and controllable method for producing 8C-like cells (8CLCs) from human pluripotent stem cells. Single-cell analysis identified key molecular events and gene networks associated with this conversion. Loss-of-function experiments identified fundamental roles for DPPA3, a master regulator of DNA methylation in oocytes3, and TPRX1, a eutherian totipotent cell homeobox (ETCHbox) family transcription factor that is absent in mice4. DPPA3 induces DNA demethylation throughout the 8CLC conversion process, whereas TPRX1 is a key executor of 8CLC gene networks. We further demonstrate that 8CLCs can produce embryonic and extraembryonic lineages in vitro or in vivo in the form of blastoids5 and complex teratomas. Our approach provides a resource to uncover the molecular process of early human embryogenesis.
Assuntos
Embrião de Mamíferos , Desenvolvimento Embrionário , Células-Tronco Pluripotentes , Zigoto , Humanos , Proteínas Cromossômicas não Histona/genética , Embrião de Mamíferos/citologia , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/genética , Zigoto/citologiaRESUMO
Parkinson's disease (PD) is one of the most common neurodegenerative disorders and is characterized by the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra. Loss of function mutations in PARK2 cause familial PD in an autosomal recessive manner. PARK2 encodes an E3 ubiquitin ligase that is involved in regulation of mitochondrial homeostasis. However, the mechanistic links between PARK2 mutations and dopaminergic neuron degeneration are unclear. Here, we have generated three patient-derived induced pluripotent stem cell (iPSC) lines from the same donor with mutant PARKIN (p. C253Y). These well characterized cell lines will facilitate the study of PARKIN function in disease relevant cell types in vitro.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Transtornos Parkinsonianos , Neurônios Dopaminérgicos , Humanos , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Mutations in the Leucine rich repeat kinase 2 (LRRK2) gene are found in both familial and sporadic Parkinson's disease (PD), and are also associated with immune-related disorders including Crohn's disease (CD) and leprosy. We have generated two homozygous LRRK2 knockout human induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 in a well-characterized human iPSC clone. The LRRK2 knockout cell lines retained normal morphology, gene expression, and the capacity to differentiate into cell types of the three germ layers. These cell lines are valuable for elucidating the role of LRRK2 in innate immunity and PD.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Sistemas CRISPR-Cas/genética , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação , Doença de Parkinson/genéticaRESUMO
Mutations occurring in the gene body of PARK7 (encoding DJ-1/PARK7) cause autosomal recessive early-onset parkinsonism (AREP). These mutations produce a loss of function and have been reported to lead to dopaminergic neuron degeneration in the substantia nigra. However, the underlying mechanisms are largely unknown. Here, we report the generation of a patient-derived induced pluripotent stem cell (iPSC) line carrying mutant DJ-1 (p.L10P). This cell line is a valuable tool for in vitro Parkinson's disease (PD) modeling and mechanistic studies.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Dopamina , Neurônios Dopaminérgicos , Humanos , Doença de Parkinson/genética , Proteína Desglicase DJ-1/genética , Substância NegraRESUMO
Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that ß-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by ß-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.
RESUMO
Familial Parkinson's disease (PD) can be caused by deleterious mutations in PINK1 (encoding PINK1) in an autosomal recessive manner. Functional studies suggest that PINK1 works as a regulator of mitochondrial homeostasis. However, how loss of PINK1 induces dopaminergic neuron degeneration is still unclear. Here, we have generated a patient-derived induced pluripotent stem cell (iPSC) line with mutant PINK1 (p. I368N). This cell line will facilitate PD disease modeling in vitro and can be used for generating isogenic cell lines through gene correction.
Assuntos
Diferenciação Celular , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Mutação , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteínas Quinases/genética , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
Loss of function mutations in PARK2 (encoding PARKIN) cause autosomal recessive Parkinson's disease (PD), which often manifests at a juvenile age. Molecular and biochemical studies show that PARKIN functions as an E3 ubiquitin ligase controlling mitochondrial homeostasis. Yet, the exact mechanisms are unclear due to the use of sub-optimal models including cancer cells and fibroblasts. We have generated a PARK2 knockout (KO) isogenic cell line using a well-characterized induced pluripotent stem cell (iPSC) clone with good differentiation potential. This cell line lacks the expression of all PARKIN isoforms and is valuable for elucidating the role of PARK2 mutations in PD.
Assuntos
Diferenciação Celular , Mutação da Fase de Leitura , Células-Tronco Pluripotentes Induzidas/patologia , Túbulos Renais/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Ubiquitina-Proteína Ligases/genética , Adulto , Células Cultivadas , Feminino , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Túbulos Renais/metabolismo , Isoformas de Proteínas , Adulto JovemRESUMO
Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.
Assuntos
Hipercolesterolemia/diagnóstico , Hiperlipoproteinemia Tipo II/diagnóstico , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Quimera/metabolismo , Modelos Animais de Doenças , Humanos , Hipercolesterolemia/patologia , Hiperlipoproteinemia Tipo II/patologia , Camundongos , MutaçãoRESUMO
In the version of this Article originally published, in Fig. 2c, the '+' sign and 'OSKM' were superimposed in the label '+OSKM'. In Fig. 4e, in the labels, all instances of 'Ant' should have been 'Anti-'. And, in Fig. 7a, the label '0.0' was misplaced; it should have been on the colour scale bar. These figures have now been corrected in the online versions.
RESUMO
Somatic cell reprogramming by exogenous factors requires cooperation with transcriptional co-activators and co-repressors to effectively remodel the epigenetic environment. How this interplay is regulated remains poorly understood. Here, we demonstrate that NCoR/SMRT co-repressors bind to pluripotency loci to create a barrier to reprogramming with the four Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC), and consequently, suppressing NCoR/SMRT significantly enhances reprogramming efficiency and kinetics. The core epigenetic subunit of the NCoR/SMRT complex, histone deacetylase 3 (HDAC3), contributes to the effects of NCoR/SMRT by inducing histone deacetylation at pluripotency loci. Among the Yamanaka factors, recruitment of NCoR/SMRT-HDAC3 to genomic loci is mostly facilitated by c-MYC. Hence, we describe how c-MYC is beneficial for the early phase of reprogramming but deleterious later. Overall, we uncover a role for NCoR/SMRT co-repressors in reprogramming and propose a dual function for c-MYC in this process.
Assuntos
Reprogramação Celular , Epigênese Genética , Células-Tronco Embrionárias Murinas/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Acetilação , Animais , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Correpressor 1 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Familial hypercholesterolemia (FH) causes elevation of low-density lipoprotein cholesterol (LDL-C) in blood and carries an increased risk of early-onset cardiovascular disease. A caveat for exploration of new therapies for FH is the lack of adequate experimental models. We have created a comprehensive FH stem cell model with differentiated hepatocytes (iHeps) from human induced pluripotent stem cells (iPSCs), including genetically engineered iPSCs, for testing therapies for FH. We used FH iHeps to assess the effect of simvastatin and proprotein convertase subtilisin/kexin type 9 (PCSK9) antibodies on LDL-C uptake and cholesterol lowering in vitro. In addition, we engrafted FH iHeps into the liver of Ldlr-/-/Rag2-/-/Il2rg-/- mice, and assessed the effect of these same medications on LDL-C clearance and endothelium-dependent vasodilation in vivo. Our iHep models recapitulate clinical observations of higher potency of PCSK9 antibodies compared with statins for reversing the consequences of FH, demonstrating the utility for preclinical testing of new therapies for FH patients.
Assuntos
Diferenciação Celular , Quimera/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Animais , LDL-Colesterol/metabolismo , Heterozigoto , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hiperlipoproteinemia Tipo II/tratamento farmacológico , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Linhagem , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/genéticaRESUMO
The prevalence and incidence of Parkinson's disease (PD) is increasing due to a prolonged life expectancy. This highlights the need for a better mechanistic understanding and new therapeutic approaches. However, traditional in vitro and in vivo experimental models to study PD are suboptimal, thus hampering the progress in the field. The epigenetic reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) offers a unique way to overcome this problem, as these cells share many properties of embryonic stem cells (ESCs) including the potential to be transformed into different lineages. PD modeling with iPSCs is nowadays facilitated by the growing availability of high-efficiency neural-specific differentiation protocols and the possibility to correct or induce mutations as well as creating marker cell lines using designer nucleases. These technologies, together with steady advances in human genetics, will likely introduce profound changes in the way we interpret PD and develop new treatments. Here, we summarize the different PD iPSCs reported so far and discuss the challenges for disease modeling using these cell lines.