RESUMO
Current knowledge of the transcriptional regulation of human pluripotency is incomplete, with lack of interspecies conservation observed. Single-cell transcriptomics analysis of human embryos previously enabled us to identify transcription factors, including the zinc-finger protein KLF17, that are enriched in the human epiblast and naïve human embryonic stem cells (hESCs). Here, we show that KLF17 is expressed coincident with the known pluripotency-associated factors NANOG and SOX2 across human blastocyst development. We investigate the function of KLF17 using primed and naïve hESCs for gain- and loss-of-function analyses. We find that ectopic expression of KLF17 in primed hESCs is sufficient to induce a naïve-like transcriptome and that KLF17 can drive transgene-mediated resetting to naïve pluripotency. This implies a role for KLF17 in establishing naïve pluripotency. However, CRISPR-Cas9-mediated knockout studies reveal that KLF17 is not required for naïve pluripotency acquisition in vitro. Transcriptome analysis of naïve hESCs identifies subtle effects on metabolism and signalling pathways following KLF17 loss of function, and possible redundancy with other KLF paralogues. Overall, we show that KLF17 is sufficient, but not necessary, for naïve pluripotency under the given in vitro conditions.
Assuntos
Blastocisto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Fatores de Transcrição/metabolismo , Humanos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genéticaRESUMO
Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.
Assuntos
Desenvolvimento Embrionário/genética , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Animais , Blastocisto/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem da Célula , Ectoderma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Camadas Germinativas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Masculino , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/deficiência , Especificidade por Substrato , Zigoto/metabolismoRESUMO
This corrects the article DOI: 10.1038/nature24033.
RESUMO
Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.
Assuntos
Diferenciação Celular , Variações do Número de Cópias de DNA , Proteína Proto-Oncogênica N-Myc , Crista Neural , Neuroblastoma , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Crista Neural/metabolismo , Crista Neural/patologia , Feminino , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Aberrações Cromossômicas , Células-Tronco Embrionárias Humanas/metabolismo , Transcriptoma , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
CRISPR-Cas9 mutagenesis facilitates the investigation of gene function in a number of developmental and cellular contexts. Human pluripotent stem cells (hPSCs), either embryonic or induced, are a tractable cellular model to investigate molecular mechanisms involved in early human development and cell fate decisions. hPSCs also have broad potential in regenerative medicine to model, investigate, and ameliorate diseases. Here, we provide an optimized protocol for efficient CRISPR-Cas9 genome editing of hPSCs to investigate the functional role of genes by engineering null mutations. We emphasize the importance of screening single guide RNAs (sgRNAs) to identify those with high targeting efficiency for generation of clonally derived null mutant hPSC lines. We provide important considerations for targeting genes that may have a role in hPSC maintenance. We also present methods to evaluate the on-target mutation spectrum and unintended karyotypic changes. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selecting and ligating sgRNAs into expression plasmids Basic Protocol 2: Validation of sgRNA via in vitro transcription and cleavage assay Basic Protocol 3: Nucleofection of primed human embryonic stem cells Basic Protocol 4: MiSeq analysis of indel mutations Basic Protocol 5: Single cell cloning of targeted hPSCs Basic Protocol 6: Karyotyping of targeted hPSCs.
Assuntos
Sistemas CRISPR-Cas , Células-Tronco Pluripotentes , Sistemas CRISPR-Cas/genética , Edição de Genes , Humanos , Mutação com Perda de Função , RNA Guia de Cinetoplastídeos/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.
Assuntos
Autorrenovação Celular/fisiologia , Células-Tronco Embrionárias Humanas/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Ativinas/metabolismo , Animais , Blastocisto/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/química , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Endoderma/citologia , Endoderma/metabolismo , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Inativação do Cromossomo X/fisiologiaRESUMO
With the advent of efficient, easy-to-use genome editing by CRISPR-Cas9, editing human embryos is now possible, providing tremendous opportunities to study gene function and cell fate in early human development. The technique can also be used to modify the human germline. Unresolved questions about pre-implantation human development could be addressed by basic research using CRISPR-Cas9. In this Perspective, we discuss advances in human genome editing and consider ethical questions and potential clinical implications of this technology.