RESUMEN
The first lineage choice in human embryo development separates trophectoderm from the inner cell mass. Naïve human embryonic stem cells are derived from the inner cell mass and offer possibilities to explore how lineage integrity is maintained. Here, we discover that polycomb repressive complex 2 (PRC2) maintains naïve pluripotency and restricts differentiation to trophectoderm and mesoderm lineages. Through quantitative epigenome profiling, we found that a broad gain of histone H3 lysine 27 trimethylation (H3K27me3) is a distinct feature of naïve pluripotency. We define shared and naïve-specific bivalent promoters featuring PRC2-mediated H3K27me3 concomitant with H3K4me3. Naïve bivalency maintains key trophectoderm and mesoderm transcription factors in a transcriptionally poised state. Inhibition of PRC2 forces naïve human embryonic stem cells into an 'activated' state, characterized by co-expression of pluripotency and lineage-specific transcription factors, followed by differentiation into either trophectoderm or mesoderm lineages. In summary, PRC2-mediated repression provides a highly adaptive mechanism to restrict lineage potential during early human development.
Asunto(s)
Células Madre Embrionarias Humanas , Complejo Represivo Polycomb 2 , Diferenciación Celular/genética , Desarrollo Embrionario , Histonas/genética , Células Madre Embrionarias Humanas/metabolismo , Humanos , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismoRESUMEN
Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.
Asunto(s)
Células Epiteliales/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Madre Embrionarias Humanas/citología , Epitelio Pigmentado de la Retina/citología , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Citotoxicidad Inmunológica , Xenoinjertos , Células Madre Embrionarias Humanas/metabolismo , Humanos , Inmunomodulación , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleótido Simple/genética , Linfocitos T/metabolismo , Transactivadores/metabolismo , Microglobulina beta-2/metabolismoRESUMEN
Understanding the genetic underpinning of early human development is of great interest not only for basic developmental and stem cell biology but also for regenerative medicine, infertility treatments, and better understanding the causes of congenital disease. Our current knowledge has mainly been generated with the use of laboratory animals, especially the mouse. While human and mouse early development present morphological resemblance, we know that the timing of the events as well as the cellular and genetic mechanisms that control fundamental processes are distinct between the species. The rapid technological development of single-cell sequencing and genome editing together with novel stem cell models of the early human embryo has made it feasible and relevant to perform functional genetic studies directly in human cells and embryos. In this review we will discuss these latest advances where combined transcriptional analysis and genome engineering has begun to shed new insights into the key processes of zygotic genome activation, lineage specification, X-chromosome inactivation and postimplantation development including primordial germ cell specification in the human embryo.
Asunto(s)
Diferenciación Celular/genética , Desarrollo Embrionario/genética , Genoma Humano/genética , Inactivación del Cromosoma X/genética , Embrión de Mamíferos , Células Germinativas/crecimiento & desarrollo , Humanos , CigotoRESUMEN
Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.