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A molecular roadmap for the emergence of early-embryonic-like cells in culture.

ABSTRACT: Unlike pluripotent cells, which generate only embryonic tissues, totipotent cells can generate a full organism, including extra-embryonic tissues. A rare population of cells resembling 2-cell-stage embryos arises in pluripotent embryonic stem (ES) cell cultures. These 2-cell-like cells display molecular features of totipotency and broader developmental plasticity. However, their specific nature and the process through which they arise remain outstanding questions. Here we identified intermediate cellular states and molecular determinants during the emergence of 2-cell-like cells. By deploying a quantitative single-cell expression approach, we identified an intermediate population characterized by expression of the transcription factor ZSCAN4 as a precursor of 2-cell-like cells. By using a small interfering RNA (siRNA) screen, we identified epigenetic regulators of 2-cell-like cell emergence, including the non-canonical PRC1 complex PRC1.6 and the EP400-TIP60 complex. Our data shed light on the mechanisms that underlie exit from the ES cell state toward the formation of early-embryonic-like cells in culture and identify key epigenetic pathways that promote this transition.

SUBMITTER: Rodriguez-Terrones D

PROVIDER: S-EPMC5755687 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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A molecular roadmap for the emergence of early-embryonic-like cells in culture.

Rodriguez-Terrones Diego D Gaume Xavier X Ishiuchi Takashi T Weiss Amélie A Kopp Arnaud A Kruse Kai K Penning Audrey A Vaquerizas Juan M JM Brino Laurent L Torres-Padilla Maria-Elena ME

Nature genetics 20171218 1

Unlike pluripotent cells, which generate only embryonic tissues, totipotent cells can generate a full organism, including extra-embryonic tissues. A rare population of cells resembling 2-cell-stage embryos arises in pluripotent embryonic stem (ES) cell cultures. These 2-cell-like cells display molecular features of totipotency and broader developmental plasticity. However, their specific nature and the process through which they arise remain outstanding questions. Here we identified intermediate ...[more]

PMID: 29255263

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Project description:BackgroundEmbryonic Sertoli cells (eSCs) play an important role in sex determination and in male gonad development which makes them a very useful cell type for therapeutic applications. However, the deriving mechanism of Sertoli cells has been unclear and challenging to create a large number of quality eSCs. Therefore, this study aimed to create the eSCs induced from mouse embryonic stem (mES) cells by regulating defined factors and to explore the relevant regulatory mechanism.MethodsSix inducing factors, Sry, Sox9, SF1, WT1, GATA4, and Dmrt1, were respectively transduced into mES cells by lentiviral infection according to the experimental design. The test groups were identified by development stage-specific markers, AMH, Emx2, SF1, and FasL, using flow cytometry. Induced eSCs were determined by FasL and AMH biomarkers under immunofluorescence, immunocytochemistry, and flow cytometry. Moreover, the pluripotency markers, gonad development-related markers, epithelial markers and mesenchymal markers in test groups were transcriptionally determined by qPCR.ResultsIn this study, the co-overexpression of all the six factors effectively produced a large population of eSCs from mES cells in 35 days of culturing. These eSCs were capable of forming tubular-like and ring-like structures with functional performance. The results of flow cytometry indicated that the upregulation of GATA4 and WT1 contributed to the growth of somatic cells in the coelomic epithelium regarded as the main progenitor cells of eSCs. Whereas, SF1 facilitated the development of eSC precursor cells, and Sry and Sox9 promoted the determination of male development. Moreover, the overexpression of Dmrt1 was essential for the maintenance of eSCs and some of their specific surface biomarkers such as FasL. The cellular morphology, biomarker identification, and transcriptomic analysis aided in exploring the regulatory mechanism of deriving eSCs from mES cells.ConclusionConclusively, we have elucidated a differentiation roadmap of eSCs derived from mES cells with a relevant regulatory mechanism. Through co-overexpression of all these six factors, a large population of eSCs was successfully induced occupying 24% of the whole cell population (1 × 105 cells/cm2). By adopting this approach, a mass of embryonic Sertoli cells can be generated for the purpose of co-culture technique, organ transplantation, gonadal developmental and sex determination researches.

Project description:Human embryonic stem (hES) cells have unique abilities to divide indefinitely without differentiating and potential to differentiate into more than 200 cell types. These properties make hES cells an ideal model system for understanding early human development and for regenerative medicine. Molecular mechanisms including cellular signaling and transcriptional regulation play important roles in hES cell differentiation. However, very little information is available on posttranscriptional regulation of hES cell pluripotency, self-renewal, and early decisions about cell fate. microRNAs (miRNAs), 22-nt long non-coding small RNAs found in plants and animals, regulate gene expression by targeting mRNAs for cleavage or translation repression. In hES cells we found that 276 miRNAs were expressed; of these, a set of 30 miRNAs had significantly changed expression during differentiation. Using a representative example, miR-302b, we show that miRNAs in human ES cells assemble into a bona fide RISC that contains Ago2 and can specifically cleave perfectly matched target RNA. Our results demonstrate that human ES cell differentiation is accompanied by changes in the expression of a unique set of miRNAs, providing a glimpse of a new molecular circuitry that may regulate early development in humans. Chromosomes 19 and X contained 98 and 40 miRNA genes, respectively, indicating that majority of miRNA genes in hES cells were expressed from these two chromosomes. Strikingly, distribution analysis of miRNA gene loci across six species including dog, rat, mouse, rhesus, chimpanzee, and human showed that miRNA genes encoded in chromosome 19 were drastically increased in chimpanzees and humans while miRNA gene loci on other chromosomes were decreased as compared with dog, rat, and mouse. Comparative genomic studies showed 99% conservation of chromosome 19 miRNA genes between chimpanzees and humans. Together, these findings reveal the evolutionary emergence, approximately 5 million years ago, of miRNAs involved in regulating early human development. One could imagine that this burst of miRNA gene clusters at specific chromosomes was part of an evolutionary event during species divergence.

Project description:BACKGROUND: Important knowledge about the role of vitamin A in vertebrate heart development has been obtained using the vitamin A-deficient avian in ovo model which enables the in vivo examination of very early stages of vertebrate heart morphogenesis. These studies have revealed the critical role of the vitamin A-active form, retinoic acid (RA) in the regulation of several developmental genes, including the important growth regulatory factor, transforming growth factor-beta2 (TGF?2), involved in early events of heart morphogenesis. However, this in ovo model is not readily available for elucidating details of molecular mechanisms determining RA activity, thus limiting further examination of RA-regulated early heart morphogenesis. In order to obtain insights into RA-regulated gene expression during these early events, a reliable in vitro model is needed. Here we describe a cell culture that closely reproduces the in ovo observed regulatory effects of RA on TGF?2 and on several developmental genes linked to TGF? signaling during heart morphogenesis. RESULTS: We have developed an avian heart forming region (HFR) cell based in vitro model that displays the characteristics associated with vertebrate early heart morphogenesis, i.e. the expression of Nkx2.5 and GATA4, the cardiogenesis genes, of vascular endothelial growth factor (VEGF-A), the vasculogenesis gene and of fibronectin (FN1), an essential component in building the heart, and the expression of the multifunctional genes TGF?2 and neogenin (NEO). Importantly, we established that the HFR cell culture is a valid model to study RA-regulated molecular events during heart morphogenesis and that the expression of TGF?2 as well as the expression of several TGF?2-linked developmental genes is regulated by RA. CONCLUSIONS: Our findings reported here offer a biologically relevant experimental in vitro system for the elucidation of RA-regulated expression of TGF?2 and other genes involved in vertebrate early cardiovascular morphogenesis.

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A molecular roadmap for the emergence of early-embryonic-like cells in culture.

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A molecular roadmap for the emergence of early-embryonic-like cells in culture.

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