Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells

Recapitulating early human development with 8C-like cells.

with details of the nature of the infringement. We will investigate the claim and if justified, we will take the appropriate steps.

Defining Human Pluripotency.

Chromosome instability and aneuploidies occur very frequently in human embryos, impairing proper embryogenesis and leading to cell cycle arrest, loss of cell viability, and developmental failures in 50–80% of cleavage-stage embryos. This high frequency of cellular extinction events represents a significant experimental obstacle challenging analyses of individual cells isolated from human preimplantation embryos. We carried out single cell expression profiling of 241 individual cells recovered from 32 human embryos during the early and late stages of viable human blastocyst (VHB) differentiation. Classification of embryonic cells was performed solely based on expression patterns of human pluripotency-associated transcripts (HPAT), which represent a family of primate-specific transposable element-derived lincRNAs highly expressed in human embryonic stem cells and regulating nuclear reprogramming and pluripotency induction. We then validated our findings by analyzing transcriptomes of 1,708 individual cells recovered from more than 100 human embryos and 259 mouse cells from more than 40 mouse embryos at different stages of preimplantation embryogenesis. HPAT's expression-guided spatiotemporal reconstruction of human embryonic development inferred from single-cell expression analysis of VHB differentiation enabled identification of telomerase-positive embryonic cells co-expressing key pluripotency regulatory genes and genetic markers of three major lineages. Follow-up validation analyses confirmed the emergence in human embryos prior to lineage segregation of telomerase-positive cells co-expressing genetic markers of multiple lineages. Observations reported in this contribution support the hypothesis of a developmental pathway of creation embryonic lineages and extraembryonic tissues from telomerase-positive pre-lineage cells manifesting multi-lineage precursor phenotype.

Single cell genomics reveals activation signatures of endogenous SCAR's networks in aneuploid human embryos and clinically intractable malignant tumors

Plants produce valuable natural products used for a wide variety of industrial applications. Thus, there is enormous interest in elucidating the biosynthetic pathways that are responsible for the production of these compounds. Identification of the genes that comprise these biosynthetic pathways has been enabled by gene-to-metabolite networks that are generated from transcriptomic and metabolomic datasets. Recent advances in both single-cell RNA-seq (scRNA-seq) and single-cell mass spectrometry metabolomics (scMS) have enabled the measurement of either gene expression or metabolite levels in individual cells. However, these datasets can only be used to indirectly correlate gene expression levels with metabolite concentrations at the single-cell level. In this proof-of-concept study, performed on cells derived from the leaves of the medicinal plant Catharanthus roseus, we demonstrate that both scRNA-seq and scMS can be applied to the same plant cell, thereby enabling direct comparisons between gene expression and metabolite levels. Protoplasts are sorted into 96-well plates using a microfluidics-based robot and then lysed under conditions that are suitable for both scMS and SMART-seq single-cell protocols. This multiplexing approach reveals both qualitative and quantitative correlations between metabolite levels and biosynthetic gene expression in individual cells. This integrated approach sheds light on the underlying processes driving complex plant biosynthesis.

Hematopoietic development from human embryonic stem cell lines

We aimed to investigate if Cyclin E1 (CCNE1) plays a role in human embryogenesis, in particular during the early developmental stages characterized by a short cell cycle. CCNE1 is expressed in plenipotent human embryonic cells and plays a critical role during hESC derivation via the naïve state and, potentially, normal embryo development. A short cell cycle due to a truncated G1 phase has been associated with the high developmental capacity of embryonic cells. CCNE1 is a critical G1/S transition regulator. CCNE1 overexpression can cause shortening of the cell cycle and it is constitutively expressed in mouse embryonic stem cells and cancer cells. We investigated expression of CCNE1 in human preimplantation embryo development and embryonic stem cells (hESC). Functional studies included CCNE1 overexpression in hESC and CCNE1 downregulation in the outgrowths formed by plated human blastocysts. Analysis was performed by immunocytochemistry and quantitative real-time PCR. Mann-Whitney statistical test was applied. The CCNE1 protein was ubiquitously and constitutively expressed in the plenipotent cells of the embryo from the 4-cell stage up to and including the full blastocyst. During blastocyst expansion, CCNE1 was downregulated in the trophectoderm (TE) cells. CCNE1 shortly co-localized with NANOG in the inner cell mass (ICM) of expanding blastocysts, mimicking the situation in naïve hESC. In the ICM of expanded blastocysts, which corresponds with primed hESC, CCNE1 defined a subpopulation of cells different from NANOG/POU5F1-expressing pluripotent epiblast (EPI) cells and GATA4/SOX17-expressing primitive endoderm (PrE) cells. This CCNE1-positive cell population was associated with visceral endoderm based on transthyretin expression and marked the third cell lineage within the ICM, besides EPI and PrE, which had never been described before. We also investigated the role of CCNE1 by plating expanded blastocysts for hESC derivation. As a result, all the cells including TE cells re-gained CCNE1 and, consequently, NANOG expression, resembling the phenotype of naïve hESC. The inhibition of CCNE1 expression with siRNA blocked proliferation and caused degeneration of those plated cells. The study is based on a limited number of good-quality human embryos donated to research. Our study sheds light on the processes underlying the high developmental potential of early human embryonic cells. The CCNE1-positive plenipotent cell type corresponds with a phenotype that enables early human embryos to recover after fragmentation, cryodamage or (single cell) biopsy on day 3 for preimplantation genetic diagnosis. Knowledge on the expression and function of genes responsible for this flexibility will help us to better understand the undifferentiated state in stem cell biology and might enable us to improve technologies in assisted reproduction. NA STUDY FUNDING AND COMPETING INTERESTS: This research is supported by grants from the Fund for Scientific Research - Flanders (FWO-Vlaanderen), the Methusalem (METH) of the VUB and Scientific Research Fond Willy Gepts of UZ Brussel. There are no competing interests.

The Fas-Fas ligand (L) system is one of the major signalling pathways to induce apoptosis in various cells and tissues. The aim of this study was to investigate the expression of the Fas-Fas L system in rat and human oocytes and preimplantation embryos. We determined the expression of Fas and Fas L mRNA of rat oocytes and embryos up to the blastocyst stage, and of human embryos at the 2- or 4-cell stage, using reverse transcription polymerase chain reaction (PCR) and nested PCR techniques. Moreover, we investigated the expression of Fas mRNA in human fragmented embryos. In rat embryos, Fas mRNA was expressed at the 2-cell stage only, whereas Fas L mRNA was expressed in oocytes, and at the pronuclear (1-cell) and 2-cell stages. In human embryos, Fas mRNA was expressed at the 4-cell stage only, whereas Fas L mRNA was expressed at both 2- and 4-cell stages. Human fragmented embryos expressed both Fas and Fas L mRNA. Because simultaneous expression of Fas and Fas L mRNA occurred in 2-cell rat embryos and in 4-cell human embryos, the Fas-Fas L system might be involved in the apoptotic pathway in the early embryos of these species.

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.

Lack of checkpoint control in human pluripotent stem cells: a cause for genomic instability?

Editorial FocusA recipe for pluripotency: the correct sp(l)ices make all the difference. Focus on "Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency"Paul J. GokhalePaul J. GokhaleCentre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, United KingdomPublished Online:15 Jun 2014https://doi.org/10.1152/ajpcell.00116.2014This is the final version - click for previous versionMoreSectionsPDF (55 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations human pluripotent stem cells (PSCs), either directly isolated from embryos or induced by exogenous factors, have not only opened up the possibility of deriving genetically normal cell types in large numbers, but they also offer a window into normally inaccessible human embryogenesis. The understanding and control of human PSC self-renewal and differentiation rely on a thorough molecular understanding of control of the stem cell state: how cells exit the pluripotent state, and how cells are specified to particular fates. Much of the understanding of PSC biology has come from studies in the mouse. However, it has recently become apparent that PSCs from mouse and human, previously thought of as equivalent types, are, in fact, quite different entities. Mouse embryonic stem cells (ESCs) can be grown on feeders with leukemia inhibitory factor or by restriction of differentiation and, thus, heterogeneity using chemical inhibitors (2i dual-inhibition conditions) (4). PSCs have also been derived from postimplantation mouse embryos; such cells are referred to as epiblast stem cells (EpiSCs). These cells share many of the characteristics of human ESCs (hESCs), including basic gene expression and growth factor requirements. However, there are some differences between human and mouse EpiSCs, such as the tendency to express markers of the primitive streak stage of development (6). To complicate the picture further, hESCs appear to be complex mixtures of cell states and seem to be more heterogeneous than mouse primed or naive ESCs (2a).Much research has been conducted over the past decade into the mechanisms by which pluripotency is controlled. Significant insights into the regulation of PSCs have been obtained from the investigation of transcription factors and their associated networks. Most of these studies have treated the mouse as a paradigm for the human system, which is likely to be a misleading assumption. Three transcription factors, octamer-binding transcription factor 4 [OCT4, POU domain class 5 transcription factor 1 (POU5F1)], sex-determining region Y-box 2 (SOX2), and NANOG, have emerged as central players in the control of pluripotency (3). These studies, however, have tended to use mixtures of cells, and, given the apparent heterogeneity in human PSC (hPSC) cultures, further refinement of the gene regulatory networks is required.The realization that splice variation may play a role in the control of the pluripotent phenotype in humans adds a further layer of complexity (1, 11). In the human system, OCT4 (POU5F1) can be expressed as different splice variants: OCT4A, OCT4B, and OCT4B1 (9). OCT4A is the direct ortholog of the mouse Oct4 transcript and is critical for self-renewal of hPSCs (9). The roles of OCT4B and OCT4B1 are much less well understood, despite their expression at the transcript level in hPSCs. Previous observations of subcellular location are suggestive of functional differences between OCT4A and OCT4B (2), but proper functional analysis is lacking. The presence of six transcribed pseudogenes makes primer design and interpretation of PCR results for OCT4 expression tricky. Thus previous studies of the function of OCT4 in hPSCs have not distinguished between the similar OCT4A and OCT4B and, thus, could not assess the role of the alternate splicing in hPSC self-renewal and differentiation. In light of the apparent complexity of the human system, researchers must begin the task of disentangling heterogeneity and splice variation to fully understand the control of the pluripotent state in humans.The study by Tsai et al. (8) in this issue of American Journal of Physiology-Cell Physiology is a first step to come to grips with the role that alternate splicing of OCT4 may play in the maintenance of pluripotency. Tsai et al. focused on the role of OCT4A, as overexpression of OCT4B does not result in increased protein production, suggesting posttranslational reduction of OCT4B levels in hESCs. The ability to specifically overexpress or knock down OCT4A yielded subtle differences between this study and previous efforts that did not discriminate between OCT4 isoforms. Specifically, targeting OCT4A for knockdown resulted in reduction of the transcription factors SOX2 and NANOG and increases in the transcription factors paired box 6 (PAX6), neural cell adhesion molecule (NCAM), and fibroblast growth factor 5 (FGF5). Most previous studies have tended to report that the effect of OCT4 knockdown in humans is similar to the mouse paradigm, namely, trophectoderm and endoderm differentiation. The specific upregulation of expression of PAX6 and NCAM, markers of neuroectoderm lineages, in the study of Tsai et al. is novel. The induction of PAX6 and NCAM upon OCT4A knockdown suggests that the relative levels of OCT4 splice variants may play a role in lineage specification in the human system. However, differences in phenotypes observed by Tsai et al. and in studies using a nonspecific OCT4 knockdown may be influenced by the signaling environment, not just which splice variant is knocked down; for example, bone morphogenetic protein 4 concentration specifies ectodermal vs. extraembryonic lineages in cells with low OCT4 levels (10).The increase in expression of FGF5 following OCT4A knockdown is interesting, as this classical marker of the mouse epiblast has proved to be very difficult to detect in differentiating hESC cultures. There may be many reasons for the lack of FGF5 detection in previous studies, but, at the very least, the results presented by Tsai et al. (8) suggest that the OCT4A knockdown cells may pass through an FGF5-positive "epiblast-like" stage during differentiation. In contrast, overexpression of OCT4A, which initially seemed to induce lineage genes, appeared to stabilize the self-renewing state, resulting in cells with higher single-cell replating ability (Fig. 1). This effect may be a function of the fact that the protein levels of OCT4A, SOX2, and NANOG did not appear to increase, even though a proportion of the OCT4A expression was being driven from a transgene. This stabilization of the self-renewing state, despite OCT4A overexpression, again suggests the presence of subtle posttranslational control. The ability of OCT4A-overexpressing cells to exist as a stable entity is interesting, as data from the mouse suggest that overexpression of Oct4 drives differentiation (5), and there is a narrow range of OCT4 expression that permits pluripotent differentiation (7). Whether the OCT4A-overexpressing cells are compromised in their ability to differentiate or whether in humans the system is able to compensate for increased OCT4A expression remains to be tested.Fig. 1.Human pluripotent stem cells exist in a range of interconverting self-renewing and differentiated cell states. Wild-type (WT) human pluripotent stem cells exist in a range of "self-renewing" states, with only a small fraction in a clonogenic state, i.e., able to recreate the whole culture from a single cell. Increasing expression of octamer-binding transcription factor 4A (OCT4A) stabilizes the self-renewing states and increases the occupancy of the clonogenic states. Reduction of expression, specifically of OCT4A, results in ectoderm differentiation.Download figureDownload PowerPointOverall, the results presented by Tsai et al. (8) and other studies (1, 11) point to an emerging role for differential splicing in the control of the decisions that PSCs make between self-renewal and differentiation. It seems likely that much more attention to exactly which splice variants are expressed and which variants are being manipulated in experimental settings is needed, if we are to fully understand the control mechanisms that operate in PSCs in humans. The observation that increased OCT4A can stabilize a more clonogenic state in humans (the poor single-cell plating efficiency being a major drawback to handling human compared with mouse PSCs) may enable researchers in the future to establish what extrinsic controls, such as growth factors or inhibitors, could be applied to cultures to keep them in a clonogenic state and, thus, minimize stresses during expansion.DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the author.AUTHOR CONTRIBUTIONSP.J.G. prepared the figure; P.J.G. drafted the manuscript; P.J.G. edited and revised the manuscript; P.J.G. approved the final version of the manuscript.REFERENCES1. Alagaratnam S, Harrison N, Bakken AC, Hoff AM, Jones M, Sveen A, Moore HD, Andrews PW, Lothe RA, Skotheim RI. Transforming pluripotency: an exon-level study of malignancy-specific transcripts in human embryonal carcinoma and embryonic stem cells. Stem Cells Dev 22: 1136–1146, 2013.Crossref | PubMed | ISI | Google Scholar2. Cauffman G, Liebaers I, Van Steirteghem A, Van de Velde H. POU5F1 isoforms show different expression patterns in human embryonic stem cells and preimplantation embryos. Stem Cells 24: 2685–2691, 2006.Crossref | PubMed | ISI | Google Scholar2a. Enver T, Pera M, Peterson C, Andrews PW. Stem cell states, fates, and the rules of attraction. Cell Stem Cell 8: 387–397, 2009.Crossref | ISI | Google Scholar3. Mallanna SK, Rizzino A. Systems biology provides new insights into the molecular mechanisms that control the fate of embryonic stem cells. J Cell Physiol 227: 27–34, 2012.Crossref | PubMed | ISI | Google Scholar4. Nichols J, Smith A. Naive and primed pluripotent states. Cell Stem Cell 4: 487–492, 2009.Crossref | PubMed | ISI | Google Scholar5. Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24: 372–376, 2000.Crossref | PubMed | ISI | Google Scholar6. Pera MF, Tam PP. Extrinsic regulation of pluripotent stem cells. Nature 465: 713–720, 2010.Crossref | PubMed | ISI | Google Scholar7. Radzisheuskaya A, Chia Gle B, dos Santos RL, Theunissen TW, Castro LF, Nichols J, Silva JC. A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages. Nat Cell Biol 15: 579–590, 2013.Crossref | PubMed | ISI | Google Scholar8. Tsai SC, Chang DF, Hong CM, Xia P, Senadheera D, Trump L, Mishra S, Lutzko C. Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency. Am J Physiol Cell Physiol (March 13, 2014). doi:10.1152/ajpcell.00205.2013.Link | ISI | Google Scholar9. Wang X, Dai J. Isoforms of OCT4 contribute to the confusing diversity in stem cell biology. Stem Cells 28: 885–893, 2010.Crossref | PubMed | ISI | Google Scholar10. Wang Z, Oron E, Nelson B, Razis S, Ivanova N. Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell 10: 440–454, 2012.Crossref | PubMed | ISI | Google Scholar11. Wu CS, Yu CY, Chuang CY, Hsiao M, Kao CF, Kuo HC, Chuang TJ. Integrative transcriptome sequencing identifies trans-splicing events with important roles in human embryonic stem cell pluripotency. Genome Res 24: 25–36, 2014.Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: P. J. Gokhale, Centre for Stem Cell Biology, Dept. of Biomedical Science, Univ. of Sheffield, Western Bank, Sheffield S10 2TN, UK (e-mail: p.[email protected]ac.uk). Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation Cited ByInduced overexpression of OCT4A in human embryonic stem cells increases cloning efficiencySteven C. Tsai, David F. Chang, Chang-Mu Hong, Ping Xia, Dinithi Senadheera, Lisa Trump, Suparna Mishra, and Carolyn Lutzko15 June 2014 | American Journal of Physiology-Cell Physiology, Vol. 306, No. 12 More from this issue > Volume 306Issue 12June 2014Pages C1106-C1107 Copyright & PermissionsCopyright © 2014 the American Physiological Societyhttps://doi.org/10.1152/ajpcell.00116.2014PubMed24740536History Published online 15 June 2014 Published in print 15 June 2014 Metrics

NANOG and OCT4 are required for the maintenance of pluripotency in embryonic stem cells (ESCs). These proteins are also expressed in the inner cell mass (ICM) of the mouse pre-implantation embryo. Immunohistochemistry was used to show the presence of NANOG and OCT4 protein, and in situ hybridization was used to localize NANOG mRNA in human embryos from two-cell to blastocyst stage, and in human ESCs (hESCs). Nanog and Oct4 were co-localized in human embryos from morula and blastocyst stages. NANOG mRNA was detected in a group of cells in the morula, in cells of the ICM of blastocysts, and evenly in hESCs. All non-differentiated hESCs expressed NANOG and OCT4 protein. Pluripotent cells expressing NANOG and Oct4 were eccentrically localized, probably in polarized cells in a human compacted morula, which appears to be different from expression in murine embryos. In this study, we demonstrate that whole mount in situ hybridization is amenable to localization of mRNAs in human development, as in other species.

Aneuploidy is commonly seen in human preimplantation embryos, most particularly at the cleavage stage because of genome activation by third cell division. Aneuploid embryos have been used for the derivation of normal embryonic stem cell (ESC) lines and developmental modeling. This review addresses aneuploidies in human preimplantation embryos and human ESCs and the potential of self-correction of these aberrations. Diploid-aneuploid mosaicism is the most frequent abnormality observed; hence, embryos selected by preimplantation genetic diagnosis at the cleavage or blastocyst stage could be partly abnormal. Differentiation is known as the barrier for eliminating mosaic embryos by death and/or decreased division of abnormal cells. However, some mosaicisms, such as copy number variations could be compatible with live birth. Several reasons have been proposed for self-correction of aneuploidies during later stages of development, including primary misdiagnosis, allocation of the aneuploidy in the trophectoderm, cell growth advantage of diploid cells in mosaic embryos, lagging of aneuploid cell division, extrusion or duplication of an aneuploid chromosome, and the abundance of DNA repair gene products. Although more studies are needed to understand the mechanisms of self-correction as a rare phenomenon, most likely, it is related to overcoming mosaicism.

In the last decade medical science has started to make the first steps towards a paradigm shift from repair to regeneration [1]. The potential use of a range of stem cells in regenerative medicine is currently one of the most intensively researched areas worldwide [2]. In particular, embryonic stem cells (ESC) and more recently Induced Pluripotent Stemcells (iPS) [3] have provoked a lot of interest as a potential source of stem cells in the treatment of diseases such as Alzheimer’s and Parkinson’s diseases. Nevertheless, these hopes have not yet been fulfilled with either ESC or iPS. Murine ESC were first described in 1981 [4] and subsequent studies showed the regenerative potential of murine stem cells derived from both preand postimplantation embryos [5–7]. This early murine work was followed by the development of human ESC in 1998 [8]. Both murine and human pre-implantation ESC are derived from the inner cell mass (ICM) of the blastocyst at day 5/6 of growth. The ICM consists of primitive ectoderm cells, which subsequently develop into epiblast cells in postimplantation embryos. Despite these ontogenic similarities human and murine ESC do not resemble each other in many aspects [9]. For example, murine ESC rely on leukemia inhibitory factor (LIF) and bone morphogenic protein-4 (BMP4) for maintenance of pluripotency [10, 11], whilst human ESC are dependent on TGFβ/Activin/Nodal pathway activity and fibroblast growth factor-2 (FGF2) [12–17] and thus resemble epiblast stem cells (EpiSC). Usually EpiSC cannot be reverted to a state such that they respond to LIF and BMP4. Furthermore, primordial germ cells, which originate from pluripotent epiblast cells in mice, respond in a similar way to LIF and BMP4, such as murine ESC derived from the ICM [18]. Additionally, murine ESC can be cultured as single cells while human ESC are particularly sensitive to dissociation and should be cultured in clumps [19]. There are clearly many basic concepts still to be explored in the biology of ESC of various species. Thorough understanding of these cells is still in its’ infancy despite 20 years of basic research. Bearing this is mind there should be an air of caution when proposing future clinical applications. Can or should we move forward into thinking about clinical applications when the fundamentals Capsule Human cord blood stem cells have a realistic and proven potential for the treatment of various diseases compared to embryonic stem cells / iPS cells.