Capturing totipotency in human cells through spliceosomal repression.

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

Acquisition of 2C-like totipotency through defined maternal-effect factors.

Fully grown oocytes have the natural ability to transform 2 terminally differentiated gametes into a totipotent zygote representing the acquisition of totipotency. This process wholly depends on maternal-effect factors (MFs). MFs stored in the eggs are therefore likely to be able to induce cellular reprogramming to a totipotency state. Here we report the generation of totipotent-like stem cells from mESCs using 4MFs Hsf1, Zar1, Padi6, and Npm2, designated as MFiTLSCs. MFiTLSCs exhibited a unique and inherent capability to differentiate into embryonic and extraembryonic derivatives. Transcriptomic analysis revealed that MFiTLSCs are enriched with 2-cell-specific genes that appear to synergistically induce a transcriptional repressive state, in that parental genomes are remodeled to a poised transcriptional repression state while totipotency is established following fertilization. This method to derive MFiTLSCs could help advance the understanding of fate determinations of totipotent stem cells in a physiological context and establish a foundation for the development of oocyte biology-based reprogramming technology.

Induction of mouse totipotent stem cells by a defined chemical cocktail.

In mice, only the zygotes and blastomeres from 2-cell embryos are authentic totipotent stem cells (TotiSCs) capable of producing all the differentiated cells in both embryonic and extraembryonic tissues and forming an entire organism1. However, it remains unknown whether and how totipotent stem cells can be established in vitro in the absence of germline cells. Here we demonstrate the induction and long-term maintenance of TotiSCs from mouse pluripotent stem cells using a combination of three small molecules: the retinoic acid analogue TTNPB, 1-azakenpaullone and the kinase blocker WS6. The resulting chemically induced totipotent stem cells (ciTotiSCs), resembled mouse totipotent 2-cell embryo cells at the transcriptome, epigenome and metabolome levels. In addition, ciTotiSCs exhibited bidirectional developmental potentials and were able to produce both embryonic and extraembryonic cells in vitro and in teratoma. Furthermore, following injection into 8-cell embryos, ciTotiSCs contributed to both embryonic and extraembryonic lineages with high efficiency. Our chemical approach to totipotent stem cell induction and maintenance provides a defined in vitro system for manipulating and developing understanding of the totipotent state and the development of multicellular organisms from non-germline cells.

Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells.

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are 'true' totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely 'cluster 3', as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

CTCF is a barrier for 2C-like reprogramming.

Totipotent cells have the ability to generate embryonic and extra-embryonic tissues. Interestingly, a rare population of cells with totipotent-like potential, known as 2 cell (2C)-like cells, has been identified within ESC cultures. They arise from ESC and display similar features to those found in the 2C embryo. However, the molecular determinants of 2C-like conversion have not been completely elucidated. Here, we show that the CCCTC-binding factor (CTCF) is a barrier for 2C-like reprogramming. Indeed, forced conversion to a 2C-like state by the transcription factor DUX is associated with DNA damage at a subset of CTCF binding sites. Depletion of CTCF in ESC efficiently promotes spontaneous and asynchronous conversion to a 2C-like state and is reversible upon restoration of CTCF levels. This phenotypic reprogramming is specific to pluripotent cells as neural progenitor cells do not show 2C-like conversion upon CTCF-depletion. Furthermore, we show that transcriptional activation of the ZSCAN4 cluster is necessary for successful 2C-like reprogramming. In summary, we reveal an unexpected relationship between CTCF and 2C-like reprogramming.

Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

In multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

Retinoic acid signaling is critical during the totipotency window in early mammalian development.

Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such '2-cell-like-cells' (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

Mouse totipotent stem cells captured and maintained through spliceosomal repression.

Since establishment of the first embryonic stem cells (ESCs), in vitro culture of totipotent cells functionally and molecularly comparable with in vivo blastomeres with embryonic and extraembryonic developmental potential has been a challenge. Here we report that spliceosomal repression in mouse ESCs drives a pluripotent-to-totipotent state transition. Using the splicing inhibitor pladienolide B, we achieve stable in vitro culture of totipotent ESCs comparable at molecular levels with 2- and 4-cell blastomeres, which we call totipotent blastomere-like cells (TBLCs). Mouse chimeric assays combined with single-cell RNA sequencing (scRNA-seq) demonstrate that TBLCs have a robust bidirectional developmental capability to generate multiple embryonic and extraembryonic cell lineages. Mechanically, spliceosomal repression causes widespread splicing inhibition of pluripotent genes, whereas totipotent genes, which contain few short introns, are efficiently spliced and transcriptionally activated. Our study provides a means for capturing and maintaining totipotent stem cells.

Evaluating totipotency using criteria of increasing stringency.

Totipotency is the ability of a single cell to give rise to all of the differentiated cell types that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies on a variety of assays of variable stringency. Here, we describe criteria to define totipotency. We explain how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in mice, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbour increased totipotent potential relative to conventional embryonic stem cells under in vitro and in vivo conditions.

TRF2-mediated telomere protection is dispensable in pluripotent stem cells.

In mammals, telomere protection is mediated by the essential protein TRF2, which binds chromosome ends and ensures genome integrity1,2. TRF2 depletion results in end-to-end chromosome fusions in all cell types that have been tested so far. Here we find that TRF2 is dispensable for the proliferation and survival of mouse embryonic stem (ES) cells. Trf2-/- (also known as Terf2) ES cells do not exhibit telomere fusions and can be expanded indefinitely. In response to the deletion of TRF2, ES cells exhibit a muted DNA damage response that is characterized by the recruitment of γH2AX-but not 53BP1-to telomeres. To define the mechanisms that control this unique DNA damage response in ES cells, we performed a CRISPR-Cas9-knockout screen. We found a strong dependency of TRF2-null ES cells on the telomere-associated protein POT1B and on the chromatin remodelling factor BRD2. Co-depletion of POT1B or BRD2 with TRF2 restores a canonical DNA damage response at telomeres, resulting in frequent telomere fusions. We found that TRF2 depletion in ES cells activates a totipotent-like two-cell-stage transcriptional program that includes high levels of ZSCAN4. We show that the upregulation of ZSCAN4 contributes to telomere protection in the absence of TRF2. Together, our results uncover a unique response to telomere deprotection during early development.

From pluripotency to totipotency: an experimentalist's guide to cellular potency.

Embryonic stem cells (ESCs) are derived from the pre-implantation mammalian blastocyst. At this point in time, the newly formed embryo is concerned with the generation and expansion of both the embryonic lineages required to build the embryo and the extra-embryonic lineages that support development. When used in grafting experiments, embryonic cells from early developmental stages can contribute to both embryonic and extra-embryonic lineages, but it is generally accepted that ESCs can give rise to only embryonic lineages. As a result, they are referred to as pluripotent, rather than totipotent. Here, we consider the experimental potential of various ESC populations and a number of recently identified in vitro culture systems producing states beyond pluripotency and reminiscent of those observed during pre-implantation development. We also consider the nature of totipotency and the extent to which cell populations in these culture systems exhibit this property.

Live Imaging of embryogenic structures in Brassica napus microspore embryo cultures highlights the developmental plasticity of induced totipotent cells.

KEY MESSAGE: In vitro embryo development is highly plastic; embryo cell fate can be re-established in tissue culture through different pathways. In most angiosperms, embryo development from the single-celled zygote follows a defined pattern of cell divisions in which apical (embryo proper) and basal (root and suspensor) cell fates are established within the first cell divisions. By contrast, embryos that are induced in vitro in the absence of fertilization show a less regular initial cell division pattern yet develop into histodifferentiated embryos that can be converted into seedlings. We used the Brassica napus microspore embryogenesis system, in which the male gametophyte is reprogrammed in vitro to form haploid embryos, to identify the developmental fates of the different types of embryogenic structures found in culture. Using time-lapse imaging of LEAFY COTYLEDON1-expressing cells, we show that embryogenic cell clusters with very different morphologies are able to form haploid embryos. The timing of surrounding pollen wall (exine) rupture is a major determinant of cell fate in these clusters, with early exine rupture leading to the formation of suspensor-bearing embryos and late rupture to suspensorless embryos. In addition, we show that embryogenic callus, which develops into suspensor-bearing embryos, initially expresses transcripts associated with both basal- and apical-embryo cell fates, suggesting that these two cell fates are fixed later in development. This study reveals the inherent plasticity of in vitro embryo development and identifies new pathways by which embryo cell fate can be established.

From Single Cell to Plants: Mesophyll Protoplasts as a Versatile System for Investigating Plant Cell Reprogramming.

Plants are sessile organisms that have a remarkable developmental plasticity, which ensures their optimal adaptation to environmental stresses. Plant cell totipotency is an extreme example of such plasticity, whereby somatic cells have the potential to form plants via direct shoot organogenesis or somatic embryogenesis in response to various exogenous and/or endogenous signals. Protoplasts provide one of the most suitable systems for investigating molecular mechanisms of totipotency, because they are effectively single cell populations. In this review, we consider the current state of knowledge of the mechanisms that induce cell proliferation from individual, differentiated somatic plant cells. We highlight initial explant metabolic status, ploidy level and isolation procedure as determinants of successful cell reprogramming. We also discuss the importance of auxin signalling and its interaction with stress-regulated pathways in governing cell cycle induction and further stages of plant cell totipotency.

Lessons from expanded potential of embryonic stem cells: Moving toward totipotency.

Embryonic stem cells possess fascinating capacity of self-renewal and developmental potential, leading to significant progress in understanding the molecular basis of pluripotency, disease modeling, and reprogramming technology. Recently, 2-cell-like embryonic stem cells (ESCs) and expanded potential stem cells or extended pluripotent stem cells (EPSCs) generated from early-cleavage embryos display some features of totipotent embryos. These cell lines provide valuable in vitro models to study underlying principles of totipotency, cell plasticity, and lineage segregation. In this review, we summarize the current progress in this filed and highlight the application potentials of these cells in the future.

BNC1 regulates cell heterogeneity in human pluripotent stem cell-derived epicardium.

The murine developing epicardium heterogeneously expresses the transcription factors TCF21 and WT1. Here, we show that this cell heterogeneity is conserved in human epicardium, regulated by BNC1 and associated with cell fate and function. Single cell RNA sequencing of epicardium derived from human pluripotent stem cells (hPSC-epi) revealed that distinct epicardial subpopulations are defined by high levels of expression for the transcription factors BNC1 or TCF21. WT1+ cells are included in the BNC1+ population, which was confirmed in human foetal hearts. THY1 emerged as a membrane marker of the TCF21 population. We show that THY1+ cells can differentiate into cardiac fibroblasts (CFs) and smooth muscle cells (SMCs), whereas THY1- cells were predominantly restricted to SMCs. Knocking down BNC1 during the establishment of the epicardial populations resulted in a homogeneous, predominantly TCF21high population. Network inference methods using transcriptomic data from the different cell lineages derived from the hPSC-epi delivered a core transcriptional network organised around WT1, TCF21 and BNC1. This study unveils a list of epicardial regulators and is a step towards engineering subpopulations of epicardial cells with selective biological activities.

Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cells.

Totipotency refers to the ability of a cell to generate all of the cell types of an organism. Unlike pluripotency, the establishment of totipotency is poorly understood. In mouse embryonic stem cells, Dux drives a small percentage of cells into a totipotent state by expressing 2-cell-embryo-specific transcripts. To understand how this transition takes place, we performed single-cell RNA-seq, which revealed a two-step transcriptional reprogramming process characterized by downregulation of pluripotent genes in the first step and upregulation of the 2-cell-embryo-specific elements in the second step. To identify factors controlling the transition, we performed a CRISPR-Cas9-mediated screen, which revealed Myc and Dnmt1 as two factors preventing the transition. Mechanistic studies demonstrate that Myc prevents downregulation of pluripotent genes in the first step, while Dnmt1 impedes 2-cell-embryo-specific gene activation in the second step. Collectively, the findings of our study reveal insights into the establishment and regulation of the totipotent state in mouse embryonic stem cells.

Capturing Totipotent Stem Cells.

Minority subpopulations within embryonic stem cell cultures display an expanded developmental potential similar to that of early embryo blastomeres or the early inner cell mass. The ability to isolate and culture totipotent cells capable of giving rise to the entire conceptus would enhance our capacity to study early embryo development, and might enable more efficient generation of chimeric animals for research and organ production for transplantation. Here we review the biological and molecular characterization of cultured cells with developmental potential similar to totipotent blastomeres, and assess recent progress toward the capture and stabilization of the totipotent state in vitro.

Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition.

Chromatin is reprogrammed after fertilization to produce a totipotent zygote with the potential to generate a new organism. The maternal genome inherited from the oocyte and the paternal genome provided by sperm coexist as separate haploid nuclei in the zygote. How these two epigenetically distinct genomes are spatially organized is poorly understood. Existing chromosome conformation capture-based methods are not applicable to oocytes and zygotes owing to a paucity of material. To study three-dimensional chromatin organization in rare cell types, we developed a single-nucleus Hi-C (high-resolution chromosome conformation capture) protocol that provides greater than tenfold more contacts per cell than the previous method. Here we show that chromatin architecture is uniquely reorganized during the oocyte-to-zygote transition in mice and is distinct in paternal and maternal nuclei within single-cell zygotes. Features of genomic organization including compartments, topologically associating domains (TADs) and loops are present in individual oocytes when averaged over the genome, but the presence of each feature at a locus varies between cells. At the sub-megabase level, we observed stochastic clusters of contacts that can occur across TAD boundaries but average into TADs. Notably, we found that TADs and loops, but not compartments, are present in zygotic maternal chromatin, suggesting that these are generated by different mechanisms. Our results demonstrate that the global chromatin organization of zygote nuclei is fundamentally different from that of other interphase cells. An understanding of this zygotic chromatin 'ground state' could potentially provide insights into reprogramming cells to a state of totipotency.

Evolutionary origin and functional divergence of totipotent cell homeobox genes in eutherian mammals.

BACKGROUND: A central goal of evolutionary biology is to link genomic change to phenotypic evolution. The origin of new transcription factors is a special case of genomic evolution since it brings opportunities for novel regulatory interactions and potentially the emergence of new biological properties. RESULTS: We demonstrate that a group of four homeobox gene families (Argfx, Leutx, Dprx, Tprx), plus a gene newly described here (Pargfx), arose by tandem gene duplication from the retinal-expressed Crx gene, followed by asymmetric sequence evolution. We show these genes arose as part of repeated gene gain and loss events on a dynamic chromosomal region in the stem lineage of placental mammals, on the forerunner of human chromosome 19. The human orthologues of these genes are expressed specifically in early embryo totipotent cells, peaking from 8-cell to morula, prior to cell fate restrictions; cow orthologues have similar expression. To examine biological roles, we used ectopic gene expression in cultured human cells followed by high-throughput RNA-seq and uncovered extensive transcriptional remodelling driven by three of the genes. Comparison to transcriptional profiles of early human embryos suggest roles in activating and repressing a set of developmentally-important genes that spike at 8-cell to morula, rather than a general role in genome activation. CONCLUSIONS: We conclude that a dynamic chromosome region spawned a set of evolutionarily new homeobox genes, the ETCHbox genes, specifically in eutherian mammals. After these genes diverged from the parental Crx gene, we argue they were recruited for roles in the preimplantation embryo including activation of genes at the 8-cell stage and repression after morula. We propose these new homeobox gene roles permitted fine-tuning of cell fate decisions necessary for specification and function of embryonic and extra-embryonic tissues utilised in mammalian development and pregnancy.