Culture Of Embryonic Stem Cells Research Articles

Three-Dimensional Printing of Hydrogel Scaffolds with Hierarchical Structure for Scalable Stem Cell Culture.

The expansion and harvest of stem cells at clinically relevant scales is critical for cell-based therapies. These approaches need to be robust and cost-effective, support the functional maintenance of desired cell behaviors, and allow for simple harvest. Here, we introduce a real-time monitoring 3D printing approach to fabricate scaffolds with quadruple hierarchical structure that meet these design goals for stem cell expansion. Specifically, a versatile strategy was developed to produce scaffolds from alginate and gelatin with approximately 102 μm interconnected macropores, 300 μm microfilaments, 1.3 mm hollow channels, and centimeter-scale overall dimensions. The scaffolds exhibited good pattern fidelity and stable mechanical properties (compressive modulus value was 22-fold that of hydrogels from the same materials), facilitating uniform and efficient cell seeding with high viability (98.9%). The utility of the scaffold was shown with the 3D culture of HepaRG cells and embryonic stem cells (ESCs) with aggregated morphology, and significantly enhanced cell proliferation was observed compared to those of cultures on flat surfaces, obtaining approximately 2 × 108 cells within a single culture. Interestingly, the functional behavior of the cells was dependent on the cell type, as ESCs maintained their pluripotency, while HepaRG cells improved their hepatic differentiation. Cells were harmlessly harvested through chelating the calcium ions in the cross-linked alginate and de-cross-linking the scaffolds, indicating the potential of this study for scalable stem cell culture for numerous downstream applications.

Zebrafish embryonic explants undergo genetically encoded self-assembly.

Embryonic stem cell cultures are thought to self-organize into embryoid bodies, able to undergo symmetry-breaking, germ layer specification and even morphogenesis. Yet, it is unclear how to reconcile this remarkable self-organization capacity with classical experiments demonstrating key roles for extrinsic biases by maternal factors and/or extraembryonic tissues in embryogenesis. Here, we show that zebrafish embryonic tissue explants, prepared prior to germ layer induction and lacking extraembryonic tissues, can specify all germ layers and form a seemingly complete mesendoderm anlage. Importantly, explant organization requires polarized inheritance of maternal factors from dorsal-marginal regions of the blastoderm. Moreover, induction of endoderm and head-mesoderm, which require peak Nodal-signaling levels, is highly variable in explants, reminiscent of embryos with reduced Nodal signals from the extraembryonic tissues. Together, these data suggest that zebrafish explants do not undergo bona fide self-organization, but rather display features of genetically encoded self-assembly, where intrinsic genetic programs control the emergence of order.

Small Molecules that Promote Self-Renewal of Stem Cells and Somatic Cell Reprogramming.

The ground state of embryonic stem cells (ESCs) is closely related to the development of regenerative medicine. Particularly, long-term culture of ESCs in vitro, maintenance of their undifferentiated state, self-renewal and multi-directional differentiation ability is the premise of ESCs mechanism and application research. Induced pluripotent stem cells (iPSC) reprogrammed from mouse embryonic fibroblasts (MEF) cells into cells with most of the ESC characteristics show promise towards solving ethical problems currently facing stem cell research. However, integration into chromosomal DNA through viral-mediated genes may activate proto oncogenes and lead to risk of cancer of iPSC. At the same time, iPS induction efficiency needs to be further improved to reduce the use of transcription factors. In this review, we discuss small molecules that promote self-renewal and reprogramming, including growth factor receptorinhibitors, GSK-3β and histone deacetylase inhibitors, metabolic regulators, pathway modulators as well as EMT/MET regulation inhibitors to enhance maintenance of ESCs and enable reprogramming. Additionally, we summarize the mechanism of action of small molecules on ESC self-renewal and iPSC reprogramming. Finally, we will report on the progress in identification of novel and potentially effective agents as well as selected strategies that show promise in regenerative medicine. On this basis, development of more small molecule combinations and efficient induction of chemically induced pluripotent stem cell (CiPSC) is vital for stem cell therapy. This will significantly improve research in pathogenesis, individualized drug screening, stem cell transplantation, tissue engineering and many other aspects.

Advances in Isolation and Culture of Chicken Embryonic Stem Cells In Vitro.

Chicken embryonic stem cells (cESCs) isolated from the egg at the stage X hold great promise for cell therapy, tissue engineering, pharmaceutical, and biotechnological applications. They are considered to be pluripotent cells with the capacity to self-renewal and differentiate into specialized cells. However, long-term maintenance of cESCs cannot be realized now, which impedes the establishment of cESC line and limits their applications. Therefore, the separation locations, isolation methods, and culture conditions especially the supplements and action mechanisms of cytokines, including leukemia inhibitory factor, fibroblast growth factor, transforming growth factor beta, bone morphogenic protein, and activin for cESCs in vitro, have been reviewed here. These defined strategies will contribute to identify the key mechanism on the self-renewal of cESCs, facilitate to optimize system that supports the derivation and longtime maintenance of cESCs, establish the cESC line, and develop the biobank of genetic resources in chicken.

A Protocol for Transcriptome-Wide Inference of RNA Metabolic Rates in Mouse Embryonic Stem Cells.

The relative ease of mouse Embryonic Stem Cells (mESCs) culture and the potential of these cells to differentiate into any of the three primary germ layers: ectoderm, endoderm and mesoderm (pluripotency), makes them an ideal and frequently used ex vivo system to dissect how gene expression changes impact cell state and differentiation. These efforts are further supported by the large number of constitutive and inducible mESC mutants established with the aim of assessing the contributions of different pathways and genes to cell homeostasis and gene regulation. Gene product abundance is controlled by the modulation of the rates of RNA synthesis, processing, and degradation. The ability to determine the relative contribution of these different RNA metabolic rates to gene expression control using standard RNA-sequencing approaches, which only capture steady state abundance of transcripts, is limited. In contrast, metabolic labeling of RNA with 4-thiouridine (4sU) coupled with RNA-sequencing, allows simultaneous and reproducible inference of transcriptome wide synthesis, processing, and degradation rates. Here we describe, a detailed protocol for 4sU metabolic labeling in mESCs that requires short 4sU labeling times at low concentration and minimally impacts cellular homeostasis. This approach presents a versatile method for in-depth characterization of the gene regulatory strategies governing gene steady state abundance in mESC.

Evaluating the cytotoxicity of graphene oxide using embryonic stem cells-derived cells.

Graphene oxide (GO) has several potential biomedical applications and therefore cytotoxic evaluation of GO is very important. However, the two most common in vitro models for testing cytotoxicity-primary human cells and immortalized cell lines-suffer serious limitations, namely limited supplies of cells and unrealistic cellular responses, respectively. Here, we demonstrate the use of embryonic stem cell (ESC)-derived cells to study GO cytotoxicity. We tested the use of retinal pigment epithelium (RPE) cells derived from three-dimensional human ESC cultures ("ESC-RPE" cells) as a model of GO cytotoxicity by exposing them to varying concentrations of GO nanosheets. For comparison, we also performed the same test with primary human retinal pigment epithelium cells ("hRPE"), and with cells derived from a human RPE cell line ("ARPE19" cells). We found that cytotoxicity metrics (viability, apoptosis, intracellular reactive oxygen species, and mitochondrial membrane potential) were very similar in ESC-RPE cells and hRPE cells, and those in ARPE19 cells were very different. We conclude that cell models of GO cytotoxicity derived from ESCs are an excellent alternative to primary human cells, without the limitations of tissue availability.

Activin A and BMP4 Signaling Expands Potency of Mouse Embryonic Stem Cells in Serum-Free Media.

SummaryInhibitors of Mek1/2 and Gsk3β, known as 2i, and, together with leukemia inhibitory factor, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency (2i/L-ESCs). However, recent reports show that prolonged Mek1/2 suppression impairs developmental potential of ESCs, and is rescued by serum (S/L-ESCs). Here, we show that culturing ESCs in Activin A and BMP4, and in the absence of MEK1/2 inhibitor (ABC/L medium), establishes advanced stem cells derived from ESCs (esASCs). We demonstrate that esASCs contributed to germline lineages, full-term chimeras and generated esASC-derived mice by tetraploid complementation. We show that, in contrast to 2i/L-ESCs, esASCs display distinct molecular signatures and a stable hypermethylated epigenome, which is reversible and similar to serum-cultured ESCs. Importantly, we also derived novel ASCs (blASCs) from blastocysts in ABC/L medium. Our results provide insights into the derivation of novel ESCs with DNA hypermethylation from blastocysts in chemically defined medium.

Microfluidic platform for 3D cell culture with live imaging and clone retrieval.

Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge. Combining imaging with precise cell retrieval is of particular interest when studying highly dynamic or transient, asynchronous, or heterogeneous cell biological and developmental processes. Here, we present a method to encapsulate live cells in a 3D hydrogel matrix, via hydrogel bead compartmentalisation. Using a small-scale screen, we optimised matrix conditions for the culture and multilineage differentiation of mouse embryonic stem cells. Moreover, we designed a custom microfluidic platform that is compatible with live imaging. With this platform we can long-term culture and subsequently extract individual cells-in-beads by media flow only, obviating the need for enzymatic cell removal from the platform. Specific beads may be extracted from the platform in isolation, without disrupting the adjacent beads. We show that we can differentiate mouse embryonic stem cells, monitor reporter expression by live imaging, and retrieve individual beads for functional assays, correlating reporter expression with functional response. Overall, we present a highly flexible 3D cell encapsulation and microfluidic platform that enables both monitoring of cellular dynamics and retrieval for molecular and functional assays.

Effects of vitamin A and retinoic acid on mouse embryonic stem cells and their differentiating progeny.

Embryonic development is controlled by retinoids, and one approach that has been used to investigate the mechanisms for retinoid actions in developmental processes has been to study the effects of adding retinoids to cultures of pluripotent embryonic stem cells (ESC). To date, most in vitro retinoid research has been directed at deciphering the actions of all-trans retinoic acid (atRA). atRA is a derivative of all-trans retinol (a.k.a. vitamin A, VA), which mammals can generate via an enzyme-catalyzed pathway. atRA's effects on development result from its (1) activation of receptor complexes (RARs and RXRs) in the nucleus which then bind to and activate RA response elements (RAREs) in genes and (2) its interactions with processes that are initiated in the cytoplasm. While much work has focused on the impact of atRA on cell differentiation, VA, itself, has been shown to exert effects on the maintenance of ESC identity that are not dependent upon classic RA-signaling pathways. In this chapter, we present results from our laboratory and others using well-documented approaches for investigating the effects of retinoids on the differentiation of ESC in vitro and introduce a novel method that uses chemically-defined growth conditions. The merits of each approach are discussed.

Derivation of LIF-Independent Embryonic Stem Cells Using Inducible OCT4 Expression.

Pluripotent mouse embryonic stem (ES) cells, which are derived from the inner cell mass (ICM) of preimplantation stage embryos, are capable of self-renewing indefinitely in the presence of the external signal leukemia inhibitory factor (LIF), activation of Wnt signaling through inhibition of GSK3, and inhibition of MAP kinase/ERK kinase signaling. The OCT4 transcription factor is expressed highly in pluripotent cells and is a central transcriptional regulator of the pluripotent state. Here, we describe a protocol to culture ES cells in LIF-independent and serum-free media using an inducible OCT4 (iOCT4) ES cell model system. This protocol is sufficient to sustain ES cell self-renewal in vitro in defined conditions in the absence of external signals. LIF-independent iOCT4 ES cells are fully capable of differentiating following deactivation of the inducible OCT4 transgene.

56 Mammalian pre-implantation embryos at the single cell level: The bovine as a model for early human embryonic development

Single-cell transcriptomics and gene editing on human pre-implantation embryos have proved that mechanisms previously identified and well characterised in mouse pre-implantation development may not hold true in the human embryo. However, ethical and legal concerns limit the availability of surplus human embryos for research, resulting in the need for novel animal models. Bovine embryos share morphological and temporal resemblance with human early development; still, key molecular and cellular developmental mechanisms need to be further explored. In the present study, we performed a comparative single-cell RNA sequencing analysis across multiple pre-implantational developmental stages of invitro-derived bovine, human (Petropoulos et al. 2016 Cell 165, 1012-1026; https://doi.org/10.1016/j.cell.2016.03.023), and mouse embryos (Cheng et al. 2019 Cell Rep. 26, 2593-2607, https://doi.org/10.1016/j.celrep.2019.02.031; Posfai et al. 2017 eLife 6, e22906, https://doi.org/10.7554/eLife.22906; Chen et al. 2016 Genome Res. 26, 1342-1354). In total 497 blastomeres corresponding to embryonic days (E) E4-E8 were prepared using Smart sEqn 2, Illumina HiSEqn 2500 sequencing with 50-bp single-end reads. Embryonic stage was defined by cell numbers and morphology as multicellular stage (Mu, ~E4), morulae (M; E5), early blastocysts (EB, ~6), mid-expanded blastocysts, (MB, ~E7), and late expanded/hatched blastocysts (LB, ~E8). We found top differentially expressed genes elucidating how lineage specification and pluripotency is controlled in the early bovine embryo. Our transcriptomic data showed that the first lineage segregation in the pre-implantation bovine embryo occurs after cavitation at E6 in the early blastocyst, suggesting a similarity with the early human blastocyst. In addition, E7 showed distinctive and more mature inner cell mass (ICM) and trophectoderm (TE) profiles. Different from the mouse, where the first lineage segregation of TE-ICM is suggested to occur at the morula stage due to CDX2-OCT4 antagonism, we found that the expression of OCT4 protein cattle expanded blastocysts is not restricted to the ICM, similar to what has been reported to occur in the human embryo. Interestingly, we also found that the second lineage segregation, which segregates the epiblast from the primitive endoderm within the ICM, starts in bovine at E8 expanded hatched late blastocysts, suggesting a potential difference with humans, where TE-ICM and epiblast-primitive endoderm has previously been reported to occur almost concurrently. We are now complementing our findings with genome editing in bovine embryos by generating knockout embryos of different target genes part of an evolutionarily conserved signaling pathway to study their effects in the early pre-implantation embryo. We aim to elucidate how early lineage specification and pluripotency establishment occurs while offering theoretical support for efficient derivation and culture of bovine embryonic stem cells.

GENERATION OF MOSAIC STEM CELL DERIVED INNER EAR ORGANOIDS TO DETERMINE PARACRINE EFFECTS OF TMPRSS3

Background and Hypothesis: The carefully timed treatment of mouse embryonic stem cell (mESC) cultures with small molecules results in mESC differentiation into 3D organoids containing all components of the inner ear such as hair cells (HCs), support cells and neurons. Loss of TMPRSS3 function, which is a transmembrane extracellular protease, has been previously shown to lead to rapid HC degeneration between culture days 36 (D36) and D38 in 3D organoids. Mosaic organoids would allow for analysis of developmental dynamics, cell-cell interactions and even therapeutic rescue efficiency. We hypothesized that we could develop an inner ear mosaic organoid containing cells with and without TMPRSS3 and that the Tmprsss3KO mosaic organoids would have greater hair cell survival than Tmprss3KO-only organoids due to the compensatory effect of intact TMPRSS3 on control hair cells within the same vesicle.
 Experimental Design or Project Methods: Two mESC cell lines (R1E background) were previously generated using CRISPR-Cas9n. Control reporter line (tdTomato) expresses a tdTomato reporter gene in all cells under the CAG promoter at the ROSA26 locus. Tmprss3 knockout line (Tmprss3KO) contains a 2A-nGFP cassette with a premature stop codon into exon 2 of the Tmprss3 gene. We generated Tmprss3KO mosaic (Tmprss3KO:tdTomato) and control mosaic (R1E:tdTomato) organoids. The aggregates were analyzed on days 25, 33 and 38 after being fixed, sectioned and stained for tdTomato, SOX2, MYO7A, cleaved CASPASE3, and BK channels using immunohistochemistry.
 Results: We have successfully used mESCs to generate Tmprss3KO mosaic inner ear organoids with similar efficiency to that of control mosaic organoids. Preliminary data suggests that the hair cell survival rates were similar across all vesicle types in the Tmprss3KO mosaic organoids. Additionally, the Tmprss3KO mosaic organoids had a significantly decreased overall BK channel expression by D38. 
 Conclusion and Potential Impact: The successful generation of mosaic organoids achieved here under both conditions sets the stage for future studies of intercellular interactions and therapeutics in this domain. More replicates are necessary to make a definitive conclusion about the effectiveness of control cells on Tmprss3KO rescue.

Targeted Mutations in the Mouse via Embryonic Stem Cells.

Genetic modification of mouse embryonic stem (ES) cells is a powerful technology that enabled the generation of a plethora of mutant mouse lines to study gene function and mammalian biology. Here we describe ES cell culture and transfection techniques used to manipulate the ES cell genome to obtain targeted ES cell clones. We include the standard gene targeting approach as well as the application of the CRISPR/Cas9 system that can improve the efficiency of homologous recombination in ES cells by introducing a double-strand DNA break at the target site.

The ZT Biopolymer: A Self-Assembling Protein Scaffold for Stem Cell Applications.

The development of cell culture systems for the naturalistic propagation, self-renewal and differentiation of cells ex vivo is a high goal of molecular engineering. Despite significant success in recent years, the high cost of up-scaling cultures, the need for xeno-free culture conditions, and the degree of mimicry of the natural extracellular matrix attainable in vitro using designer substrates continue to pose obstacles to the translation of cell-based technologies. In this regard, the ZT biopolymer is a protein-based, stable, scalable, and economical cell substrate of high promise. ZT is based on the naturally occurring assembly of two human proteins: titin-Z1Z2 and telethonin. These protein building blocks are robust scaffolds that can be conveniently functionalized with full-length proteins and bioactive peptidic motifs by genetic manipulation, prior to self-assembly. The polymer is, thereby, fully encodable. Functionalized versions of the ZT polymer have been shown to successfully sustain the long-term culturing of human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), and murine mesenchymal stromal cells (mMSCs). Pluripotency of hESCs and hiPSCs was retained for the longest period assayed (4 months). Results point to the large potential of the ZT system for the creation of a modular, pluri-functional biomaterial for cell-based applications.

An automated microfluidic device for time-lapse imaging of mouse embryonic stem cells.

Long-term, time-lapse imaging studies of embryonic stem cells (ESCs) require a controlled and stable culturing environment for high-resolution imaging. Microfluidics is well-suited for such studies, especially when the media composition needs to be rapidly and accurately altered without disrupting the imaging. Current studies in plates, which can only add molecules at the start of an experiment without any information on the levels of endogenous signaling before the exposure, are incompatible with continuous high-resolution imaging and cell-tracking. Here, we present a custom designed, fully automated microfluidic chip to overcome these challenges. A unique feature of our chip includes three-dimensional ports that can connect completely sealed on-chip valves for fluid control to individually addressable cell culture chambers with thin glass bottoms for high-resolution imaging. We developed a robust protocol for on-chip culturing of mouse ESCs for minimum of 3 days, to carry out experiments reliably and repeatedly. The on-chip ESC growth rate was similar to that on standard culture plates with same initial cell density. We tested the chips for high-resolution, time-lapse imaging of a sensitive reporter of ESC lineage priming, Nanog-GFP, and HHex-Venus with an H2B-mCherry nuclear marker for cell-tracking. Two color imaging of cells was possible over a 24-hr period while maintaining cell viability. Importantly, changing the media did not affect our ability to track individual cells. This system now enables long-term fluorescence imaging studies in a reliable and automated manner in a fully controlled microenvironment.

Zinc Maintains Embryonic Stem Cell Pluripotency and Multilineage Differentiation Potential via AKT Activation.

Embryonic stem cells (ESCs) possess remarkable abilities, as they can differentiate into all cell types (pluripotency) and be self-renewing, giving rise to two identical cells. These characteristics make ESCs a powerful research tool in fundamental embryogenesis as well as candidates for use in regenerative medicine. Significant efforts have been devoted to developing protocols to control ESC fate, including soluble and complex cocktails of growth factors and small molecules seeking to activate/inhibit key signaling pathways for the maintenance of pluripotency states or activate differentiation. Here we describe a novel method for the effective maintenance of mouse ESCs, avoiding the supplementation of complex inhibitory cocktails or cytokines, e.g., LIF. We show that the addition of zinc to ESC cultures leads to a stable pluripotent state that shares biochemical, transcriptional and karyotypic features with the classical LIF treatment. We demonstrate for the first time that ESCs maintained in long-term cultures with added zinc, are capable of sustaining a stable ESCs pluripotent phenotype, as well as differentiating efficiently upon external stimulation. We show that zinc promotes long-term ESC self-renewal (>30 days) via activation of ZIP7 and AKT signaling pathways. Furthermore, the combination of zinc with LIF results in a synergistic effect that enhances LIF effects, increases AKT and STAT3 activity, promotes the expression of pluripotency regulators and avoids the expression of differentiation markers.

Temporal and partial inhibition of GLI1 in neural stem cells (NSCs) results in the early maturation of NSC derived oligodendrocytes in vitro

BackgroundOligodendrocytes are a type of glial cells that synthesize the myelin sheath around the axons and are critical for the nerve conduction in the CNS. Oligodendrocyte death and defects are the leading causes of several myelin disorders such as multiple sclerosis, progressive multifocal leukoencephalopathy, periventricular leukomalacia, and several leukodystrophies. Temporal activation of the Sonic Hedgehog (SHH) pathway is critical for the generation of oligodendrocyte progenitors, and their differentiation and maturation in the brain and spinal cord during embryonic development in mammals.MethodsOur protocol utilized adherent cultures of human induced pluripotent stem cells (iPSC) and human embryonic stem cells (hESCs) with a green fluorescent protein (GFP) reporter knocked into one allele of the OLIG2 gene locus, dual SMAD inhibition, and transient partial inhibition of glioma-associated oncogene 1 (GLI1) by the small molecule GANT61 during the formation of the SOX2/PAX6-positive neural stem cells (NSCs). The SHH pathway was later restimulated by a Smoothened agonist purmorphamine to induce the generation of OLIG2 glial precursors. One hundred ninety-two individual oligodendrocyte precursor cells (OPCs) from GANT61 and control group were analyzed by single-cell RNA sequencing (RNA-Seq).ResultsWe demonstrate here that transient and partial inhibition of the SHH pathway transcription factor GLI1 in NSCs by a small molecule inhibitor GANT61 was found to generate OPCs that were more migratory and could differentiate earlier toward myelin-producing oligodendrocytes. Single-cell transcriptomic analysis (RNA-Seq) showed that GANT61-NSC-derived oligodendrocyte precursor cells (OPCs) had differential activation of some of the genes in the cytoskeleton rearrangement pathways that are involved in OPC motility and induction of maturation. At the protein level, this was also associated with higher levels of myelin-specific genes in the GANT61 group compared to controls. GANT61-NSC-derived OPCs were functional and could generate compact myelin in vitro and in vivo after transplantation in myelin-deficient shiverer mice.ConclusionsThis is a small molecule-based in vitro protocol that leads to the faster generation of functional oligodendrocytes. The development of protocols that lead to efficient and faster differentiation of oligodendrocytes from progenitors provides important advances toward the development of autologous neural stem cell-based therapies using human iPSCs.

Defining Human Pluripotency.

Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.

Glutamine independence is a selectable feature of pluripotent stem cells.

Most rapidly proliferating mammalian cells rely on the oxidation of exogenous glutamine to support cell proliferation. We previously found that culture of mouse embryonic stem cells (ESCs) in the presence of inhibitors against MEK and GSK3β to maintain pluripotency reduces cellular reliance on glutamine for tricarboxylic acid (TCA) cycle anaplerosis, enabling ESCs to proliferate in the absence of exogenous glutamine. Here we show that reduced dependence on exogenous glutamine is a generalizable feature of pluripotent stem cells. Enhancing self-renewal, through either overexpression of pluripotency-associated transcription factors or altered signal transduction, decreases the utilization of glutamine-derived carbons in the TCA cycle. As a result, cells with the highest potential for self-renewal can be enriched by transient culture in glutamine-deficient media. During pluripotent cell culture or reprogramming to pluripotency, transient glutamine withdrawal selectively leads to the elimination of non-pluripotent cells. These data reveal that reduced dependence on glutamine anaplerosis is an inherent feature of self-renewing pluripotent stem cells and reveal a simple, non-invasive mechanism to select for mouse and human pluripotent stem cells within a heterogeneous population during both ESC passage and induced pluripotent cell reprogramming.

DPPA2/4 and SUMO E3 ligase PIAS4 opposingly regulate zygotic transcriptional program.

The molecular mechanism controlling the zygotic genome activation (ZGA) in mammals remains poorly understood. The 2-cell (2C)-like cells spontaneously emerging from cultures of mouse embryonic stem cells (ESCs) share some key transcriptional and epigenetic programs with 2C-stage embryos. By studying the transition of ESCs into 2C-like cells, we identified developmental pluripotency associated 2 and 4 (Dppa2/4) as important regulators controlling zygotic transcriptional program through directly up-regulating the expression of double homeobox (Dux). In addition, we found that DPPA2 protein is sumoylated and its activity is negatively regulated by small ubiquitin-like modifier (Sumo) E3 ligase protein inhibitor of activated STAT 4 (PIAS4). PIAS4 is down-regulated during ZGA process and during transitioning of ESCs into 2C-like cells. Depleting Pias4 or overexpressing Dppa2/4 is sufficient to activate 2C-like transcriptional program, whereas depleting Dppa2/4 or forced expression of Pias4 or Sumo2–Dppa2 inhibits 2C-like transcriptional program. Furthermore, ectopic expression of Pias4 or Sumo2–Dppa2 impairs early mouse embryo development. In summary, our study identifies key molecular rivals consisting of transcription factors and a Sumo2 E3 ligase that regulate zygotic transcriptional program upstream of Dux.