Inheritance of H3K9 methylation regulates genome architecture in Drosophila early embryos.

Constitutive heterochromatin is essential for transcriptional silencing and genome integrity. The establishment of constitutive heterochromatin in early embryos and its role in early fruitfly development are unknown. Lysine 9 trimethylation of histone H3 (H3K9me3) and recruitment of its epigenetic reader, heterochromatin protein 1a (HP1a), are hallmarks of constitutive heterochromatin. Here, we show that H3K9me3 is transmitted from the maternal germline to the next generation. Maternally inherited H3K9me3, and the histone methyltransferases (HMT) depositing it, are required for the organization of constitutive heterochromatin: early embryos lacking H3K9 methylation display de-condensation of pericentromeric regions, centromere-centromere de-clustering, mitotic defects, and nuclear shape irregularities, resulting in embryo lethality. Unexpectedly, quantitative CUT&Tag and 4D microscopy measurements of HP1a coupled with biophysical modeling revealed that H3K9me2/3 is largely dispensable for HP1a recruitment. Instead, the main function of H3K9me2/3 at this developmental stage is to drive HP1a clustering and subsequent heterochromatin compaction. Our results show that HP1a binding to constitutive heterochromatin in the absence of H3K9me2/3 is not sufficient to promote proper embryo development and heterochromatin formation. The loss of H3K9 HMTs and H3K9 methylation alters genome organization and hinders embryonic development.

Lamina-Dependent Stretching and Unconventional Chromosome Compartments in Early C. elegans Embryos.

Current models suggest that chromosome domains segregate into either an active (A) or inactive (B) compartment. B-compartment chromatin is physically separated from the A compartment and compacted by the nuclear lamina. To examine these models in the developmental context of C. elegans embryogenesis, we undertook chromosome tracing to map the trajectories of entire autosomes. Early embryonic chromosomes organized into an unconventional barbell-like configuration, with two densely folded B compartments separated by a central A compartment. Upon gastrulation, this conformation matured into conventional A/B compartments. We used unsupervised clustering to uncover subpopulations with differing folding properties and variable positioning of compartment boundaries. These conformations relied on tethering to the lamina to stretch the chromosome; detachment from the lamina compacted, and allowed intermingling between, A/B compartments. These findings reveal the diverse conformations of early embryonic chromosomes and uncover a previously unappreciated role for the lamina in systemic chromosome stretching.

Unlocking the potential of SY-stem cells.

The sixth SY-Stem Symposium, jointly organized by the Research Institute of Molecular Pathology and the Institute of Molecular Biotechnology took place in Vienna in March 2024. Again, aspiring new group leaders were given a stage to present their work and vision of their labs. To round up the excellent program, the scientific organizers included renowned keynote speakers. Here, we provide a summary of the talks covering topics such as early embryogenesis, nervous system development and disease, regeneration and the latest technologies.

RNA 5-Methylcytosine Facilitates the Maternal-to-Zygotic Transition by Preventing Maternal mRNA Decay.

The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the maternal environment is converted to an environment of embryonic-driven development through dramatic reprogramming. However, how maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) modification in zebrafish early embryos, we found that m5C-modified maternal mRNAs display higher stability than non-m5C-modified mRNAs during MZT. We discovered that Y-box binding protein 1 (Ybx1) preferentially recognizes m5C-modified mRNAs through π-π interactions with a key residue, Trp45, in Ybx1's cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Together with the mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates an unexpected mechanism of RNA m5C-regulated maternal mRNA stabilization during zebrafish MZT, highlighting the critical role of m5C mRNA modification in early development.

The dynamic proteome in Arabidopsis thaliana early embryogenesis.

The morphology of the flowering plant is established during early embryogenesis. In recent years, many studies have focused on transcriptional profiling in plant embryogenesis, but the dynamic landscape of the Arabidopsis thaliana proteome remains elusive. In this study, Arabidopsis embryos at 2/4-cell, 8-cell, 16-cell, 32-cell, globular and heart stages were collected for nanoproteomic analysis. In total, 5386 proteins were identified. Of these, 1051 proteins were universally identified in all developmental stages and a range of 27 to 2154 proteins was found to be stage specific. These proteins could be grouped into eight clusters according to their expression levels. Gene Ontology enrichment analysis showed that genes involved in ribosome biogenesis and auxin-activated signalling were enriched during early embryogenesis, indicating that active translation and auxin signalling are important events in Arabidopsis embryo development. Combining RNA-sequencing data with the proteomics analysis, the correlation between mRNA and protein was evaluated. An overall positive correlation was found between mRNA and protein. This work provides a comprehensive landscape of the Arabidopsis proteome in early embryogenesis. Some important proteins/transcription factors identified through network analysis may serve as potential targets for future investigation.

Modeling X chromosome inactivation using t5iLA naive human pluripotent stem cells.

BACKGROUND: X chromosome inactivation (XCI) is a critical epigenetic event for dosage compensation of X-linked genes in female mammals, ensuring developmental stability. A robust in vitro model is required for mimicking XCI during the early stages of embryonic development. This methodology article introduces an advanced framework for the in-depth study of XCI using human pluripotent stem cells (hPSCs). By focusing on the transition between naive and primed pluripotent states, we highlight the role of long non-coding RNA X-inactive specific transcript (XIST) and epigenetic alterations in mediating XCI. RESULTS: Our methodology enables the distinction between naive and primed hESCs based on XIST expression and the activity of X-linked reporters, facilitating the investigation of XCI initiation and maintenance. Through detailed experimental procedures, we demonstrate the utility of our hESC lines in modeling the process of human XCI, including the establishment of conditions for random XCI induction and the analysis of X chromosome reactivation. METHODS: The study outlines a comprehensive approach for characterizing the X chromosome status in hPSCs, employing dual fluorescent reporter hESC lines. These reporter lines enable real-time tracking of XCI dynamics through differentiation processes. We detailed protocols for the induction of X chromosome reactivation and inactivation, as well as the X status characterization methods including cultivation of hESCs, flow cytometric analysis, RNA fluorescence in situ hybridization (FISH), and transcriptome sequencing, providing a step-by-step guide for researchers to investigate XCI mechanisms in vitro. CONCLUSIONS: This article provides a detailed, reproducible methodology for studying XCI mechanisms in vitro, employing hPSCs as a model system. It presents a significant advance in our ability to investigate XCI, offering potential applications in developmental biology, disease modeling, and regenerative medicine. By facilitating the study of XCI dynamics, this methodological framework paves the way for deeper understanding and manipulation of this fundamental biological process.

Locational and functional characterization of PI4KB in the mouse embryo.

Phosphatidylinositol 4-kinase beta (PI4KB) is a member of the PI4K family, which is mainly enriched and functions in the Golgi apparatus. The kinase domain of PI4KB catalyzes the phosphorylation of phosphatidylinositol to form phosphatidylinositol 4-phosphate, a process that regulates various sub-cellular events, such as non-vesicular cholesterol and ceramide transport, protein glycosylation, and vesicle transport, as well as cytoplasmic division. In this study, a strain of PI4KB knockout mouse, immunofluorescence, reverse transcription polymerase chain reaction and microinjection were used to characterize the cytological location and biological function of PI4KB in the mouse embryos. we found that knocking down Pi4kb in mouse embryos resulted in embryonic lethality at around embryonic day (E) 7.5. Additionally, we observed dramatic fluctuations in PI4KB expression during the development of preimplantation embryos, with high expression in the 4-cell and morula stages. PI4KB colocalized with the Golgi marker protein TGN46 in the perinuclear and cytoplasmic regions in early blastomeres. Postimplantation, PI4KB was highly expressed in the epiblast of E7.5 embryos. Treatment of embryos with PI4KB inhibitors was found to inhibit the development of the morula into a blastocyst and the normal progression of cytoplasmic division during the formation of a 4-cell embryo. These findings suggest that PI4KB plays an important role in mouse embryogenesis by regulating various intracellular vital functions of embryonic cells.

SAC during early cell divisions: Sacrificing fidelity over timely division, regulated differently across organisms: Chromosome alignment and segregation are left unsupervised from the onset of development until checkpoint activity is acquired, varying from species to species.

Early embryogenesis is marked by a frail Spindle Assembly Checkpoint (SAC). The time of SAC acquisition varies depending on the species, cell size or a yet to be uncovered developmental timer. This means that for a specific number of divisions, biorientation of sister chromatids occurs unsupervised. When error-prone segregation is an issue, an aneuploidy-selective apoptosis system can come into play to eliminate chromosomally unbalanced cells resulting in healthy newborns. However, aneuploidy content can be too great to overcome, endangering viability. SAC generates a diffusible signal to lengthen time spent in mitosis if needed, ensuring correct chromosome segregation, a fundamental factor in the generation of euploid cells. Thus, it remains puzzling what benefit could come from delaying SAC acquisition till later in the development. In this review, we describe what is known on SAC acquisition in distinct species and highlight pending research as well as potential applications for such knowledge.

Revisiting poly(A)-binding proteins: Multifaceted regulators during gametogenesis and early embryogenesis.

Post-transcriptional regulation faces a distinctive challenge in gametes. Transcription is limited when the germ cells enter the division phase due to condensed chromatin, while gene expression during gamete maturation, fertilization, and early cleavage depends on existing mRNA post-transcriptional coordination. The dynamics of the 3'-poly(A) tail play crucial roles in defining mRNA fate. The 3'-poly(A) tail is covered with poly(A)-binding proteins (PABPs) that help to mediate mRNA metabolism and recent work has shed light on the number and function of germ cell-specific expressed PABPs. There are two structurally different PABP groups distinguished by their cytoplasmic and nuclear localization. Both lack catalytic activity but are coupled with various roles through their interaction with multifunctional partners during mRNA metabolism. Here, we present a synopsis of PABP function during gametogenesis and early embryogenesis and describe both conventional and current models of the functions and regulation of PABPs, with an emphasis on the physiological significance of how germ cell-specific PABPs potentially affect human fertility.

Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider.

BACKGROUND: The process of early development varies across the species-rich phylum Arthropoda. Owing to the limited research strategies for dissecting lineage-specific processes of development in arthropods, little is known about the variations in early arthropod development at molecular resolution. The Theridiidae spider, Parasteatoda tepidariorum, has its genome sequenced and could potentially contribute to dissecting early embryonic processes. RESULTS: We present genome-wide identification of candidate genes that exhibit locally restricted expression in germ disc forming stage embryos of P. tepidariorum, based on comparative transcriptomes of isolated cells from different regions of the embryo. A subsequent pilot screen by parental RNA interference identifies three genes required for body axis formation. One of them is a GATA-like gene that has been fast evolving after duplication and divergence from a canonical GATA family gene. This gene is designated fuchi nashi (fuchi) after its knockdown phenotypes, where the cell movement toward the formation of a germ disc was reversed. fuchi expression occurs in cells outside a forming germ disc and persists in the endoderm. Transcriptome and chromatin accessibility analyses of fuchi pRNAi embryos suggest that early fuchi activity regulates chromatin state and zygotic gene activation to promote endoderm specification and pattern formation. We also show that there are many uncharacterized genes regulated by fuchi. CONCLUSIONS: Our genome-based research using an arthropod phylogenetically distant from Drosophila identifies a lineage-specific, fast-evolving gene with key developmental roles in one of the earliest, genome-wide regulatory events, and allows for molecular exploration of the developmental variations in early arthropod embryos.

Impact of spraying eggs with betaine after exposure to short-term thermal stress during early embryogenesis on pre and post-hatch performance of Japanese quail.

It is essential to understand and manage environmental factors for good quail production and welfare. One of the most important environmental stressors that hinder quail productivity is heat stress. This study aimed to evaluate the impact of spraying Japanese quail (Coturnix coturnix japonica) eggs with betaine after exposure to short-term high temperature during early embryogenesis on pre and post-hatch performance of quail. A total of 750 eggs were equally divided into two groups. Eggs in the first group were incubated at normal incubation temperature (37.5 °C/NIT), while those in the second group were incubated at high incubation temperature (39.0 °C/HIT) for 3 h daily from day 4-6 of incubation. Eggs in both groups were subjected to five treatments, NC (negative control), PC sprayed distilled water (positive control), while B0.5, B1, and B2 treatments were sprayed with distilled water supplemented with 500, 1000, and 2000 mg betaine/L, respectively. The chick weight at hatch, slaughter weight, and first egg weight was significantly impaired by the HIT treatment. The HIT group revealed a significant increase in cloacal temperature, H/L ratio, liver enzymes, triglyceride, and cholesterol and a significant decrease in hatchability, T3 hormone, and blood protein levels than the NIT group. Regarding betaine effects, the embryonic mortality rates, hatchability, hatched chick weight, and oviduct percentage in groups treated with 1000 or 2000 mg betaine/L were significantly improved compared with the control. Also, spraying betaine at 1000 or 2000 mg/L significantly increased blood protein and triiodothyronine (T3) hormone levels and significantly decrease liver enzyme levels and total feed consumption compared with the untreated group. The right/total ventricle ratio (RV/TV) of quail in HIT group was significantly increased, while betaine treatment significantly decreased this ratio. Considering these results, it is strongly suggested that spraying of betaine on eggs at 2000 mg/L optimizes Japanese quail performance.

Research progress on the study of transcriptome-wide poly(A) tails in mammalian oocytes and early embryos.

During mammalian oocyte-to-embryo transition, before zygotic genome activation, the transcription in oocytes and embryos is silenced, so the post-transcriptional regulation of mRNA plays an essential role in this process. Poly(A) tail is an important post-transcriptional modification that affects mRNA metabolism and translation efficiency. With the development of sequencing technology and analysis tools, especially the methods based on third-generation sequencing technology, the length and composition of poly(A) tails can be accurately measured, greatly expanding our understanding of poly(A) tails in mammalian early embryonic development. This review focuses on the achievements of poly(A) tail sequencing methods and the research progress of poly(A) tail in regulating oocyte-to-embryo transition, discussing the future applications for the investigation of mammalian early embryonic development and infertility related diseases.

Mitochondrial DNA Copy Number in Cleavage Stage Human Embryos-Impact on Infertility Outcome.

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between mitochondrial DNA (mtDNA) content of cleavage-stage preimplantation embryos and their developmental potential. A total of 69 couples underwent IVF treatment (averaged women age: 36.5, SD 4.9) and produced a total of 314 embryos. A single blastomere was biopsied from each embryo at the cleavage stage (day-3 post-fertilization) subjected to low-pass next generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number amount was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 6.3 ± 7.5 versus 7.1 ± 5.8, p < 0.004; U Mann−Whitney test), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (6.6 ± 4.8 vs. 8.5 ± 13.6, p 0.09), sex (6.6 ± 4.1 vs. 6.2 ± 6.8, p 0.16), maternal age (6.9 ± 7.8 vs. 6.7 ± 4.5, p 0.14) or its ability to implant (7.4 ± 6.6 vs. 5.1 ± 4.6, p 0.18). The mtDNA content cannot serve as a useful biomarker at this point in development. However, further studies investigating both quantitative and qualitative aspects of mtDNA are still required to fully evaluate the relationship between mitochondrial DNA and human reproduction.

In vitro investigation of mammalian early embryonic development.

Mammalian embryonic development starts from a fertilized egg, which cleaves to form morula and blastocyst. At the same time, the early embryo is transported from the fallopian tube to the uterus for implantation. After implantation, the embryo undergoes gastrulation and forms a gastrula, further developing a new individual. The development of embryo in the uterus causes the difficulties in sampling and observation, hindering the understanding of mammalian embryonic development. Therefore, it is necessary to develop the technology to overcome the barrier of in vivo embryonic development. In December 2021, "Embryo 'husbandry' opens windows into early development" was selected as one of Science's 2021 breakthroughs. This review focuses on the achievements of in vitro mammalian embryos and discusses their limitations and the future applications for the investigation of mammalian embryonic development and human related diseases.

The subcortical maternal complex: emerging roles and novel perspectives.

Since its recent discovery, the subcortical maternal complex (SCMC) is emerging as a maternally inherited and crucial biological structure for the initial stages of embryogenesis in mammals. Uniquely expressed in oocytes and preimplantation embryos, where it localizes to the cell subcortex, this multiprotein complex is essential for early embryo development in the mouse and is functionally conserved across mammalian species, including humans. The complex has been linked to key processes leading the transition from oocyte to embryo, including meiotic spindle formation and positioning, regulation of translation, organelle redistribution, and epigenetic reprogramming. Yet, the underlying molecular mechanisms for these diverse functions are just beginning to be understood, hindered by unresolved interplay of SCMC components and variations in early lethal phenotypes. Here we review recent advances confirming involvement of the SCMC in human infertility, revealing an unexpected relationship with offspring health. Moreover, SCMC organization is being further revealed in terms of novel components and interactions with additional cell constituents. Collectively, this evidence prompts new avenues of investigation into possible roles during the process of oogenesis and the regulation of maternal transcript turnover during the oocyte to embryo transition.

Formation of a multi-layered 3-dimensional structure of the heterochromatin compartment during early mammalian development.

During embryogenesis in mammals, the 3-dimensional (3D) genome organization changes globally in parallel with transcription changes in a cell-type specific manner. This involves the progressive formation of heterochromatin, the best example of which is the inactive X chromosome (Xi) in females, originally discovered as a compact 3D structure at the nuclear periphery known as the Barr body. The heterochromatin formation on the autosomes and the Xi is tightly associated with the differentiation state and the developmental potential of cells, making it an ideal readout of the cellular epigenetic state. At a glance, the heterochromatin appears to be uniform. However, recent studies are beginning to reveal a more complex picture, with multiple hierarchical levels co-existing within the heterochromatin compartment. Such hierarchical levels appear to exist in the heterochromatin compartment on autosomes as well as on the Xi. Here, we review recent progress in our understanding of the 3D genome organization changes during the period of differentiation surrounding pluripotency in vivo and in vitro, with a focus on the heterochromatin compartment. We first look at the whole genome, then focus on the Xi, and discuss their differences and similarities. Finally, we present a unified view of how the heterochromatin compartment is formed and regulated during early development. In particular, we emphasize that there are multiple layers within the heterochromatic compartment on both the autosomes and the Xi, with regulatory mechanisms common and specific to each layer.

Identification of Signaling Pathways for Early Embryonic Lethality and Developmental Retardation in Sephs1-/- Mice.

Selenophosphate synthetase 1 (SEPHS1) plays an essential role in cell growth and survival. However, the underlying molecular mechanisms remain unclear. In the present study, the pathways regulated by SEPHS1 during gastrulation were determined by bioinformatical analyses and experimental verification using systemic knockout mice targeting Sephs1. We found that the coagulation system and retinoic acid signaling were most highly affected by SEPHS1 deficiency throughout gastrulation. Gene expression patterns of altered embryo morphogenesis and inhibition of Wnt signaling were predicted with high probability at E6.5. These predictions were verified by structural abnormalities in the dermal layer of Sephs1-/- embryos. At E7.5, organogenesis and activation of prolactin signaling were predicted to be affected by Sephs1 knockout. Delay of head fold formation was observed in the Sephs1-/- embryos. At E8.5, gene expression associated with organ development and insulin-like growth hormone signaling that regulates organ growth during development was altered. Consistent with these observations, various morphological abnormalities of organs and axial rotation failure were observed. We also found that the gene sets related to redox homeostasis and apoptosis were gradually enriched in a time-dependent manner until E8.5. However, DNA damage and apoptosis markers were detected only when the Sephs1-/- embryos aged to E9.5. Our results suggest that SEPHS1 deficiency causes a gradual increase of oxidative stress which changes signaling pathways during gastrulation, and afterwards leads to apoptosis.

Tricarboxylic acid cycle activity suppresses acetylation of mitochondrial proteins during early embryonic development in Caenorhabditis elegans.

The tricarboxylic acid (TCA) cycle (or citric acid cycle) is responsible for the complete oxidation of acetyl-CoA and formation of intermediates required for ATP production and other anabolic pathways, such as amino acid synthesis. Here, we uncovered an additional mechanism that may help explain the essential role of the TCA cycle in the early embryogenesis of Caenorhabditis elegans. We found that knockdown of citrate synthase (cts-1), the initial and rate-limiting enzyme of the TCA cycle, results in early embryonic arrest, but that this phenotype is not because of ATP and amino acid depletions. As a possible alternative mechanism explaining this developmental deficiency, we observed that cts-1 RNAi embryos had elevated levels of intracellular acetyl-CoA, the starting metabolite of the TCA cycle. Of note, we further discovered that these embryos exhibit hyperacetylation of mitochondrial proteins. We found that supplementation with acetylase-inhibiting polyamines, including spermidine and putrescine, counteracted the protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Contrary to the hypothesis that spermidine acts as an acetyl sink for elevated acetyl-CoA, the levels of three forms of acetylspermidine, N1-acetylspermidine, N8-acetylspermidine, and N1,N8-diacetylspermidine, were not significantly increased in embryos treated with exogenous spermidine. Instead, we demonstrated that the mitochondrial deacetylase sirtuin 4 (encoded by the sir-2.2 gene) is required for spermidine's suppression of protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Taken together, these results suggest the possibility that during early embryogenesis, acetyl-CoA consumption by the TCA cycle in C. elegans prevents protein hyperacetylation and thereby protects mitochondrial function.

Oocyte-specific maternal Slbp2 is required for replication-dependent histone storage and early nuclear cleavage in zebrafish oogenesis and embryogenesis.

Stem-loop binding protein (SLBP) is required for replication-dependent histone mRNA metabolism in mammals. Zebrafish possesses two slbps, and slbp1 is necessary for retinal neurogenesis. However, the detailed expression and function of slbp2 in zebrafish are still unknown. In this study, we first identified zebrafish slbp2 as an oocyte-specific maternal factor and then generated a maternal-zygotic slbp2 F3 homozygous mutant (MZslbp2Δ4-/-) using CRISPR/Cas9. The depletion of maternal Slbp2 disrupted early nuclear cleavage, which resulted in developmental arrest at the MBT stage. The developmental defects could be rescued in slbp2 transgenic MZslbp2Δ4-/- embryos. However, homozygous mutant MZslbp1Δ1-/- developed normally, indicating slbp1 is dispensable for zebrafish early embryogenesis. Through comparative proteome and transcriptome profiling between WT and MZslbp2Δ4-/- embryos, we identified many differentially expressed proteins and genes. In comparison with those in WT embryos, four replication-dependent histones, including H2a, H2b, H3, and H4, all reduced their expression, while histone variant h2afx significantly increased in MZslbp2Δ4-/- embryos at the 256-cell stage and high stage. Zebrafish Slbp2 can bind histone mRNA stem-loop in vitro, and the defects of MZslbp2Δ4-/- embryos can be partially rescued by overexpression of H2b. The current data indicate that maternal Slbp2 plays a pivotal role in the storage of replication-dependent histone mRNAs and proteins during zebrafish oogenesis.

Transition of inner cell mass to embryonic stem cells: mechanisms, facts, and hypotheses.

Embryonic stem cells (ESCs) are immortal stem cells that own multi-lineage differentiation potential. ESCs are commonly derived from the inner cell mass (ICM) of pre-implantation embryos. Due to their tremendous developmental capacity and unlimited self-renewal, ESCs have diverse biomedical applications. Different culture media have been developed to procure and maintain ESCs in a state of naïve pluripotency, and to preserve a stable genome and epigenome during serial passaging. Chromatin modifications such as DNA methylation and histone modifications along with microRNA activity and different signaling pathways dynamically contribute to the regulation of the ESC gene regulatory network (GRN). Such modifications undergo remarkable changes in different ESC media and determine the quality and developmental potential of ESCs. In this review, we discuss the current approaches for derivation and maintenance of ESCs, and examine how differences in culture media impact on the characteristics of pluripotency via modulation of GRN during the course of ICM outgrowth into ESCs. We also summarize the current hypotheses concerning the origin of ESCs and provide a perspective about the relationship of these cells to their in vivo counterparts (early embryonic cells around the time of implantation). Finally, we discuss generation of ESCs from human embryos and domesticated animals, and offer suggestions to further advance this fascinating field.