The first two blastomeres contribute unequally to the human embryo.

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.

Human embryo live imaging reveals nuclear DNA shedding during blastocyst expansion and biopsy.

Proper preimplantation development is essential to assemble a blastocyst capable of implantation. Live imaging has uncovered major events driving early development in mouse embryos; yet, studies in humans have been limited by restrictions on genetic manipulation and lack of imaging approaches. We have overcome this barrier by combining fluorescent dyes with live imaging to reveal the dynamics of chromosome segregation, compaction, polarization, blastocyst formation, and hatching in the human embryo. We also show that blastocyst expansion mechanically constrains trophectoderm cells, causing nuclear budding and DNA shedding into the cytoplasm. Furthermore, cells with lower perinuclear keratin levels are more prone to undergo DNA loss. Moreover, applying trophectoderm biopsy, a mechanical procedure performed clinically for genetic testing, increases DNA shedding. Thus, our work reveals distinct processes underlying human development compared with mouse and suggests that aneuploidies in human embryos may not only originate from chromosome segregation errors during mitosis but also from nuclear DNA shedding.

A tridimensional atlas of the developing human head.

The evolution and development of the head have long captivated researchers due to the crucial role of the head as the gateway for sensory stimuli and the intricate structural complexity of the head. Although significant progress has been made in understanding head development in various vertebrate species, our knowledge of early human head ontogeny remains limited. Here, we used advanced whole-mount immunostaining and 3D imaging techniques to generate a comprehensive 3D cellular atlas of human head embryogenesis. We present detailed developmental series of diverse head tissues and cell types, including muscles, vasculature, cartilage, peripheral nerves, and exocrine glands. These datasets, accessible through a dedicated web interface, provide insights into human embryogenesis. We offer perspectives on the branching morphogenesis of human exocrine glands and unknown features of the development of neurovascular and skeletomuscular structures. These insights into human embryology have important implications for understanding craniofacial defects and neurological disorders and advancing diagnostic and therapeutic strategies.

Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids.

In vitro stem cell models that replicate human gastrulation have been generated, but they lack the essential extraembryonic cells needed for embryonic development, morphogenesis, and patterning. Here, we describe a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, termed peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. Although peri-gastruloids are not viable due to the exclusion of trophoblasts, they recapitulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and undergoing early neurulation and organogenesis. Single-cell RNA-sequencing unveiled transcriptomic similarities between advanced human peri-gastruloids and primary peri-gastrulation cell types found in humans and non-human primates. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.

Parental genome unification is highly error-prone in mammalian embryos.

Most human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but its origin remains elusive. Human zygotes that cluster their nucleoli at the pronuclear interface are thought to be more likely to develop into healthy euploid embryos. Here, we show that the parental genomes cluster with nucleoli in each pronucleus within human and bovine zygotes, and clustering is required for the reliable unification of the parental genomes after fertilization. During migration of intact pronuclei, the parental genomes polarize toward each other in a process driven by centrosomes, dynein, microtubules, and nuclear pore complexes. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other, yet separated. Parental genome clustering ensures the rapid unification of the parental genomes on nuclear envelope breakdown. However, clustering often fails, leading to chromosome segregation errors and micronuclei, incompatible with healthy embryo development.

Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.

Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.

Transcription factor-based transdifferentiation of human embryonic to trophoblast stem cells.

During the first week of development, human embryos form a blastocyst composed of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here, we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of the transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection, we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from primed human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles, and directed differentiation potential, similar to existing human TSCs. Systematic omission of each, or combinations of factors, revealed the crucial importance of GATA2 and GATA3 for iTSC transdifferentiation. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases.

Human embryoids: A new strategy of recreating the first steps of embryonic development in vitro.

Molecular mechanisms surrounding early human embryonic events such as blastocyst formation, implantation, and the specification of the body axes are some of the most attractive research questions of developmental biology today. A knowledge on the detailed signaling landscape underlying these critical events in the human could impact the way we treat early pregnancy disorders and infertility, and considerably advance our abilities to make precise human tissues in a lab. However, owing to ethical, technical, and policy restrictions, research on early human embryo development historically stalled behind animal models. The rapid progress in 3D culture of human embryonic stem cells over the past years created an opportunity to overcome this critical challenge. We review recently developed strategies of making 3D models of the human embryo built from embryonic stem cells, which we refer to as embryoids. We focus on models aimed at reconstituting the 3D epithelial characteristics of the early human embryo, namely the intra/extraembryonic signaling crosstalk, tissue polarity, and embryonic cavities. We identify distinct classes of embryoids based on whether they explicitly include extraembryonic tissues and we argue for the merit of compromising on certain aspects of embryo mimicry in balancing the experimental feasibility with ethical considerations. Human embryoids open gates toward a new field of synthetic human embryology, allowing to study the long inaccessible stages of early human development at unprecedented detail.

Why study human embryo development?

Understanding the processes and mechanisms underlying early human embryo development has become an increasingly active and important area of research. It has potential for insights into important clinical issues such as early pregnancy loss, origins of congenital anomalies and developmental origins of adult disease, as well as fundamental insights into human biology. Improved culture systems for preimplantation embryos, combined with the new tools of single cell genomics and live imaging, are providing new insights into the similarities and differences between human and mouse development. However, access to human embryo material is still restricted and extended culture of early embryos has regulatory and ethical concerns. Stem cell-derived models of different phases of human development can potentially overcome these limitations and provide a scalable source of material to explore the early postimplantation stages of human development. To date, such models are clearly incomplete replicas of normal development but future technological improvements can be envisaged. The ethical and regulatory environment for such studies remains to be fully resolved.

Suppression of YAP safeguards human naïve pluripotency.

Propagation of human naïve pluripotent stem cells (nPSCs) relies on the inhibition of MEK/ERK signalling. However, MEK/ERK inhibition also promotes differentiation into trophectoderm (TE). Therefore, robust self-renewal requires suppression of TE fate. Tankyrase inhibition using XAV939 has been shown to stabilise human nPSCs and is implicated in TE suppression. Here, we dissect the mechanism of this effect. Tankyrase inhibition is known to block canonical Wnt/ß-catenin signalling. However, we show that nPSCs depleted of ß-catenin remain dependent on XAV939. Rather than inhibiting Wnt, we found that XAV939 prevents TE induction by reducing activation of YAP, a co-factor of TE-inducing TEAD transcription factors. Tankyrase inhibition stabilises angiomotin, which limits nuclear accumulation of YAP. Upon deletion of angiomotin-family members AMOT and AMOTL2, nuclear YAP increases and XAV939 fails to prevent TE induction. Expression of constitutively active YAP similarly precipitates TE differentiation. Conversely, nPSCs lacking YAP1 or its paralog TAZ (WWTR1) resist TE differentiation and self-renewal efficiently without XAV939. These findings explain the distinct requirement for tankyrase inhibition in human but not in mouse nPSCs and highlight the pivotal role of YAP activity in human naïve pluripotency and TE differentiation. This article has an associated 'The people behind the papers' interview.

Transcriptome analysis reveals that defects in cell cycle regulation contribute to preimplantation embryo arrest.

Patients with preimplantation embryo arrest (PREMBA) often experience assisted reproductive failure primarily due to the lack of transferable embryos, and the molecular mechanisms underlying PREMBA remain unclear. In our study, the embryos from five women with recurrent preimplantation embryo arrest and three women with tubal factor infertility were used for single-embryo transcriptome sequencing. Meanwhile, the transcriptomes of normal human preimplantation embryos obtained from GSE36552 were utilized to perform a comparative analysis with the transcriptomes of PREMBA embryos. Our results showed dysregulation of the cell cycle phase transition might be a potential pathogenic factor contributing to PREMBA. Through integrated analysis of the differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA), we identified a number of hub genes using the protein-protein interaction network. The top 5 hub genes were as follows: CCNB2, BUB1B, CDC25A, CCNB3, and PLK3. The expression of hub genes was validated in PREMBA embryos and donated embryos using RT-qPCR. The knockdown of Ccnb2 in mouse zygotes led to an increase in embryo fragmentation, a rise in apoptosis, and a reduction in blastocyst formation. Furthermore, silencing the expression of CDC25A in HEK293T cells resulted in a decrease in cell proliferation and an increase in apoptosis, providing further support for our findings. Our findings could predict the development outcomes of preimplantation embryos and be used as potential therapeutic targets to prevent recurrent failures of IVF/ICSI attempts.

The non-coding genome in early human development - Recent advancements.

Not that long ago, the human genome was discovered to be mainly non-coding, that is comprised of DNA sequences that do not code for proteins. The initial paradigm that non-coding is also non-functional was soon overturned and today the work to uncover the functions of non-coding DNA and RNA in human early embryogenesis has commenced. Early human development is characterized by large-scale changes in genomic activity and the transcriptome that are partly driven by the coordinated activation and repression of repetitive DNA elements scattered across the genome. Here we provide examples of recent novel discoveries of non-coding DNA and RNA interactions and mechanisms that ensure accurate non-coding activity during human maternal-to-zygotic transition and lineage segregation. These include studies on small and long non-coding RNAs, transposable element regulation, and RNA tailing in human oocytes and early embryos. High-throughput approaches to dissect the non-coding regulatory networks governing early human development are a foundation for functional studies of specific genomic elements and molecules that has only begun and will provide a wider understanding of early human embryogenesis and causes of infertility.

Transcriptome comparisons of trophoblasts from regenerative cell models with peri-implantation human embryos.

Mechanisms controlling trophoblast proliferation and differentiation during embryo implantation are poorly understood. Human trophoblast stem cells (TSC) and BMP4/A83-01/PD173074-treated pluripotent stem cell-derived trophoblast cells (BAP) are two widely employed, contemporary models to study trophoblast development and function, but how faithfully they mimic early trophoblast cells has not been fully examined. We evaluated the transcriptomes of trophoblast cells from BAP and TSC and directly compared them with those from peri-implantation human embryos during extended embryo culture (EEC) between embryonic day 8 to 12. The BAP and TSC grouped closely with trophoblast cells from EEC within each trophoblast sublineage following dimensional analysis and unsupervised hierarchical clustering. However, subtle differences in transcriptional programs existed within each trophoblast sublineage. We also validated the presence of six genes in peri-implantation human embryos by immunolocalization. Our analysis reveals that both BAP and TSC models have features of peri-implantation trophoblasts, while maintaining minor transcriptomic differences, and thus serve as valuable tools for studying implantation in lieu of human embryos.

Cardiac development demystified by use of the HDBR atlas.

Much has been learned over the last half century regarding the molecular and genetic changes that take place during cardiac development. As yet, however, these advances have not been translated into knowledge regarding the marked changes that take place in the anatomical arrangements of the different cardiac components. As such, therefore, many aspects of cardiac development are still described on the basis of speculation rather than evidence. In this review, we show how controversial aspects of development can readily be arbitrated by the interested spectator by taking advantage of the material now gathered together in the Human Developmental Biology Resource; HDBR. We use the material to demonstrate the changes taking place during the formation of the ventricular loop, the expansion of the atrioventricular canal, the incorporation of the systemic venous sinus, the formation of the pulmonary vein, the process of atrial septation, the remodelling of the pharyngeal arches, the major changes occurring during formation of the outflow tract, the closure of the embryonic interventricular communication, and the formation of the ventricular walls. We suggest that access to the resource makes it possible for the interested observer to arbitrate, for themselves, the ongoing controversies that continue to plague the understanding of cardiac development.

Bronchial tree of the human embryo: Examination based on a mammalian model.

The symmetry of the right and left bronchi, proposed in a previous comparative anatomical study as the basic model of the mammalian bronchial tree, was examined to determine if it applied to the embryonic human bronchial tree. Imaging data of 41 human embryo specimens at Carnegie stages (CS) 16-23 (equivalent to 6-8 weeks after fertilization) belonging to the Kyoto collection were obtained using phase-contrast X-ray computed tomography. Three-dimensional bronchial trees were then reconstructed from these images. Bronchi branching from both main bronchi were labeled as dorsal, ventral, medial, or lateral systems based on the branching position with numbering starting cranially. The length from the tracheal bifurcation to the branching point of the labeled bronchus was measured, and the right-to-left ratio of the same labeled bronchus in both lungs was calculated. In both lungs, the human embryonic bronchial tree showed symmetry with an alternating pattern of dorsal and lateral systems up to segmental bronchus B9 as the basic shape, with a more peripheral variation. This pattern is similar to that described in adult human lungs. Bronchial length increased with the CS in all labeled bronchi, whereas the right-to-left ratio was constant at approximately 1.0. The data demonstrated that the prototype of the human adult bronchial branching structure is formed and maintained in the embryonic stage. The morphology and branching position of all lobar bronchi and B6, B8, B9, and the subsegmental bronchus of B10 may be genetically determined. On the other hand, no common structures between individual embryos were found in the peripheral branches after the subsegmental bronchus of B10, suggesting that branch formation in this region is influenced more by environmental factors than by genetic factors.

Ontogenic transformation of the ankle from the initial mediolateral arrangement of the calcaneus and talus: A histological study of human embryos and early fetuses.

The human calcaneus is robust and provides a prominent heel for effective bipedal locomotion, although the adjacent talus has no muscle attachments. However, there is incomplete information about the morphological changes in these prominent bones during embryo development. We examined serial histological sections of 23 human embryos and early-term fetuses (approximately 5-10 weeks' gestational age [GA]). At a GA of 5 weeks, the precartilage talus was parallel to and on the medial side of the calcaneus, which had a prolate spheroid shape and consisted of three masses. At a GA of 6 weeks, the cartilaginous talus extended along the proximodistal axis, and the tuber calcanei became long and bulky, with a small sustentaculum talus at the "distal" side. At a GA of 6 to 8 weeks, the sustentaculum had a medial extension below the talus so that the talus "rode over" the calcaneus. In contrast, the talus had a more complex shape, depending on the growth of adjacent bones. At a GA of 9 to 10 weeks, the talus was above the calcaneus, but the medial part still faced the plantar subcutaneous tissue because of the relatively small sustentaculum. Therefore, the final morphology appeared after an additional several weeks. Muscle activity seemed to facilitate growth of the tuber calcanei, but growth of the other parts of calcaneus, including the sustentaculum, seemed to depend on active proliferation at the different sites of cartilage. Multiple tendons and ligaments seemed to fix the talus so that it remained close to the calcaneus.

Longitudinal profiling of human androgenotes through single-cell analysis unveils paternal gene expression dynamics in early embryo development.

STUDY QUESTION: How do transcriptomics vary in haploid human androgenote embryos at single cell level in the first four cell cycles of embryo development? SUMMARY ANSWER: Gene expression peaks at the fourth cell cycle, however some androcytes exhibit unique transcriptional behaviors. WHAT IS KNOWN ALREADY: The developmental potential of an embryo is determined by the competence of the oocyte and the sperm. However, studies of the contribution of the paternal genome using pure haploid androgenotes are very scarce. STUDY DESIGN, SIZE, DURATION: This study was performed analyzing the single-cell transcriptomic sequencing of 38 androcytes obtained from 10 androgenote bioconstructs previously produced in vitro (de Castro et al., 2023). These results were analyzed through different bioinformatics software such as g: Profiler, GSEA, Cytoscape, and Reactome. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single cell sequencing was used to obtain the transcriptomic profiles of the different androcytes. The results obtained were compared between the different cycles studied using the DESeq2 program and functional enrichment pathways using g: Profiler, Cytoscape, and Reactome. MAIN RESULTS AND THE ROLE OF CHANCE: A wave of paternally driven transcriptomic activation was found during the third-cell cycle, with 1128 upregulated and 225 downregulated genes and the fourth-cell cycle, with 1373 upregulated and 286 downregulated genes, compared to first-cell cycle androcytes. Differentially expressed routes related to cell differentiation, DNA-binding transcription, RNA biosynthesis and RNA polymerase II transcription regulatory complex, and cell death were found in the third and fourth with respect to the first-cell cycle. Conversely, in the fourth cell cycle, 153 downregulated and 332 upregulated genes were found compared with third cell cycle, associated with differentially expressed processes related to E-box binding and zinc finger protein 652 (ZNF652) transcription factor. Further, significant overexpression of LEUTX, PRAMEF1, DUXA, RFPL4A, TRIM43, and ZNF675 found in androgenotes, compared to biparental embryos, highlights the paternal contributions to zygote genome activation. LARGE SCALE DATA: All raw sequencing data are available through the Gene Expression Omnibus (GEO) under accessions number: GSE216501. LIMITATIONS, REASONS FOR CAUTION: Extrapolation of biological events from uniparental constructs to biparental embryos should be done with caution. Maternal and paternal genomes do not act independently of each other in a natural condition. The absence of one genome may affect gene transcription of the other. In this sense, the haploid condition of the bioconstructs could mask the transcriptomic patterns of the single cells. WIDER IMPLICATIONS OF THE FINDINGS: The results obtained demonstrated the level of involvement of the human paternal haploid genome in the early stages of embryo development as well as its evolution at the transcriptomic level, laying the groundwork for the use of these bioconstructs as reliable models to dispel doubts about the genetic role played by the paternal genome in the early cycles of embryo development. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Instituto de Salud Carlos III (ISCIII) through the project 'PI22/00924', co-funded by European Regional Development Fund (ERDF); 'A way to make Europe'. F.D. was supported by the Spanish Ministry of Economy and Competitiveness through the Miguel Servet program (CPII018/00002). M.J.E. was supported by Instituto de Salud Carlos III (PI19/00577 [M.J.E.]) and FI20/00086. P.dC. was supported by a predoctoral grant for training in research into health (PFIS PI19/00577) from the Instituto de Salud Carlos III. All authors declare having no conflict of interest with regard to this trial.

Detection of Minute virus of mice strains in different cell lines: Implications for adventitious agent testing.

Minute virus of mice (MMV) has contaminated biotechnological processes in the past and specific MMV testing is therefore recommended, if the production cell line is known to be permissive for this virus. Testing is widely done using cell-culture-based adventitious virus assays, yet MMV strains may differ in their in vitro cell tropism. Here, we investigated the growth characteristics of different MMV strains on A9 and 324K cells and identified significant differences in susceptibility of these widely used indicator cell lines to infection by different strains of MMV, which has implications for MMV detectability during routine testing of biotechnology process harvests. An MMV-specific polymerase chain reaction was evaluated as a more encompassing method and was shown as suitable replacement for cell culture-based detection of the different MMV strains, with the additional benefit that detection is more rapid and can be extended to other rodent parvoviruses that might contaminate biotechnological processes. Although no MMV contamination event of human-derived cell lines has happened in the past, biotechnological processes that are based on these also need to consider MMV-specific testing, as, for example, HEK293, a human-derived cell line commonly used in biopharmaceutical manufacturing, was shown as susceptible to productive MMV infection in the current work.

Retained chromosomal integrity following CRISPR-Cas9-based mutational correction in human embryos.

Human germline gene correction by targeted nucleases holds great promise for reducing mutation transmission. However, recent studies have reported concerning observations in CRISPR-Cas9-targeted human embryos, including mosaicism and loss of heterozygosity (LOH). The latter has been associated with either gene conversion or (partial) chromosome loss events. In this study, we aimed to correct a heterozygous basepair substitution in PLCZ1, related to infertility. In 36% of the targeted embryos that originated from mutant sperm, only wild-type alleles were observed. By performing genome-wide double-digest restriction site-associated DNA sequencing, integrity of the targeted chromosome (i.e., no deletions larger than 3 Mb or chromosome loss) was confirmed in all seven targeted GENType-analyzed embryos (mutant editing and absence of mutation), while short-range LOH events (shorter than 10 Mb) were clearly observed by single-nucleotide polymorphism assessment in two of these embryos. These results fuel the currently ongoing discussion on double-strand break repair in early human embryos, making a case for the occurrence of gene conversion events or partial template-based homology-directed repair.

Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos.

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.