Early Human Development Research Articles

Reprogramming the future: Capitalizing on in vitro embryo culture by advancing stem cell technologies in the fight against rare genetic disorders.

Capitalizing on breakthroughs in reproductive genetics, the utilization of in vitro embryo culture and stem cell technologies heralds a transformative era in addressing global challenges posed by rare genetic diseases. These cutting-edge practices illuminate the intricacies of early human development, elucidate the mechanisms behind rare diseases, and guide the development of potential therapies. Balancing this remarkable innovation with necessary ethical considerations, these technologies have the potential to revolutionize the trajectory of rare genetic disorders, transforming the landscape of diagnosis, treatment, and genetic counseling while offering renewed hope for affected individuals and families worldwide.

TIME-LAPSE technology in modern embryological practice

This article describes the basic principles of TIME-LAPSE technology. We report the use of this technology in modern embryological practice and compare it with the conventional cultivation system. Herein, we overview modern incubators equipped with the TIME-LAPSE system, including domestic ones, with their advantages and disadvantages listed. The main events of early human embryo development are described and rare events noted, such as abnormal pronucleus formation and disappearance, multinucleation of blastomeres, reverse cleavage, or “direct cleavage” into three blastomeres, which can only be visualized using TIME-LAPSE technology, but not with traditional visual assessment of embryo morphology. The use of artificial intelligence in conjunction with TIME-LAPSE technology for objective assessment and ranking of embryos based on their morphokinetic characteristics is given a thorough consideration. In conclusion, the article presents data on the impact of using technology on the effectiveness of IVF programs. Moreover, we herein describe such unobvious advantages of the TIME-LAPSE system as the ability to train and transfer video data between participants in the IVF protocol.

Typical neonicotinoids and organophosphate esters, but not their metabolites, adversely impact early human development by activating BMP4 signaling

Recent studies have highlighted the presence of potentially harmful chemicals, such as neonicotinoids (NEOs) and organophosphate esters (OPEs), in everyday items. Despite their potential threats to human health, these dangers are often overlooked. In a previous study, we discovered that NEOs and OPEs can negatively impact development, but liver metabolism can help mitigate their harmful effects. In our current research, our objective was to investigate the toxicity mechanisms associated with NEOs, OPEs, and their liver metabolites using a human embryonic stem cell-based differentiation model that mimics early embryonic development. Our transcriptomics data revealed that NEOs and OPEs significantly influenced the expression of hundreds of genes, disrupted around 100 biological processes, and affected two signaling pathways. Notably, the BMP4 signaling pathway emerged as a key player in the disruption caused by exposure to these pollutants. Both NEOs and OPEs activated BMP4 signaling, potentially impacting early embryonic development. Interestingly, we observed that treatment with a human liver S9 fraction, which mimics liver metabolism, effectively reduced the toxic effects of these pollutants. Most importantly, it reversed the adverse effects dependent on the BMP4 pathway. These findings suggest that normal liver function plays a crucial role in detoxifying environmental pollutants and provides valuable experimental insights for addressing this issue.

A DNA methylation haplotype block landscape in human tissues and preimplantation embryos reveals regulatory elements defined by comethylation patterns.

DNA methylation and associated regulatory elements play a crucial role in gene expression regulation. Previous studies have focused primarily on the distribution of mean methylation levels. Advances in whole-genome bisulfite sequencing (WGBS) have enabled the characterization of DNA methylation haplotypes (MHAPs), representing CpG sites from the same read fragment on a single chromosome, and the subsequent identification of methylation haplotype blocks (MHBs), in which adjacent CpGs on the same fragment are comethylated. Using our expert-curated WGBS data sets, we report comprehensive landscapes of MHBs in 17 representative normal somatic human tissues and during early human embryonic development. Integrative analysis reveals MHBs as a distinctive type of regulatory element characterized by comethylation patterns rather than mean methylation levels. We show the enrichment of MHBs in open chromatin regions, tissue-specific histone marks, and enhancers, including super-enhancers. Moreover, we find that MHBs tend to localize near tissue-specific genes and show an association with differential gene expression that is independent of mean methylation. Similar findings are observed in the context of human embryonic development, highlighting the dynamic nature of MHBs during early development. Collectively, our comprehensive MHB landscapes provide valuable insights into the tissue specificity and developmental dynamics of DNA methylation.

MRI-based structural covariance network in early human brain development.

MRI-based structural covariance network in early human brain development.

Isoform-resolved transcriptome of the human preimplantation embryo

Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing data, which fails to capture most full-length mRNAs. Here, we generate an isoform-resolved transcriptome of early human development by performing long- and short-read RNA sequencing on 73 embryos spanning the zygote to blastocyst stages. We identify 110,212 unannotated isoforms transcribed from known genes, including highly conserved protein-coding loci and key developmental regulators. We further identify 17,964 isoforms from 5,239 unannotated genes, which are largely non-coding, primate-specific, and highly associated with transposable elements. These isoforms are widely supported by the integration of published multi-omics datasets, including single-cell 8CLC and blastoid studies. Alternative splicing and gene co-expression network analyses further reveal that embryonic genome activation is associated with splicing disruption and transient upregulation of gene modules. Together, these findings show that the human embryo transcriptome is far more complex than currently known, and will act as a valuable resource to empower future studies exploring development.

Integrative genome-scale analyses reveal post-transcriptional signatures of early human small intestinal development in a directed differentiation organoid model

BackgroundMicroRNAs (miRNAs) are important post-transcriptional gene regulators controlling cellular lineage specification and differentiation during embryonic development, including the gastrointestinal system. However, miRNA-mediated regulatory mechanisms involved in early embryonic development of human small intestine (SI) remains underexplored. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage.ResultsWe leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Small RNA-seq profiling revealed temporal dynamics of miRNA signatures across different developmental events of the model, including definitive endoderm formation, SI lineage specification and SI regional patterning. Our multi-omic, integrative analyses showed further that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut master marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development.ConclusionThe present study uncovers a novel facet of miRNAs in regulating prenatal SI development. We leveraged multi-omic, systems biology approaches to discover candidate miRNA regulators associated with early SI developmental events in a human organoid model. In this study, we highlighted miRNA-mediated post-transcriptional regulation relevant to the event of SI lineage specification. The candidate miRNA regulators that we identified for the other stages of SI development also warrant detailed characterization in the future.

MICA: a multi-omics method to predict gene regulatory networks in early human embryos.

Recent advances in single-cell omics have transformed characterisation of cell types in challenging-to-study biological contexts. In contexts with limited single-cell samples, such as the early human embryo inference of transcription factor-gene regulatory network (GRN) interactions is especially difficult. Here, we assessed application of different linear or non-linear GRN predictions to single-cell simulated and human embryo transcriptome datasets. We also compared how expression normalisation impacts on GRN predictions, finding that transcripts per million reads outperformed alternative methods. GRN inferences were more reproducible using a non-linear method based on mutual information (MI) applied to single-cell transcriptome datasets refined with chromatin accessibility (CA) (called MICA), compared with alternative network prediction methods tested. MICA captures complex non-monotonic dependencies and feedback loops. Using MICA, we generated the first GRN inferences in early human development. MICA predicted co-localisation of the AP-1 transcription factor subunit proto-oncogene JUND and the TFAP2C transcription factor AP-2γ in early human embryos. Overall, our comparative analysis of GRN prediction methods defines a pipeline that can be applied to single-cell multi-omics datasets in especially challenging contexts to infer interactions between transcription factor expression and target gene regulation.

Human pluripotent stem cell fate trajectories toward lung and hepatocyte progenitors

In this study, we interrogate molecular mechanisms underlying the specification of lung progenitors from human pluripotent stem cells (hPSCs). We employ single-cell RNA-sequencing with high temporal precision, alongside an optimized differentiation protocol, to elucidate the transcriptional hierarchy of lung specification to chart the associated single-cell trajectories. Our findings indicate that Sonic hedgehog, TGF-β, and Notch activation are essential within an ISL1/NKX2-1 trajectory, leading to the emergence of lung progenitors during the foregut endoderm phase. Additionally, the induction of HHEX delineates an alternate trajectory at the early definitive endoderm stage, preceding the lung pathway and giving rise to a significant hepatoblast population. Intriguingly, neither KDR+ nor mesendoderm progenitors manifest as intermediate stages in the lung and hepatic lineage development. Our multistep model offers insights into lung organogenesis and provides a foundation for in-depth study of early human lung development and modeling using hPSCs.

The Effects of Combined Exposure to Bisphenols and Perfluoroalkyls on Human Perinatal Stem Cells and the Potential Implications for Health Outcomes.

The present study investigates the impact of two endocrine disruptors, namely Bisphenols (BPs) and Perfluoroalkyls (PFs), on human stem cells. These chemicals leach from plastic, and when ingested through contaminated food and water, they interfere with endogenous hormone signaling, causing various diseases. While the ability of BPs and PFs to cross the placental barrier and accumulate in fetal serum has been documented, the exact consequences for human development require further elucidation. The present research work explored the effects of combined exposure to BPs (BPA or BPS) and PFs (PFOS and PFOA) on human placenta (fetal membrane mesenchymal stromal cells, hFM-MSCs) and amniotic fluid (hAFSCs)-derived stem cells. The effects of the xenobiotics were assessed by analyzing cell proliferation, mitochondrial functionality, and the expression of genes involved in pluripotency and epigenetic regulation, which are crucial for early human development. Our findings demonstrate that antenatal exposure to BPs and/or PFs may alter the biological characteristics of perinatal stem cells and fetal epigenome, with potential implications for health outcomes at birth and in adulthood. Further research is necessary to comprehend the full extent of these effects and their long-term consequences.

Meiotic and mitotic aneuploidies drive arrest of in vitro fertilized human preimplantation embryos

BackgroundThe high incidence of aneuploidy in early human development, arising either from errors in meiosis or postzygotic mitosis, is the primary cause of pregnancy loss, miscarriage, and stillbirth following natural conception as well as in vitro fertilization (IVF). Preimplantation genetic testing for aneuploidy (PGT-A) has confirmed the prevalence of meiotic and mitotic aneuploidies among blastocyst-stage IVF embryos that are candidates for transfer. However, only about half of normally fertilized embryos develop to the blastocyst stage in vitro, while the others arrest at cleavage to late morula or early blastocyst stages.MethodsTo achieve a more complete view of the impacts of aneuploidy, we applied low-coverage sequencing-based PGT-A to a large series (n = 909) of arrested embryos and trophectoderm biopsies. We then correlated observed aneuploidies with abnormalities of the first two cleavage divisions using time-lapse imaging (n = 843).ResultsThe combined incidence of meiotic and mitotic aneuploidies was strongly associated with blastocyst morphological grading, with the proportion ranging from 20 to 90% for the highest to lowest grades, respectively. In contrast, the incidence of aneuploidy among arrested embryos was exceptionally high (94%), dominated by mitotic aneuploidies affecting multiple chromosomes. In turn, these mitotic aneuploidies were strongly associated with abnormal cleavage divisions, such that 51% of abnormally dividing embryos possessed mitotic aneuploidies compared to only 23% of normally dividing embryos.ConclusionsWe conclude that the combination of meiotic and mitotic aneuploidies drives arrest of human embryos in vitro, as development increasingly relies on embryonic gene expression at the blastocyst stage.

A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial–temporal regulation of histone arginine methylation in neurodevelopment

BackgroundOxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency.ResultsWe report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial–temporal regulation of histone arginine methylation in specific brain regions.ConclusionsThis study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

Modeling early human cortical development and evaluating neurotoxicity with a forebrain organoid system

The complexity and subtlety of brain development renders it challenging to examine effects of environmental toxicants on human fetal brain development. Advances in pluripotent cell-derived organoid systems open up novel avenues for human development, disease and toxicity modeling. Here, we have established a forebrain organoid system and recapitulated early human cortical development spatiotemporally including neuroepithelium induction, apical-basal axis formation, neural progenitor proliferation and maintenance, neuronal differentiation and layer/region patterning. To explore whether this forebrain organoid system is suitable for neurotoxicity modeling, we subjected the organoids to bisphenol A (BPA), a common environmental toxicant of global presence and high epidemic significance. BPA exposure caused substantial abnormalities in key cortical developmental events, inhibited progenitor cell proliferation and promoted precocious neuronal differentiation, leading premature progenitor cell depletion and aberrant cortical layer patterning and structural organization. Consistent with an antagonistic mechanism between thyroid hormone and BPA, T3 supplementation attenuated BPA-mediated cortical developmental abnormalities. Altogether, our in vitro recapitulation of cortical development with forebrain organoids provides a paradigm for efficient neural development and toxicity modeling and related remedy testing/screening.

DMRT1 regulates human germline commitment

Germline commitment following primordial germ cell (PGC) specification during early human development establishes an epigenetic programme and competence for gametogenesis. Here we follow the progression of nascent PGC-like cells derived from human embryonic stem cells in vitro. We show that switching from BMP signalling for PGC specification to Activin A and retinoic acid resulted in DMRT1 and CDH5 expression, the indicators of migratory PGCs in vivo. Moreover, the induction of DMRT1 and SOX17 in PGC-like cells promoted epigenetic resetting with striking global enrichment of 5-hydroxymethylcytosine and locus-specific loss of 5-methylcytosine at DMRT1 binding sites and the expression of DAZL representing DNA methylation-sensitive genes, a hallmark of the germline commitment programme. We provide insight into the unique role of DMRT1 in germline development for advances in human germ cell biology and in vitro gametogenesis.

Deciphering early human pancreas development at the single-cell level

Understanding pancreas development can provide clues for better treatments of pancreatic diseases. However, the molecular heterogeneity and developmental trajectory of the early human pancreas are poorly explored. Here, we performed large-scale single-cell RNA sequencing and single-cell assay for transposase accessible chromatin sequencing of human embryonic pancreas tissue obtained from first-trimester embryos. We unraveled the molecular heterogeneity, developmental trajectories and regulatory networks of the major cell types. The results reveal that dorsal pancreatic multipotent cells in humans exhibit different gene expression patterns than ventral multipotent cells. Pancreato-biliary progenitors that generate ventral multipotent cells in humans were identified. Notch and MAPK signals from mesenchymal cells regulate the differentiation of multipotent cells into trunk and duct cells. Notably, we identified endocrine progenitor subclusters with different differentiation potentials. Although the developmental trajectories are largely conserved between humans and mice, some distinct gene expression patterns have also been identified. Overall, we provide a comprehensive landscape of early human pancreas development to understand its lineage transitions and molecular complexity.

Substrate nesting guides cyst morphogenesis of human pluripotent stem cells without 3D extracellular matrix overlay

Understanding the principles underlying the self-organization of stem cells into tissues is fundamental for deciphering human embryo development. Here, we report that, without three-dimensional (3D) extracellular matrix (ECM) overlay, human pluripotent stem cells (hPSCs) cultured on two-dimensional soft elastic substrates can self-organize into 3D cysts resembling the human epiblast sac in a stiffness-dependent manner. Our theoretical modeling predicts that this cyst organization is facilitated and guided by the spontaneous nesting of the soft substrate, which results from the adhesion-dependent mechanical interaction between cells and substrate. Such substrate nesting is sufficient for the 3D assembly and polarization of hPSCs required for cyst organization, even without 3D ECM overlay. Furthermore, we identify that the reversible substrate nesting and cyst morphogenesis also require appropriate activation of ROCK-Myosin II pathway. This indicates a unique set of tissue morphomechanical signaling mechanisms that clearly differ from the canonical cystogenic mechanism previously reported in 3D ECM. Our findings highlight an unanticipated synergy between mechanical microenvironment and mechanotransduction in controlling tissue morphogenesis and suggest a mechanics-based strategy for generation of hPSCs-derived models for early human embryogenesis. Statement of significanceSoft substrates can induce the self-organization of human pluripotent stem cells (hPSCs) into cysts without three-dimensional (3D) extracellular matrix (ECM) overlay. However, the underlying mechanisms by which soft substrate guides cystogenesis are largely unknown. This study shows that substrate nesting, resulting from cell–substrate interaction, plays an important role in cyst organization, including 3D assembly and apical–basal polarization. Additionally, actomyosin contractility mediated by the ROCK-Myosin II pathway also contributes to the substrate deformation and cyst morphology. These findings demonstrate the interplay between the mechanical microenvironment and cells in tissue morphogenesis, suggesting a mechanics-based strategy in building hPSC-derived models for early human embryo development.

Large-scale production of human blastoids amenable to modeling blastocyst development and maternal-fetal cross talk.

Recent advances in human blastoids have opened new avenues for modeling early human development and implantation. One limitation of our first protocol for human blastoid generation was relatively low efficiency. We now report an optimized protocol for the efficient generation of large quantities of high-fidelity human blastoids from naive pluripotent stem cells. This enabled proteomics analysis that identified phosphosite-specific signatures potentially involved in the derivation and/or maintenance of the signaling states in human blastoids. Additionally, we uncovered endometrial stromal effects in promoting trophoblast cell survival, proliferation, and syncytialization during co-culture with blastoids and blastocysts. Side-by-side single-cell RNA sequencing revealed similarities and differences in transcriptome profiles between pre-implantation blastoids and blastocysts, as well as post-implantation cultures, and uncovered a population resembling early migratory trophoblasts during co-culture with endometrial stromal cells. Our optimized protocol will facilitate broader use of human blastoids as an accessible, perturbable, scalable, and tractable model for human blastocysts.

Influence of infant microbiome on health and development.

The microbiome is a complex ecosystem comprising microbes, their genomes, and the surrounding environment. The microbiome plays a critical role in early human development, including maturation of the host immune system and gastrointestinal tract. Multiple factors, including diet, anti-biotic use, and other environmental exposures, influence the establishment of the microbiome during infancy. Numerous studies have identified associations between the microbiome and neonatal diseases, including necrotizing enterocolitis, sepsis, and malnutrition. Furthermore, there is compelling evidence that perturbation of the microbiome in early life can have lasting developmental effects that increase an individual's risk for immune and metabolic diseases in later life. Supplementation of the microbiome with probiotics reduces the risk of necrotizing enterocolitis and sepsis in at-risk infants. This review focuses on the structure and function of the infant microbiome, the environmental and clinical factors that influence its assembly, and its impact on infant health and development.

Replication stress causes delayed mitotic entry and chromosome 12 fragility at the ANKS1B large neuronal gene in human induced pluripotent stem cells.

Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural chromosomal instability, including recurrent aberrations of chromosome 12. In differentiated cells, replication stress-sensitive genomic regions, including common fragile sites, are widely mapped through mitotic chromosome break induction by mild aphidicolin treatment, an inhibitor of replicative polymerases. IPSCs exhibit lower apoptotic threshold and higher repair capacity hindering fragile site mapping. Caffeine potentiates genotoxic effects and abrogates G2/M checkpoint delay induced by chemical and physical mutagens. Using 5-ethynyl-2'-deoxyuridine (EdU) for replication labeling, we characterized the mitotic entry dynamics of asynchronous iPSCs exposed to aphidicolin and/or caffeine. Under the adjusted timing of replication stress exposure accounting revealed cell cycle delay, higher metaphase chromosome breakage rate was observed in iPSCs compared to primary lymphocytes. Using differential chromosome staining and subsequent locus-specific fluorescent in situ hybridization, we mapped the FRA12L fragile site spanning the large neuronal ANKS1B gene at 12q23.1, which may contribute to recurrent chromosome 12 missegregation and rearrangements in iPSCs. Publicly available data on the ANKS1B genetic alterations and their possible functional impact are reviewed. Our study provides the first evidence of common fragile site induction in iPSCs and reveals potential somatic instability of a clinically relevant gene during early human development and in vitro cell expansion.

Yolk sac cell atlas reveals multiorgan functions during human early development.

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.