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.

Spatiotemporal analysis of human intestinal development at single-cell resolution.

Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.

Human Pluripotency Is Initiated and Preserved by a Unique Subset of Founder Cells.

The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.

A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart.

The process of cardiac morphogenesis in humans is incompletely understood. Its full characterization requires a deep exploration of the organ-wide orchestration of gene expression with a single-cell spatial resolution. Here, we present a molecular approach that reveals the comprehensive transcriptional landscape of cell types populating the embryonic heart at three developmental stages and that maps cell-type-specific gene expression to specific anatomical domains. Spatial transcriptomics identified unique gene profiles that correspond to distinct anatomical regions in each developmental stage. Human embryonic cardiac cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of embryonic cardiac gene expression. In situ sequencing was then used to refine these results and create a spatial subcellular map for the three developmental phases. Finally, we generated a publicly available web resource of the human developing heart to facilitate future studies on human cardiogenesis.

Challenges of Organoid Research.

Organoids are 3D cell culture systems derived from human pluripotent stem cells that contain tissue resident cell types and reflect features of early tissue organization. Neural organoids are a particularly innovative scientific advance given the lack of accessibility of developing human brain tissue and intractability of neurological diseases. Neural organoids have become an invaluable approach to model features of human brain development that are not well reflected in animal models. Organoids also hold promise for the study of atypical cellular, molecular, and genetic features that underscore neurological diseases. Additionally, organoids may provide a platform for testing therapeutics in human cells and are a potential source for cell replacement approaches to brain injury or disease. Despite the promising features of organoids, their broad utility is tempered by a variety of limitations yet to be overcome, including lack of high-fidelity cell types, limited maturation, atypical physiology, and lack of arealization, features that may limit their reliability for certain applications.

Branching topology of the human embryo transcriptome revealed by Entropy Sort Feature Weighting.

Analysis of single cell transcriptomics (scRNA-seq) data is typically performed after subsetting to highly variable genes (HVGs). Here, we show that Entropy Sorting provides an alternative mathematical framework for feature selection. On synthetic datasets, continuous Entropy Sort Feature Weighting (cESFW) outperforms HVG selection in distinguishing cell-state-specific genes. We apply cESFW to six merged scRNA-seq datasets spanning human early embryo development. Without smoothing or augmenting the raw counts matrices, cESFW generates a high-resolution embedding displaying coherent developmental progression from eight-cell to post-implantation stages and delineating 15 distinct cell states. The embedding highlights sequential lineage decisions during blastocyst development, while unsupervised clustering identifies branch point populations obscured in previous analyses. The first branching region, where morula cells become specified for inner cell mass or trophectoderm, includes cells previously asserted to lack a developmental trajectory. We quantify the relatedness of different pluripotent stem cell cultures to distinct embryo cell types and identify marker genes of naïve and primed pluripotency. Finally, by revealing genes with dynamic lineage-specific expression, we provide markers for staging progression from morula to blastocyst.

An interactive resource of molecular signalling in the developing human haematopoietic stem cell niche.

The emergence of definitive human haematopoietic stem cells (HSCs) from Carnegie Stage (CS) 14 to CS17 in the aorta-gonad-mesonephros (AGM) region is a tightly regulated process. Previously, we conducted spatial transcriptomic analysis of the human AGM region at the end of this period (CS16/CS17) and identified secreted factors involved in HSC development. Here, we extend our analysis to investigate the progression of dorso-ventral polarised signalling around the dorsal aorta over the entire period of HSC emergence. Our results reveal a dramatic increase in ventral signalling complexity from the CS13-CS14 transition, coinciding with the first appearance of definitive HSCs. We further observe stage-specific changes in signalling up to CS17, which may underpin the step-wise maturation of HSCs described in the mouse model. The data-rich resource is also presented in an online interface enabling in silico analysis of molecular interactions between spatially defined domains of the AGM region. This resource will be of particular interest for researchers studying mechanisms underlying human HSC development as well as those developing in vitro methods for the generation of clinically relevant HSCs from pluripotent stem cells.

Approaches to benchmark and characterize in vitro human model systems.

In vitro human models, such as gastruloids and organoids, are complex three-dimensional (3D) structures often consist of cells from multiple germ layers that possess some attributes of a developing embryo or organ. To use these models to interrogate human development and organogenesis, these 3D models must accurately recapitulate aspects of their in vivo counterparts. Recent advances in single-cell technologies, including sequencing and spatial approaches, have enabled efforts to better understand and directly compare organoids with native tissues. For example, single-cell genomic efforts have created cell and organ atlases that enable benchmarking of in vitro models and can also be leveraged to gain novel biological insights that can be used to further improve in vitro models. This Spotlight discusses the state of current in vitro model systems, the efforts to create large publicly available atlases of the developing human and how these data are being used to improve organoids. Limitations and perspectives on future efforts are also discussed.

Human assembloids.

Deconstructing and then reconstructing developmental processes ex vivo is crucial to understanding how organs assemble and how physiology can be disrupted in disease. Human 3D stem cell-derived systems, such as organoids, have facilitated this pursuit; however, they often do not capture inter-tissue or inter-lineage cellular interactions that give rise to emergent tissue properties during development. Assembloids are self-organizing 3D cellular systems that result from the integration of multiple organoids or the combination of organoids with missing cell types or primary tissue explants. Here, we outline the concept and types of assembloids and present their applications for studying the nervous system and other tissues. We describe tools that are used to probe and manipulate assembloids and delineate current challenges and the potential for this new approach to interrogate development and disease.

An integrated atlas of human placental development delineates essential regulators of trophoblast stem cells.

The trophoblast lineage safeguards fetal development by mediating embryo implantation, immune tolerance, nutritional supply and gas exchange. Human trophoblast stem cells (hTSCs) provide a platform to study lineage specification of placental tissues; however, the regulatory network controlling self-renewal remains elusive. Here, we present a single-cell atlas of human trophoblast development from zygote to mid-gestation together with single-cell profiling of hTSCs. We determine the transcriptional networks of trophoblast lineages in vivo and leverage probabilistic modelling to identify a role for MAPK signalling in trophoblast differentiation. Placenta- and blastoid-derived hTSCs consistently map between late trophectoderm and early cytotrophoblast, in contrast to blastoid-trophoblast, which correspond to trophectoderm. We functionally assess the requirement of the predicted cytotrophoblast network in an siRNA-screen and reveal 15 essential regulators for hTSC self-renewal, including MAZ, NFE2L3, TFAP2C, NR2F2 and CTNNB1. Our human trophoblast atlas provides a powerful analytical resource to delineate trophoblast cell fate acquisition, to elucidate transcription factors required for hTSC self-renewal and to gauge the developmental stage of in vitro cultured cells.

Scaling patterns of cortical folding and thickness in early human brain development in comparison with primates.

Across mammalia, brain morphology follows specific scaling patterns. Bigger bodies have bigger brains, with surface area outpacing volume growth, resulting in increased foldedness. We have recently studied scaling rules of cortical thickness, both local and global, finding that the cortical thickness difference between thick gyri and thin sulci also increases with brain size and foldedness. Here, we investigate early brain development in humans, using subjects from the Developing Human Connectome Project, scanned shortly after pre-term or full-term birth, yielding magnetic resonance images of the brain from 29 to 43 postmenstrual weeks. While the global cortical thickness does not change significantly during this development period, its distribution does, with sulci thinning, while gyri thickening. By comparing our results with our recent work on humans and 11 non-human primate species, we also compare the trajectories of primate evolution with human development, noticing that the 2 trends are distinct for volume, surface area, cortical thickness, and gyrification index. Finally, we introduce the global shape index as a proxy for gyrification index; while correlating very strongly with gyrification index, it offers the advantage of being calculated only from local quantities without generating a convex hull or alpha surface.

Recent advances in understanding cell types during human gastrulation.

Gastrulation is a fundamental process during embryonic development, conserved across all multicellular animals [1]. In the majority of metazoans, gastrulation is characterised by large scale morphogenetic remodeling, leading to the conversion of an early pluripotent embryonic cell layer into the three primary 'germ layers': an outer ectoderm, inner endoderm and intervening mesoderm layer. The morphogenesis of these three layers of cells is closely coordinated with cellular diversification, laying the foundation for the generation of the hundreds of distinct specialized cell types in the animal body. The process of gastrulation has for a long time attracted tremendous attention in a broad range of experimental systems ranging from sponges to mice. In humans the process of gastrulation starts approximately 14 days after fertilization and continues for slightly over a week. However our understanding of this important process, as it pertains to human, is limited. Donations of human fetal material at these early stages are exceptionally rare, making it nearly impossible to study human gastrulation directly. Therefore, our understanding of human gastrulation is predominantly derived from animal models such as the mouse [2,3] and from studies of limited collections of fixed whole samples and histological sections of human gastrulae [4-7], some of which date back to over a century ago. More recently we have been gaining valuable molecular insights into human gastrulation using in vitro models of hESCs [8-12] and increasingly, in vitro cultured human and non-human primate embryos [13-16]. However, while methods have been developed to culture human embryos into this stage (and probably beyond), current ethical standards prohibit the culture of human embryos past 14 days again limiting our ability to experimentally probe human gastrulation. This review discusses recent molecular insights from the study of a rare CS 7 human gastrula obtained as a live sample and raises several questions arising from this recent study that it will be interesting to address in the future using emerging models of human gastrulation.

Carnegie in 4D? Stem-cell-based models of human embryo development.

How cells build embryos is still a major mystery. Many unresolved questions require the study of the processes that pattern and shape the embryo in live specimens, in toto, across spatial and temporal scales. In mammalian embryogenesis, this remains a major challenge as the embryo develops in utero, precluding easy accessibility. For human embryos, technical, ethical and legal limitations further hamper the in-depth investigation of embryogenesis, especially beyond gastrulation stages. This has resulted in an over-reliance on model organisms, particularly mice, to understand mammalian development. However, recent efforts show critical differences between rodent and primate embryos, including timing, architecture and transcriptional regulation. Thus, a human-centric understanding of embryogenesis is much needed. To empower this, novel in vitro approaches, which coax human pluripotent stem cells to form embryonic organoids that model embryo development, are pivotal. Here, we summarize these emergent technologies that recapitulate aspects of human development "in a dish". We show how these technologies can provide insights into the molecular, cellular and morphogenetic processes that fuel the formation of a fully formed fetus, and discuss the potential of these platforms to revolutionize our understanding of human development in health and disease. Despite their clear promise, we caution against over-interpreting the extent to which these in vitro platforms model the natural embryo. In particular, we discuss how fate, form and function - a tightly coupled trinity in vivo, can be disconnected in vitro. Finally, we propose how careful benchmarking of existing models, in combination with rational protocol design based on an increased understanding of in vivo developmental dynamics and insights from mouse in vitro models of embryo development, will help guide the establishment of better models of human embryo development.

Developmental principles informing human pluripotent stem cell differentiation to cartilage and bone.

Human pluripotent stem cells can differentiate into any cell type given appropriate signals and hence have been used to research early human development of many tissues and diseases. Here, we review the major biological factors that regulate cartilage and bone development through the three main routes of neural crest, lateral plate mesoderm and paraxial mesoderm. We examine how these routes have been used in differentiation protocols that replicate skeletal development using human pluripotent stem cells and how these methods have been refined and improved over time. Finally, we discuss how pluripotent stem cells can be employed to understand human skeletal genetic diseases with a developmental origin and phenotype, and how developmental protocols have been applied to gain a better understanding of these conditions.

Gender inequality and utilization of ART: an international cross-sectional and longitudinal analysis.

STUDY QUESTION: What is the association between a country's level of gender equality and access to ART, as measured through ART utilization? SUMMARY ANSWER: ART utilization is associated with a country's level of gender equality even after controlling for the level of development. WHAT IS KNOWN ALREADY: Although gender equality is recognized as an important determinant of population health, its association with fertility care, a highly gendered condition, has not been explored. STUDY DESIGN, SIZE, DURATION: A longitudinal cross-national analysis of ART utilization in 69 countries during 2002-2014 was carried out. PARTICPANTS/MATERIALS, SETTING, METHODS: The Gender Inequality Index (GII), Human Development Index (HDI), and their component indicators were modelled against ART utilization using univariate regression models as well as mixed-effects regression methods (adjusted for country, time, and economic/human development) with multiple imputation to account for missing data. MAIN RESULTS AND THE ROLE OF CHANCE: ART utilization is associated with the GII. In an HDI-adjusted analysis, a one standard deviation decrease in the GII (towards greater equality) is associated with a 59% increase in ART utilization. Gross national income per capita, the maternal mortality ratio, and female parliamentary representation were the index components most predictive of ART utilization. LIMITATIONS, REASONS FOR CAUTION: Only ART was used rather than all infertility treatments (including less costly and non-invasive treatments such as ovulation induction). This was a country-level analysis and the results cannot be generalized to smaller groups. Not all modelled variables were available for each country across 2002-2014. WIDER IMPLICATIONS OF THE FINDINGS: Access to fertility care is central to women's sexual and reproductive health, to women's rights, and to human rights. As gender equality improves, so does access to ART. This relation is likely to be reinforcing and bi-directional, with progress towards global, equitable access to fertility care also improving women's status and participation in societies. STUDY FUNDING/COMPETING INTEREST(S): External funding was not provided for this study. G.D.A. declares consulting fees from Labcorp and CooperSurgical. G.D.A. is the founder and CEO of Advanced Reproductive Care, Inc., as well as the Chair of the International Committee for Monitoring Assisted Reproductive Technologies (ICMART) and the World Endometriosis Research Foundation, both of which are unpaid roles. G.M.C. is an ICMART Board Representative, which is an unpaid role, and no payments are received from ICMART to UNSW, Sydney, or to G.M.C. to undertake this study. O.F., S.D., F.Z.-H., and E.K. report no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Sex Differences in Myocardial and Vascular Aging.

It is well known that cardiovascular disease manifests differently in women and men. The underlying causes of these differences during the aging lifespan are less well understood. Sex differences in cardiac and vascular phenotypes are seen in childhood and tend to track along distinct trajectories related to dimorphism in genetic factors as well as response to risk exposures and hormonal changes during the life course. These differences underlie sex-specific variation in cardiovascular events later in life, including myocardial infarction, heart failure, ischemic stroke, and peripheral vascular disease. With respect to cardiac phenotypes, females have intrinsically smaller body size-adjusted cardiac volumes and they tend to experience greater age-related wall thickening and myocardial stiffening with aging. With respect to vascular phenotypes, sexual dimorphism in both physiology and pathophysiology are also seen, including overt differences in blood pressure trajectories. The majority of sex differences in myocardial and vascular alterations that manifest with aging seem to follow relatively consistent trajectories from the very early to the very later stages of life. This review aims to synthesize recent cardiovascular aging-related research to highlight clinically relevant studies in diverse female and male populations that can inform approaches to improving the diagnosis, management, and prognosis of cardiovascular disease risks in the aging population at large.

X-chromosome inactivation patterns depend on age and tissue but not conception method in humans.

Female somatic X-chromosome inactivation (XCI) balances the X-linked transcriptional dosages between the sexes, randomly silencing the maternal or paternal X chromosome in each cell of 46,XX females. Skewed XCI toward one parental X has been observed in association with ageing and in some female carriers of X-linked diseases. To address the problem of non-random XCI, we quantified the XCI skew in different biological samples of naturally conceived females of different age groups and girls conceived after in vitro fertilization (IVF). Generally, XCI skew differed between saliva, blood, and buccal swabs, while saliva and blood had the most similar XCI patterns in individual females. XCI skew increased with age in saliva, but not in other tissues. We showed no significant differences in the XCI patterns in tissues of naturally conceived and IVF females. The gene expression profile of the placenta and umbilical cord blood was determined depending on the XCI pattern. The increased XCI skewing in the placental tissue was associated with the differential expression of several genes out of 40 considered herein. Notably, skewed XCI patterns (> 80:20) were identified with significantly increased expression levels of four genes: CD44, KDM6A, PHLDA2, and ZRSR2. The differences in gene expression patterns between samples with random and non-random XCI may shed new light on factors contributing to the XCI pattern outcome and indicate new paths in future research on the phenomenon of XCI skewing.

Contributions to inclusive and impactful development and psychopathology science: interrogating ecology-linked vulnerability and resilience opportunities.

Since its launch in a 1984 Special Issue of Child Development, significant contributions and insights have followed that have expanded our understanding of psychopathology and normal human growth and development. Despite these efforts, there are persistent and under-analyzed skewed patterns of vulnerability across and within groups. The persistence of a motivated forgetfulness to acknowledge citizens' uneven access to resources and supports, or as stated elsewhere, "inequality presence denial," is, at minimum, a policy, social and health practice problem. This article will examine some of these issues from the standpoint of a universal human vulnerability perspective. It also investigates sources of resistance to acknowledging and responding to the scholarship production problem of uneven representations of basic human development research versus psychopathology preoccupations by race. Collectively, findings suggest interesting "patchwork" patterns of particular cultural repertoires as ordinary social and scholarly traditions.

Correlation of geopolitics, education, democracy with COVID-19 vaccination rate.

INTRODUCTION: Vaccine hesitancy is an ongoing problem and determining the factors that increase the vaccination rate in various countries of the world might be useful for further implementation of efficient public health policies and negating anti-vaccination campaigns. MATERIALS AND METHODS: Human Development Index (HDI), Education Index (EI), Democracy Index (DI), COVID-19 vaccination rates, COVID-19 data were collected from public sources such as UNDP - Human Development Reports, UNESCO - Education Index, Economist Intelligence, WHO- COVID-19 Dashboard, Our World In Data, The Financial Times COVID-19 Dashboard. Statistical analysis such as Pearson correlation, and linear regression analyses were done to determine a relation between the above-mentioned indices and COVID-19 vaccination rates (1-dose, 2-dose, booster, and combined). RESULTS: HDI had the strongest positive correlation with the vaccination rates (1-dose- r (181) = 0.632, p < 0.001, 2-dose- r (181) = 0.671, p < 0.001, booster- r (181) = 0.718, p < 0.001, combined- 0.703, p < 0.001). EI (1-dose- r (177) = 0.560, p < 0.001, 2-dose- r (177) = 0.599, p < 0.001, booster- r (177) = 0.642, p < 0.001, combined- 0.626, p < 0.001), DI (1-dose- r (163) = 0.445, p < 0.001, 2-dose- r (163) = 0.479, p < 0.001, booster- r (163) = 0.534, p < 0.001, combined- 0.508, p < 0.001), as well as Geographic location (1-dose- η (Eta) = 0.610 p < 0.001, 2-dose- η (Eta) = 0.633 p < 0.001, booster- η (Eta) = 0.657, p < 0.001, combined- η (Eta) = 0.645, p < 0.001) had positive correlation with vaccination rates. CONCLUSION: There is a strong positive correlation of COVID-19 vaccination rates with HDI and EI.

A chronicle of the liberation struggle: the career development of Ahmed Kathrada.

Ahmed Kathrada (1929-2017), an anti-apartheid activist, political prisoner, and parliamentary politician had been at the centre of South Africa's liberation struggle for six decades and is remembered as a soft-spoken, strong-willed and incorruptible freedom fighter. This psychobiography uses the career development framework of Jeffrey Greenhaus and the ecological human development systems framework proposed by Urie Bronfenbrenner to investigate and illuminate Kathrada's life history. Kathrada was purposively selected as subject due to his extraordinary contributions to the liberation struggle. The study used primary and secondary documentary data. The findings indicate poor alignment between the age categories proposed by the Greenhaus framework and Kathrada's career development stages due to him joining the liberation movement at a very young age, his rapid career development during the early career stage and the impact of imprisonment on his career development. The findings relating to Bronfenbrenner's theoretical framework illustrate the interaction between individual and environmental factors that influenced Kathrada's life and career. Ultimately, employing two theoretical frameworks assisted in formulating a pragmatic, holistic and comprehensive psychobiography that illuminates Kathrada's life and career. The study contributes to the legacy of Kathrada and underscores the importance of longitudinal studies in the career development of extraordinary individuals.