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Embryonic cells grow in environments that provide a plethora of physical cues, including mechanical forces that shape the development of the entire embryo. Despite their prevalence, the role of these forces in embryonic development and their integration with chemical signals have been mostly neglected, and scrutiny in modern molecular embryology tilted, instead, towards the dissection of molecular pathways involved in cell fate determination and patterning. It is now possible to investigate how mechanical signals induce downstream genetic regulatory networks to regulate key developmental processes in the embryo. Here, we review the insights into mechanical control of early vertebrate development, including the role of forces in tissue patterning and embryonic axis formation. We also highlight recent in vitro approaches using individual embryonic stem cells and self-organizing multicellular models of human embryos, which have been instrumental in expanding our understanding of how mechanics tune cell fate and cellular rearrangements during human embryonic development. Cells in the embryo are subject to autonomous and external mechanical forces that help steer embryonic tissue patterning. Technical developments, such as in vitro models of early embryos, allow probing of the roles of mechanical forces in animal and human embryonic development.

Cellular dynamics and fate decision in early human embryogenesis remain largely unknown owing to the challenges of performing studies in human embryos1. Here, we explored whole-genomes of 334 single-cell colonies and targeted deep sequences of 379 bulk tissues obtained from various anatomical locations of seven recently deceased adult human donors. Using somatic mutations as an intrinsic barcode, we reconstructed early cellular phylogenies that demonstrate (1) an endogenous mutational rate that is higher in the first cell division but decreases to approximately one per cell per cell division later in life; (2) universal unequal contribution of early cells to embryo proper, resulting from early cellular bottlenecks that stochastically set aside epiblast cells within the embryo; (3) examples of varying degrees of early clonal imbalances between tissues on the left and right sides of the body, different germ layers and specific anatomical parts and organs; (4) emergence of a few ancestral cells that will substantially contribute to adult cell pools in blood and liver; and (5) presence of mitochondrial DNA heteroplasmy in the fertilized egg. Our approach also provides insights into the age-related mutational processes and loss of sex chromosomes in normal somatic cells. In sum, this study provides a foundation for future studies to complete cellular phylogenies in human embryogenesis. Adult human tissues from diverse sites around the body are used to reconstruct cellular phylogenies from early development, using somatic mutations as an internal barcode.

Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest- and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.

Limited access to embryos has hampered the study of human embryogenesis and disorders that occur during early pregnancy. Human pluripotent stem cells provide an alternative means to study human development in a dish1–7. Recent advances in partial embryo models derived from human pluripotent stem cells have enabled human development to be examined at early post-implantation stages8–14. However, models of the pre-implantation human blastocyst are lacking. Starting from naive human pluripotent stem cells, here we developed an effective three-dimensional culture strategy with successive lineage differentiation and self-organization to generate blastocyst-like structures in vitro. These structures—which we term ‘human blastoids’—resemble human blastocysts in terms of their morphology, size, cell number, and composition and allocation of different cell lineages. Single-cell RNA-sequencing analyses also reveal the transcriptomic similarity of blastoids to blastocysts. Human blastoids are amenable to embryonic and extra-embryonic stem cell derivation and can further develop into peri-implantation embryo-like structures in vitro. Using chemical perturbations, we show that specific isozymes of protein kinase C have a critical function in the formation of the blastoid cavity. Human blastoids provide a readily accessible, scalable, versatile and perturbable alternative to blastocysts for studying early human development, understanding early pregnancy loss and gaining insights into early developmental defects. An in vitro culture strategy enables the generation of blastocyst-like structures termed human blastoids from naive human pluripotent stem cells, providing a model for studying human embryogenesis.

Early post-implantation human embryonic development has been challenging to study due to both technical limitations and ethical restrictions. Proper modeling of the process is important for infertility and toxicology research. Here we provide details of the design and implementation of a microfluidic device that can be used to model human embryo development. The microfluidic human embryo model is established from human pluripotent stem cells (hPSCs), and the resulting structures exhibit molecular and cellular features resembling the progressive development of the early post-implantation human embryo. The compartmentalized configuration of the microfluidic device allows the formation of spherical hPSC clusters in prescribed locations in the device, enabling the two opposite regions of each hPSC cluster to be exposed to two different exogenous chemical environments. Under such asymmetrical chemical conditions, several early post-implantation human embryo developmental landmarks, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and gastrulating cells (or mesendoderm cells), can be robustly recapitulated using the microfluidic device. The microfluidic human embryo model is compatible with high-throughput studies, live imaging, immunofluorescence staining, fluorescent in situ hybridization, and single-cell sequencing. This protocol takes ~5 d to complete, including microfluidic device fabrication (2 d), cell seeding (1 d), and progressive development of the microfluidic model until gastrulation-like events occur (1–2 d). Human pluripotent stem cells are seeded in a microfluidic device to establish a model that resembles the progressive development of the early post-implantation human embryo.

Macrophages are the first cells of the nascent immune system to emerge during embryonic development. In mice, embryonic macrophages infiltrate developing organs, where they differentiate symbiotically into tissue-resident macrophages (TRMs)1. However, our understanding of the origins and specialization of macrophages in human embryos is limited. Here we isolated CD45+ haematopoietic cells from human embryos at Carnegie stages 11 to 23 and subjected them to transcriptomic profiling by single-cell RNA sequencing, followed by functional characterization of a population of CD45+CD34+CD44+ yolk sac-derived myeloid-biased progenitors (YSMPs) by single-cell culture. We also mapped macrophage heterogeneity across multiple anatomical sites and identified diverse subsets, including various types of embryonic TRM (in the head, liver, lung and skin). We further traced the specification trajectories of TRMs from either yolk sac-derived primitive macrophages or YSMP-derived embryonic liver monocytes using both transcriptomic and developmental staging information, with a focus on microglia. Finally, we evaluated the molecular similarities between embryonic TRMs and their adult counterparts. Our data represent a comprehensive characterization of the spatiotemporal dynamics of early macrophage development during human embryogenesis, providing a reference for future studies of the development and function of human TRMs. Single-cell RNA sequencing of haematopoietic cells from human embryos at different developmental stages sheds light on the development and specification of macrophages in different tissues.

Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available two-dimensional technological platforms1,2 that do not recapitulate the in vivo conditions3–5. Here we report a three-dimensional (3D) blastocyst-culture system that enables human blastocyst development up to the primitive streak anlage stage. These 3D embryos mimic developmental landmarks and 3D architectures in vivo, including the embryonic disc, amnion, basement membrane, primary and primate unique secondary yolk sac, formation of anterior–posterior polarity and primitive streak anlage. Using single-cell transcriptome profiling, we delineate ontology and regulatory networks that underlie the segregation of epiblast, primitive endoderm and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique and characteristic phenotypes. After implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to pluripotency upon implantation, and the transcriptome of these cells is maintained until the generation of the primitive streak anlage. These developmental processes are driven by different pluripotency factors. Together, findings from our 3D-culture approach help to determine the molecular and morphogenetic developmental landscape that occurs during human embryogenesis. A 3D culture system to model human embryonic development, together with single-cell transcriptome profiling, provides insights into the molecular developmental landscape during human post-implantation embryogenesis.

In the interphase of the cell cycle, chromatin is arranged in a hierarchical structure within the nucleus1,2, which has an important role in regulating gene expression3-6. However, the dynamics of 3D chromatin structure during human embryogenesis remains unknown. Here we report that, unlike mouse sperm, human sperm cells do not express the chromatin regulator CTCF and their chromatin does not contain topologically associating domains (TADs). Following human fertilization, TAD structure is gradually established during embryonic development. In addition, A/B compartmentalization is lost in human embryos at the 2-cell stage and is re-established during embryogenesis. Notably, blocking zygotic genome activation (ZGA) can inhibit TAD establishment in human embryos but not in mouse or Drosophila. Of note, CTCF is expressed at very low levels before ZGA, and is then highly expressed at the ZGA stage when TADs are observed. TAD organization is significantly reduced in CTCF knockdown embryos, suggesting that TAD establishment during ZGA in human embryos requires CTCF expression. Our results indicate that CTCF has a key role in the establishment of 3D chromatin structure during human embryogenesis.

Understanding human development is of fundamental biological and clinical importance. Despite its significance, mechanisms behind human embryogenesis remain largely unknown. Here, we attempt to model human early embryo development with expanded pluripotent stem cells (EPSCs) in 3-dimensions. We define a protocol that allows us to generate self-organizing cystic structures from human EPSCs that display some hallmarks of human early embryogenesis. These structures mimic polarization and cavitation characteristic of pre-implantation development leading to blastocyst morphology formation and the transition to post-implantation-like organization upon extended culture. Single-cell RNA sequencing of these structures reveals subsets of cells bearing some resemblance to epiblast, hypoblast and trophectoderm lineages. Nevertheless, significant divergences from natural blastocysts persist in some key markers, and signalling pathways point towards ways in which morphology and transcriptional-level cell identities may diverge in stem cell models of the embryo. Thus, this stem cell platform provides insights into the design of stem cell models of embryogenesis., Human early development remains largely inaccessible, owing to technical and ethical limitations of working with natural embryos. Here the authors assess the extent to which human expanded pluripotent stem cells can specify distinct cell lineages and capture aspects of early human embryogenesis.

Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development., Single cell analysis of early human embryos identifies key changes in pluripotency, the requirement of FGF signalling for embryo survival, and defines a putative anterior-like region of hypoblast cells, providing insights into how early human development is regulated.

Microglia play an essential role for central nervous system (CNS) development and homeostasis and have been implicated in the onset, progression, and clearance of numerous diseases affecting the CNS. Previous in vitro research on human microglia was restricted to post-mortem brain tissue-derived microglia, with limited availability and lack of scalability. Recently, the first protocols for the generation of microglia from human pluripotent stem cells have become available, thus enabling the implementation of powerful platforms for disease modeling, drug testing, and studies on cell transplantation. Here we give a detailed and comprehensive overview of the protocols available for generating microglia from human pluripotent stem cells, highlighting the advantages, drawbacks, and operability and placing them into the context of current knowledge of human embryonic development. We review novel insights into microglia biology and the role of microglia in neurological diseases as drawn from the new methods and provide an outlook for future lines of research involving human pluripotent stem cell-derived microglia.

MicroRNAs (miRNAs) are small non-coding RNA molecules that participate in transcriptional and post-transcriptional regulation of gene expression. miRNAs have numerous roles in cellular function including embryonic development. Human embryonic stem cells (hESCs) are capable of self-renewal and can differentiate into most of cell types including cardiomyocytes (CMs). These characteristics of hESCs make them considered as an important model for studying human embryonic development and tissue specific differentiation. In this study, we tried to demonstrate the profile of miRNA expression in cardiac differentiation from hESCs. To induce differentiation, we differentiated hESCs into CMs by direct differentiation method and characterized differentiated cells. To analyze the expression of miRNAs, we distinguished (days 4, 8, 12, 16, 20, 24, 28) and isolated RNAs from each differentiation stage. miRNA specific RT-qPCR was performed and the expression profile of miR-1, -30d, -133a, -143, -145, -378a, -499a was evaluated. The expression of all miRs was up-regulated at day 8. miR-143 and -145 expression was also up-regulated at the later stage of differentiation. Only miR-378a expression returned to undifferentiated hESC levels at the other stages of differentiation. In conclusion, we elucidated the expression profile of miRNAs during differentiation into cardiomyocytes from hESCs. Our findings demonstrate the expression of miRNAs was stage-dependent during differentiation and suggest that the differentiation into CMs can be regulated by miRNAs through direct or indirect pathway.

A 3D culture system recapitulates human development prior to gastrulation and enables transcriptomic studies.

In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis, but the underlying processes are poorly understood and controlled. Here we show that modulating the bulk cell density (BCD: cell number per culture volume) deterministically alters anteroposterior patterning of primitive streak (PS)-like priming. The BCD in conjunction with the chemical WNT pathway activator CHIR99021 results in distinct paracrine microenvironments codifying hPSCs towards definitive endoderm, precardiac or presomitic mesoderm within the first 24 h of differentiation, respectively. Global gene expression and secretome analysis reveals that TGFß superfamily members, antagonist of Nodal signalling LEFTY1 and CER1, are paracrine determinants restricting PS progression. These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. By demonstrating a decisive, functional role of the BCD, we show its utility as a method to control lineage-specific differentiation. Furthermore, these findings have profound consequences for inter-experimental comparability, reproducibility, bioprocess optimization and scale-up., Differentiation of human pluripotent stem cells (hPSC) mimics aspects of embryonic development in vitro but is poorly understood. Here, the authors identify bulk cell density as a key parameter directing transition from pluripotency to primitive streak-like priming in hPSCs via secreted LEFTY/CERBERUS.

Neurogenesis is the developmental process regulating cell proliferation of neural stem cells, determining their differentiation into glial and neuronal cells, and orchestrating their organization into finely regulated functional networks. Can this complex process be recapitulated in vitro using induced pluripotent stem cell (iPSC) technology? Can neurodevelopmental and neurodegenerative diseases be modeled using iPSCs? What is the potential of iPSC technology in neurobiology? What are the recent advances in the field of neurological diseases? Since the applications of iPSCs in neurobiology are based on the capacity to regulate in vitro differentiation of human iPSCs into different neuronal subtypes and glial cells, and the possibility of obtaining iPSC-derived neurons and glial cells is based on and hindered by our poor understanding of human embryonic development, we reviewed current knowledge on in vitro neural differentiation from a developmental and cellular biology perspective. We highlight the importance to further advance our understanding on the mechanisms controlling in vivo neurogenesis in order to efficiently guide neurogenesis in vitro for cell modeling and therapeutical applications of iPSCs technology.

The trophoblast cell lineage is specified early at the blastocyst stage, leading to the emergence of the trophectoderm and the pluripotent cells of the inner cell mass. Using a double mRNA amplification technique and a comparison with transcriptome data on pluripotent stem cells, placenta, germinal and adult tissues, we report here some essential molecular features of the human mural trophectoderm. In addition to genes known for their role in placenta (CGA, PGF, ALPPL2 and ABCG2), human trophectoderm also strongly expressed Laminins, such as LAMA1, and the GAGE Cancer/Testis genes. The very high level of ABCG2 expression in trophectoderm, 7.9-fold higher than in placenta, suggests a major role of this gene in shielding the very early embryo from xenobiotics. Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression. A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta. The induction of this network could be recapitulated in an in vitro trophoblast differentiation model. Electronic supplementary material The online version of this article (doi:10.1007/s12015-011-9301-3) contains supplementary material, which is available to authorized users.

Chromosome instability is a hallmark of tumorigenesis. This study establishes that chromosome instability is also common during early human embryogenesis. A new array-based method allowed screening of genome-wide copy number and loss of heterozygosity in single cells. This revealed not only mosaicism for whole-chromosome aneuploidies and uniparental disomies in most cleavage-stage embryos but also frequent segmental deletions, duplications and amplifications that were reciprocal in sister blastomeres, implying the occurrence of breakage-fusion-bridge cycles. This explains the low human fecundity and identifies post-zygotic chromosome instability as a leading cause of constitutional chromosomal disorders.

Primitive carotid–vertebral and carotid–basilar anastomoses are formed early during human embryogenesis at approximately 24 days. From cephalic to caudal direction, these anastomoses are cranial extensions of the primitive internal carotid, trigeminal, otic, hypoglossal and proatlantal intersegmental arteries. Normal and/or abnormal morphofunctional aspects of prenatal and postnatal forms of the proatlantal intersegmental artery, from the 24th day of gestation to postnatal eight decades, are described according to personal and literature data. Many (ab) normal carotid–vertebral anastomoses are also marked in differential diagnosis of the proatlantal intersegmental artery. The proatlantal intersegmental artery maintains the posterior circulation until the vertebral arteries are fully developed between the seventh and eighth gestational weeks. When this artery fails to obliterate, it becomes persistent one. The proatlantal intersegmental artery, most commonly, is an incidental finding or it may be of clinical significance in some patients.

Human embryonic stem cells (HESC) are pluripotent stem cell lines derived from the inner cell mass (ICM) of human blastocyst-stage embryos. They are characterized by their unlimited capacity to self-renew in culture. In addition, they have a broad developmental potential, as demonstrated by their ability to form practically any cell type in vivo and in vitro. These two features have made HESC extremely important in basic and applied research. In addition, they may serve as a powerful tool for studying human development. HESC can recapitulate embryogenesis by expressing developmentally regulated genes and by activating molecular pathways as they occur in vivo. Moreover, they can be used to analyze the effect of specific mutations on particular developmental events and may enable us to identify critical factors that play a role in the processes of cell commitment, differentiation, and adult cell reprogramming. Thus, modeling human embryogenesis by the use of HESC may allow new insights into developmental processes, which would otherwise be inaccessible for research.

The collagens constitute a large group of proteins in the extracellular matrix that can be divided into several distinct families. Collagen types XII and XIV belong to a subgroup of non-fibrillar-collagens termed (fibril-associated collagens with interrupted triple-helices) (FACIT) and may be involved in basement membrane regulation providing specific molecular bridges between fibrils and other matrix components. However, the tissue distribution of the two proteins during human embryogenesis is still unclear. As a first step toward the elucidation of their possible cell biological functions, we compared the distribution of the two collagens during human organogenesis at the light microscopical level. We detected specific differences between the expression patterns of the two molecules, which may be related to their respective function within the basement membrane zones during human embryonic development. For example, in the developing intestine, collagen type-XII was present in the basement membrane zones of epithelia and endothelia. However, collagen type-XIV was restricted to the mesothelial basement membrane zones. We conclude that both collagens might well be able to serve different functions during human embryonic development although their structures are highly similar.

NOV, located on human chromosome 8q24.1, was originally cloned following discovery of its avian homolog as a consequence of over-expression in virally induced nephroblastoma. The gene product is a secreted, modular, protein and a member of the CCN gene family. Evidence to date indicates that the expression of the wild type protein is associated with cellular quiescence in normal embryonic fibroblasts yet produces growth stimulatory effects on established murine NIH 3T3 cells. Here we report the expression of NOV in the first trimester of human embryogenesis, between 5 and 10 weeks. In situ hybridisation and immunohistochemistry reveal widespread expression in derivatives of all three germ layers. The most abundant sites of expression are in the motor neurons and floor plate of the spinal cord, adrenal cortex, fusing skeletal, and smooth muscle, the urogenital system and the developing heart. Additionally, expression is seen in the cranial ganglia, differentiating chondrocytes, gonads, and lung. The sites of expression suggest strongly that autocrine or paracrine expression of NOV is associated with the process of cell differentiation.

The recently identified nidogen-2 is a matrix protein showing homology to the well-known basement membrane molecule nidogen-1. Nidogen-1 might well serve as a link between laminin-1 and collagen type IV and thus stabilise certain basement membranes in vivo and play a major role in embryogenesis. However, the exact tissue distribution of nidogen-1 and nidogen-2 during human embryogenesis is still unclear. As a first step towards the elucidation of their possible cell biological functions during human development, we compared the distribution of both nidogens during human organogenesis at the light microscope level. Nidogen-2 and nidogen-1 were found to be ubiquitous components of basement membrane zones underneath developing epithelia of most of the major organ systems. However, in the developing intestine and the pancreas anlage, only nidogen-1 was present in the epithelial basement membrane zones of all developmental stages investigated. Our data suggest that nidogen-2 and nidogen-1, as is known for mouse development, could well participate in cell biological functions during human development. These two proteins might well be able to fulfil identical functions during human organogenesis.

The endocrine function of the thymus develops earlier than lymphocytopoietic. Thymalin is produced by epithelial cells in the thymus primordium. It is released into the blood and regulates differentiation of T lymphocytes in the liver, the initial hemopoietic organ. The hormonal and lymphopoietic functions of human thymus are united on weeks 7.5-8 of embryonic life.

Organogenesis occurs during the first 8 weeks of human embryonic development; in consequence, early human growth and development take place before and in the absence of fully developed internal organs. During this period, normal development depends on several factors, but two are imperative: nutrition and a functional transport system for the distribution of nutrients and for waste disposal. The yolk sac (YS), a highly differentiated adnexal organ, is known to accomplish this fundamental task during early pregnancy. In this review, we summarize our contribution to the understanding of early human embryology, focusing interest on analysis of the morphofunctional link that is established between the human embryo and the YS during the embryonic period. Embryos were collected from the gestational sac after salpingectomies performed on patients with singleton pregnancies occurring in the fallopian tube. Samples of YS were taken from 20 human embryos at Carnegie stages ranging from 12 to 20. The age of the embryo was estimated from data of the patient's last menstrual history and confirmed from crown-rump length measurements and morphological characteristics of the specimen. The samples were fixed in 3% glutaraldehyde and then postfixed in 2% osmium tetroxide and prepared for light, transmission, and scanning electron microscopy according to conventional techniques. The samples were examined with a Philips 301 EM and an S-4000 Hitachi field emission SEM. The yolk stalk, and the YS wall with its corresponding endodermal, mesenchymal, and mesothelial layers, were analyzed. In accordance with their morphological features, the endodermal cells are equipped with organelles to fulfill several functions that are expressed in absorption from the vitelline cavity via microvilli present into the outer cell surface, in secretion to the extracellular space, and in the synthesis of numerous proteins which are transported by the bloodstream to the embryo. The mesothelial surface is provided with cell-surface differentiation that promotes a protective coat to prevent damage from compression or friction of the YS wall against the amnios, umbilical cord, and chorionic cavity wall during growth. The mesenchyme is the main site for blood vessel formation and gives rise to a network that provides the embryo with nutrients and a means of waste disposal. A critical analysis of the role of the endodermal vesicle in the production of fluid that is accumulated in the YS, and of the role that the vitelline duct play in the exchange function between the YS and intestinal tract, is presented. We have demonstrated that the vitelline duct is not functional after week 5 because of the closure of its lumen. This finding is discussed with reference to the biological meaning of the vitelline duct and its functional period of activity, and its possible role in the physiology of exchange during the embryonic period is assessed.

Purpose : After noticing specific aberrant forms of development in preimplantation embryos from women with endometriosis, we embarked upon this study in order to examine the frequency of these events as compared with controls. Methods : A total of 235 embryos representing 30 women undergoing 56 cycles of in vitro fertilization were retrospectively analyzed on videotape and placed into categories based upon the incidence of specific morphological criteria found during observations made on the first and second days following oocyte retrieval. Results : Several of the aberrant nuclear and cytoplasmic events were found to be statistically increased in the group of embryos from women with endometriosis. Conclusions : The incidence of defined aberrant phenotypes in embryos from women with laparoscopically documented endometriosis may allow us to identify specific embryotoxic events with reference to endometriosis and other diagnoses.

Programmed cell death is a physiological process in mammalian development by which specific types of cells are eliminated, and, hence, is of fundamental importance in normal human embryogenesis. A patient is described with multiple congenital anomalies that may be explained by a disturbance of programmed cell death. Anomalies included macrocephaly, hypoplastic lacrimal ducts, narrow external ear canals, pharyngeal mucous membrane fold, unilateral cryptorchidism, cord-like vasa deferentia, and complete cutaneous syndactyly of the hands and feet.

The phenomenon of parental imprinting involves the preferential expression of one parental allele of a subset of chromosomal genes and has so far only been documented in the mouse. We show here, by exploiting sequence polymorphisms in exon nine of the human insulin-like growth factor 2 (IGF2) gene, that only the paternally-inherited allele is active in embryonic and extra-embryonic cells from first trimester pregnancies. In addition, only the paternal allele is expressed in tissues from a patient who suffered from Beckwith-Wiedemann syndrome. Thus the parental imprinting of IGF2 appears to be evolutionarily conserved from mouse to man and has implications for the generation of the Beckwith-Wiedemann syndrome.

Division of the human zygote leads to the formation of the blastocyst that contains human embryonic stem cells (hESC) which develop into the embryo. Little is known about the physiological signals that direct hESC division and differentiation during early embryogenesis. A number of growth factors, including the pregnancy-associated hormone human chorionic gonadotropin (hCG), are secreted by trophoblasts^1-3^ that lie adjacent to the embryoblast in the blastocyst, but it is not known whether these growth factors directly signal the epiblast. Here we show that hCG promotes the division of embryoblast-derived inner mass cells (hESC), and their differentiation during blastulation and neurulation. Inhibition of LH/hCG receptor (LHCGR) signaling with P-antisense oligonucleotides suppresses hESC proliferation. Similarly, hESC proliferation can be blocked using an antibody against the extracellular activation site of LHCGR, an effect that is reversed by treatment with hCG. hCG treatment rapidly upregulates steroidogenic acute regulatory protein-mediated cholesterol transport and the synthesis of progesterone, a neurogenic steroid^4,5^. Treatment of hESC colonies with progesterone induces neurulation as demonstrated by the expression of nestin and the formation of columnar neuroectodermal cells that organize into neural tube-like rosettes. Suppression of progesterone signaling by withdrawing progesterone or treating with the progesterone receptor antagonist RU-486 inhibits the differentiation of hESC colonies into embryoid bodies (blastulation) and rosettes (neurulation). These results explain the default pathway of hESC differentiation towards a neural stem cell fate in vitro. Collectively, our findings implicate trophoblastic hCG secretion and signaling via LHCGR on the adjacent embryoblast in the induction of hESC proliferation and differentiation into blastocysts and neurula. This paracrine/juxtacrine signaling by extraembryonic tissues is the commencement of trophic support by placental tissues in the growth and development of the human embryo.

The commitment of cells to the inner cell mass or trophectoderm in preimplantation blastocysts is the first segregation of cell lineages in the developing human embryo. The exogenous signals that control the fate determination between inner cell mass and trophectoderm remain obscure. Human embryonic stem cells (hESCs) provide a new model for the study of human developmental biology. We previously showed that TGF-/Activin/ Nodal signaling maintains the pluripotency of hESCs, which are derivatives of the inner cell mass of human blastocyst. Here we further demonstrate that TGF-/activin/nodal signaling also regulate the fate of hESCs. Our data also suggests the TGF-/activin/nodal signaling be the key switcher in the fate determination of cells at early stage of human embryonic development.

Murine teratocarcinoma models have been widely used in the study of several biological processes including early mammalian development and neoplasia. The relatively new human germ-cell tumor models have an important role in the study of human embryogenesis and neoplasia since human embryogenesis differs significantly from species such as mice and the histopathology of human testicular germ-cell tumors differs substantially from tumors obtained in highly inbred strains of mice. In this report we describe the use of human germ-cell lines in the study of cell differentiation; expression of early embryonic antigens; production of the major regulator of erythropoiesis, erythropoietin; and production of retrovirus-like particles.

The purpose of the present investigation is the study of the ultrastructural regularities of the formation, establishment, and development of the microcirculatory pathways in the prenatal period of human morphogenesis. Material and Methodology. Human embryos and fetuses of 4-24 weeks of development served as the experimental objects. The liver, the thyroid and thymus glands, the testes, the ovaries, skeletal muscle, the small intestine, the spleen, peritoneal lymph nodes, and the adenohypophysis were studied. The material was fixed by immersion in a 2.5% solution of glutaraldehyde in phosphate buffer (pH 7.4) for 1.5-2 h at room temperature, and was then treated in a 1% solution of osmium tetroxide for 40-50 min, washed in four portions of phosphate buffer for i0 min each, dehydrated in alcohols of increasing concentration, and embedded in Epon resin. Semithin and ultrathin sections were prepared on an LKB-8800 ultratomeo The semithin sections were stained with cresyl violet or toluidine blue. The ultrathin sections were contrasted with uranyl acetate and lead citrate, and then were studied and photographed in an UEMV-100 AK electron microscope, Results of Investisation and Discussion. At the early stages or organogenesis, the interstitial fluid moves through the intercellular channels and clefts. In the structural organization of the prevascular microcirculatory system, two components can be distinguished: interstitial compartments of varied magnitude which are separated by the contacting processes of the cells of the mesenchyme (Fig. i, a), and the intracellular channels and clefts between the specific working cells of the organ (Fig. i, b). The latter type of organization of the ultracirculatory pathways is maintained over the course of the entire prenatal ontogenesis. Thus, a compartmentalization of the structural organization of the pathways of the prevascular microcirculation are already observed at the early stages of embryonal development. At the early stages of human intrauterine development, the mesenchyme is represented by process-bearing cells, among which two types can be distinguished: stellate and spindleshaped. The stellate cells contain a round or an oval-shaped nucleus with an eccentrically placed nucleolus. The chromatin is uniformly distributed throughaut the entire volume of the karyoplasm. The nucleus is surrounded by a narrow rim of cytoplasm in which an insignificant number of organelles, single granules of glycogen, and individual drops of lipid can be discerned. There are processes in these cells of varied Size and thickness, by means of which they make contact with one another. The spindle-shaped cells are characterized by an elongated form; the nucleus is extended along the long axis of the cell. A marginal condensation of chromatin is noted. The nucleolus is located eccentrically. The nucleus is surrounde d by a narrow rim of cytoplasm; the organelles are better developed than in the stellate cells. Larger mitochondria are discerned in the cytoplasm, as well as a well-developed

The period for which oocyte-derived factors are engaged in the control of human embryonic development involves at least the first four cell cycles after fertilization. The maternal-embryonic transition in humans 8- to 16-cell embryos is a relatively vulnerable process, the failure of which entails developmental arrest of the given blastomere. The very early cellular differentiative events in human embryos, including blastomere surface polarization and segregation of the inner cell mass and trophectoderm cell lineages, appear to be dependent largely on the maternal genetic program. However, the embryonic genome is required for the formation of the blastocyst cavity, which is necessary to allow further differentiation of the first two embryonic tissues. Blastomeres with major developmental defects are removed by fragmentation and their loss is compensated by proliferation of remaining normal blastomeres. This mechanism is also mainly responsible for the regulation of ploidy through elimination of aneuploid blastomeres. The data presented suggest that embryos of individual mammalian species may differ in the timing of relevant developmental changes at the cellular and molecular levels. This should be taken into account when findings obtained on embryos of one species are used to anticipate the behavior of embryos of another species under identical conditions.

Thymocytes of human embryos at the 7–8-week stage have irregularly shaped nuclei with one to three distinct nucleoli, characterized by absence of compact chromatin or heterochromatin. The electron-dense cytoplasm of these cells contains polysomes and a few mitochondria. Receptors for sheep's red blood cells and T-antigen are absent on the surface of the cells. In 11–12-week human embryos, the T-lymphocytes are reduced in size, clumps of heterochromatin appear in their nuclei and receptors for sheep's red blood cells (79%) and T-antigen (60%) appear on the surface of the cells. Later, the quantity of compact chromatin in the nuclei of the thermocytes increases and the cells acquire their definitive properties and structure.

1. The anlage of the muscle spindle is the “spindle nerve fiber complex.” It can be found in some muscles of a 6.5-week human embryo, but more regularly in the 7–8-week embryo. 2. Initial signs of differentiation of intrafusal elements, marked by the appearance of a nuclear bag in the equatorial region of the future spindle, are observed in the rectus abdominis, triceps brachii and other muscles in the 9-week human fetus, i.e., 3 weeks sooner than the earliest time recorded in the literature (Cuajunco, 1940). 3. A period of one week from the beginning of differentiation of the intrafusal bundle is needed for differentiation of all the component parts of the spindle to begin. Formation of the spindle capsule as a stratified sheath and formation of the perilymphatic space occur later in the course of human embryogenesis. 4. The muscle spindles of the human fetus are supplied with afferent endings of the flower-spray type. 5. Asynchronism is observed in the order of appearance and rate of differentiation of the muscle spindles in muscles serving different functions.

In order to determine the correlations between the activities of the thymus and adrenal glands, a study of the periods of hormone activity was made on 155 fetuses, 9–40 weeks old. The activity of alkaline phosphatases in the intestinal epithelium of the fetus served as a test of the corticoid level in the fetal blood. Small amounts of corticoids appeared in the fetal blood for the first time after the 11–13th week, the amount practically increasing to the 20th week. From the 21–40th weeks, i.e., just before birth, the corticoid content of the blood in both series reached considerable proportions. There is no correlation between the development of the thymus gland and the appearance of corticoids in the fetal blood.

1. Production of FSH and LH by the human embryonic pituitary in vitro has been studied during the action of hypothalamic or pineal tissue on the pituitary in 125 fetuses after 17 to 40 weeks of intrauterine development. 2. The pituitary of female fetuses can secrete FSH and LH spontaneously into the medium only between the 19th and 29th week of embryogenesis; the pituitary of the male fetus can secrete only FSH spontaneously into the medium, and only between the 24th and 29th week of development. 3. Factors stimulating FSH secretion by the pituitary are found in the hypothalamus of fetuses of both seses. No sex difference in the level of their activity has been established, but the period during which they are found depends on the sex of the fetus: in female fetuses these factors are found from the 19th to the 29th week, but in male fetuses from the 24th to the 29th week of development. 4. Under experimental conditions in vitro, addition of pineal gland was a factor evoking sex differences in the level of FSH secretion: from female fetuses it stimulates secretion, while pineal gland from male fetuses inhibits it (except, perhaps, for the period between the 24th and 26th weeks of development of the male fetus). 5. In the hypothalamus of fetuses of either sex factors weakly stimulating secretion of LH by the pituitary are found. No sex differences exist in the level of their, activity, but the duration of the period when they are found is as dependent on sex of the fetus as in the case of, FSH. These factors are present in female fetuses from the 19th to the 29th week, and in male fetuses from the 24th to the 29th week of development. 6. Sex differences in the level of LH secretion are due to the appearance of reserpine-sensitive factors, activitly stimulating LH production, in the hypothalamus only of female fetuses, after the 20th week of development.

A method of cultivating and fertilizing human ova obtained from ovaries removed at operation is described. A special method of sampling active spermatozoa was used for fertilization. Zygote formation was observed 24–36 h after fertilization of the ripe ova, an embryo of the two-cell stage was formed after 36–40 h, and an embryo at the four-cell stage after 48 h. The number of blastomeres reached 8 after 60–64 h, and after approximately 100–120 h the embryos reached the morula stage. The results provide wide scope for the study of the physiology and pathology of early human embryonic development.