The development of early human lymphatic vessels as characterized by lymphatic endothelial markers.

Lymphatic vessel development studies in mice and zebrafish models have demonstrated that lymphatic endothelial cells (LECs) predominantly differentiate from venous endothelial cells via the expression of the transcription factor Prox1. However, LECs can also be generated from undifferentiated mesoderm, suggesting potential diversity in their precursor cell origins depending on the organ or anatomical location. Despite these advances, recapitulating human lymphatic malformations in animal models has been difficult, and considering lymphatic vasculature function varies widely between species, analysis of development directly in humans is needed. Here, we examined early lymphatic development in humans by analyzing the histology of 31 embryos and three 9-week-old fetuses. We found that human embryonic cardinal veins, which converged to form initial lymph sacs, produce Prox1-expressing LECs. Furthermore, we describe the lymphatic vessel development in various organs and observe organ-specific differences. These characterizations of the early development of human lymphatic vessels should help to better understand the evolution and phylogenetic relationships of lymphatic systems, and their roles in human disease.

Generation of Human Blastoids from Naive Pluripotent Stem Cells.

Under certain culture conditions, naive human pluripotent stem cells can generate human blastocyst-like structures (called human blastoids). Human blastoids serve as an accessible model for human blastocysts and are amenable for large-scale production. Here, we describe a detailed step-by-step protocol for the robust and high-efficient generation of human blastoids from naive human pluripotent stem cells.

Identification and characterization of human hematopoietic mesoderm.

The embryonic mesoderm comprises heterogeneous cell subpopulations with distinct lineage biases. It is unclear whether a bias for the human hematopoietic lineage emerges at this early developmental stage. In this study, we integrated single-cell transcriptomic analyses of human mesoderm cells from embryonic stem cells and embryos, enabling us to identify and define the molecular features of human hematopoietic mesoderm (HM) cells biased towards hematopoietic lineages. We discovered that BMP4 plays an essential role in HM specification and can serve as a marker for HM cells. Mechanistically, BMP4 acts as a downstream target of HDAC1, which modulates the expression of BMP4 by deacetylating its enhancer. Inhibition of HDAC significantly enhances HM specification and promotes subsequent hematopoietic cell differentiation. In conclusion, our study identifies human HM cells and describes new mechanisms for human hematopoietic development.

Lung fusion with the liver in chick and quail embryos.

Even in mammals with the diaphragm, the lung and liver are likely to attach mutually without separation by any structure in embryos. The aim of this study was to examine whether or not the lung attaches to the liver in embryonic development of birds without diaphragm. First, we ensured the topographical relation between the lung and liver in 12 human embryos at 5 weeks. After the serosal mesothelium was established, the human lung sometimes (3 embryos) attached tightly to the liver without interruption by the developing diaphragm in the pleuroperitoneal fold. Second, we observed the lung-liver interface in chick and quail embryos. At 3-5 days' incubation (stages 20-27), the lung and liver were fused at bilateral narrow areas just above the muscular stomach. Therein, mesenchymal cells, possibly derived from the transverse septum, were intermingled between the lung and liver. The interface tended to be larger in the quail than the chick. At and until 7 days' incubation, the fusion of the lung and liver disappeared and, instead, a membrane connected them bilaterally. The right membrane extended caudally to attach to the mesonephros and caudal vena cava. At 12 days' incubation, bilateral thick folds, containing the abdominal air sac and pleuroperitoneal muscle (striated muscle), separated the dorsally located lung from the liver. Therefore, the lung-liver fusion occurred transiently in birds. Rather than the presence of the muscular diaphragm, whether the lung and liver were fused seemed to depend on a timing and sequence of development of the mesothelial coverings of these viscera.

Survey of Japanese researchers and the public regarding the culture of human embryos in vitro beyond 14 days.

The International Society for Stem Cell Research (ISSCR) has eliminated its prohibition on research involving the culturing of human embryos beyond 14 days within the updated 2021 guidelines. We conducted a survey of Japanese researchers working in stem cell- or embryo-related research (n = 535) and the public (n = 3,000) about their attitudes toward the 14-day rule. Among the researchers, 46.2% agreed that embryos could be cultured beyond 14 days, a result that was slightly lower among the public (37.9%). Among those that disagreed with embryo culturing beyond 14 days, 9.5% of researchers and 5.1% of the public agreed with culturing embryos within 14 days. Among the public, higher comprehension levels correlated with both agreement and disagreement with the culture of embryos beyond 14 days compared with "cannot judge." Further research and pubic discourse are necessary in order to better understand the factors informing participant decisions regarding the 14-day rule.

The so-called absorption process of the pulmonary vein into the left atrium of the heart: a histological study using human embryos and fetuses.

BACKGROUND: Embryonic pulmonary veins (PVs) are believed to be absorbed into the left atrium (LA) to provide an adult morphology in which "four" veins drain separately into the atrium. MATERIALS AND METHODS: Serial histological sections were obtained from 27 human embryos and fetuses. RESULTS: Between 5 and 6 weeks, the four PVs joined together to form a trunk-like structure (initial spatium pulmonalis) that was larger than the initial LA (two-ostia pattern). The cardiac nerves ran inferiorly along the posterior aspect of the four veins, as well as the spatium. At and until 7 weeks, the cardiac nerves were concentrated to elongate the nerve fold, and the latter separated the left PV trunk from the expanding LA (left spatium). Similarly, the right PV opened to a thick and deep LA recess (right spatium). At 8-12 weeks, depending on the growth of the LA, the opening of the left and right PVs became distant, and the spatium was elongated transversely. The left spatium was enlarged to open widely to the proper left atrium in contrast to the right spatium pushed anteriorly by the right atrium. The three-ostia pattern was transiently observed because of the lost delimitation between the left spatium and proper atrium. The myocardium was thin in the left spatium behind the left atrial nerve fold, whereas the right spatium was tube-like with a thick myocardium. CONCLUSIONS: The four-ostia pattern seemed to be established at birth due to a drastically increased venous return from the lung, resulting in a flat smooth left atrial posterior wall.

Morphogenetic progression of thigh and lower leg muscles during human embryonic development.

Fetal musculoskeletal movements are first observed at approximately seven to 8 weeks of gestation. However, the separation and formation of skeletal muscles, especially the limbs, have not yet been described in detail. In this study, we elucidate the sequence of events leading to the formation of each thigh and lower leg muscle using serial sections. To observe muscle formation, 26 serial sections (50 legs) of human embryonic specimens ranging from Carnegie stages (CS) 19 to 23 were selected from the Kyoto collection stored at the Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine. As a result, we show the detailed formation and separation processes of the thigh and lower leg muscles. In the thigh, sartorius and tensor fasciae latae are separated at CS19, and the individual muscles observed in adults are identified after CS21. In the lower leg, the tibialis anterior exhibits early separation at CS20, and all muscles are identified at CS22. This study enables future research into the relationship between embryonic development and the evolution of muscle action from quadrupedal to erect bipedal walking.

Human embryos donated for human embryonic stem cell derivation.

Human embryonic stem cells (hESCs), produced from human embryos, are demonstrating: utility and promise in disease modeling; enhanced and unique understanding of early events in basic genetic or molecular or cellular or epigenetic development; novel human approaches to pharmaceutical screening; pathways toward the discoveries of disease treatments and cures; and foundational importance for regenerative medicine. The regulatory landscape is rigorous, and rightly so. Here, we discuss the current US federal and state regulatory environment. A unique approach of presenting anonymized embryo donor statements is provided to personalize the decision-making process of human embryo donation for hESC derivation. From the uses of preimplantation genetic-tested and affected human embryos to derived disease-specific hESCs, one can glean the much needed information on early human genetics and developmental biology, which are presented here. Finally, we discuss the future uses of hESCs, and other pluripotent stem cells, in general and reproductive medicine.

Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells.

A hallmark of primate postimplantation embryogenesis is the specification of extraembryonic mesoderm (EXM) before gastrulation, in contrast to rodents where this tissue is formed only after gastrulation. Here, we discover that naive human pluripotent stem cells (hPSCs) are competent to differentiate into EXM cells (EXMCs). EXMCs are specified by inhibition of Nodal signaling and GSK3B, are maintained by mTOR and BMP4 signaling activity, and their transcriptome and epigenome closely resemble that of human and monkey embryo EXM. EXMCs are mesenchymal, can arise from an epiblast intermediate, and are capable of self-renewal. Thus, EXMCs arising via primate-specific specification between implantation and gastrulation can be modeled in vitro. We also find that most of the rare off-target cells within human blastoids formed by triple inhibition (Kagawa et al., 2021) correspond to EXMCs. Our study impacts our ability to model and study the molecular mechanisms of early human embryogenesis and related defects.

A Single-Cell Characterization of Human Post-implantation Embryos Cultured In Vitro Delineates Morphogenesis in Primary Syncytialization.

Implantation of the human blastocyst is a milestone event in embryonic development. The trophoblast is the first cell lineage to differentiate during implantation. Failures in trophoblast differentiation during implantation are correlated to the defects of pregnancy and embryonic growth. However, many gaps remain in the knowledge of human embryonic development, especially regarding trophoblast morphogenesis and function. Herein, we performed single-cell RNA sequencing (scRNA-seq) analysis on human post-implantation embryos cultured in vitro. A hierarchical model was established, which was characterized by the sequential development of two primitive cytotrophoblast cell (pCTB) subtypes, two primitive syncytiotrophoblast subtypes, and migrative trophoblast cells (MTB) after the trophectoderm . Further analysis characterized cytoskeleton transition of trophoblast cells and morphogenesis, such as irregular nuclei, cell cycle arrest, and cellular aging during implantation. Moreover, we found syncytialization of hTSCs could mimic the morphogenesis, serving as a powerful tool for further understanding of the mechanism during the implantation stage of pregnancy. Our work allows for the reconstruction of trophoblast cell transcriptional transition and morphogenesis during implantation and provides a valuable resource to study pathologies in early pregnancy, such as recurrent implantation failure.

When Does the Human Embryonic Heart Start Beating? A Review of Contemporary and Historical Sources of Knowledge about the Onset of Blood Circulation in Man.

The onset of embryonic heart beating may be regarded as the defining feature for the beginning of personal human life. Clarifying the timing of the first human heartbeat, therefore, has religious, philosophical, ethical, and medicolegal implications. This article reviews the historical and contemporary sources of knowledge on the beginning of human heart activity. Special attention is given to the problem of the determination of the true age of human embryos and to the problem of visualization of the human embryonic heart activity. It is shown that historical and current textbook statements about the onset of blood circulation in man do not derive from observations on living human embryos but derive from the extrapolation of observations on animal embryos to the human species. This fact does not preclude the existence of documented observations on human embryonic heart activity: Modern diagnostic (ultrasound) and therapeutic (IVF) procedures facilitate the visualization of early embryonic heart activity in precisely dated pregnancies. Such studies showed that the human heart started its pumping action during the fourth post-fertilization week. A small number of direct observations on the heart activity of aborted human embryos were reported since the 19th century, but did not receive much recognition by embryologists.

Comparison of the 2021 International Society for Stem Cell Research (ISSCR) guidelines for "laboratory-based human stem cell research, embryo research, and related research activities" and the corresponding Japanese regulations.

This paper presents a comparison of the 2021 guidelines for stem cell research and clinical translation outlined by the International Society for Stem Cell Research (ISSCR) with the current regulations in Japan regarding the performance of such research. This paper provides a convenient English-language summary of the Japanese regulations, and illustrates the difference between the ISSCR guidelines and Japanese regulations regarding the conditions of implementation of study activities using human embryos or stem cells, for researchers outside Japan. The regulations governing the performance of research activities using human embryos or stem cells in Japan are relatively complex and comprise a range of laws and guidelines; the specific rules applied depend on the characteristics of each study. Therefore, even similar research activities may differ in terms of not only the guidelines or laws implemented, but also the procedures required. Such situations may confuse researchers.

Ethical Issues in Commercialisation of Stem Cell Therapies: Mapping the Terrain.

Bioethical debates on stem cell research have focused primarily on the moral status of human embryos. This article highlights seven distinct policy and ethical issues associated with the commercialisation of stem cell therapies, describes some of the underlying moral questions on which they turn, and argues that there is an urgent need to refocus the debate on stem cell research beyond the controversy over embryo destruction.

Bioethics in human embryology: the double-edged sword of embryo research.

There has been a significant increase in the use of assisted reproductive therapies (ARTs) over the past several decades, allowing many couples with infertility to conceive. Despite the achievements in this field, a mounting body of evidence concerning the epigenetic risks associated with ART interventions such as ovarian hormonal stimulation, intracytoplasmic sperm injection (ICSI), and in vitro culture (IVC) of oocytes and embryos has also emerged. Induced development of multiple follicles, the IVC media itself, and extended culture may alter the epigenome of both gametes and embryos, resulting in yet to be fully understood developmental, postnatal, and adult life health consequences. Investigators have attempted to decipher the molecular mechanisms mediating ART-induced epigenetic changes using either human samples or animal models with some success. As research in this field continues to expand, the ethical responsibilities of embryologists and researchers have become critically important. Here, we briefly discuss the ethical aspects of ART research, concentrating on the constraints arising from the perceived 'unnaturalness' of many of these procedures. Secondly, we focus on the bioethics and morality of human embryo research in general and how ethically acceptable model systems may be used to mimic early human embryogenesis. Lastly, we review the 14-day culture limit of human embryos and the notion that this rule could be considered of taken into account using new technologies and cues from animal models. The 'black box' of early post-implantation embryogenesis might be revealed using embryo models. As long as this distinct moral line has been drawn and closely followed, we should not fear scientific growth in embryo research. Although in vitro fertilization (IVF) is ethically acceptable, research with human embryos to improve its success raises serious ethical concerns that are in need of constant revisiting.Glossary index: Moral status: the ascription of obligations and rights to embryos on the basis of sentience; Sentience: the capacity of the developing embryo to experience feelings and sensations, such as the awareness of pain; Ectogenesis: the growth of the embryo in an artificial environment outside the mother's body.

On the origins and fate of chromosomal abnormalities in human preimplantation embryos: an unsolved riddle.

About 8 out of 10 human embryos obtained in vitro harbour chromosomal abnormalities of either meiotic or mitotic origin. Abnormalities of mitotic origin lead to chromosomal mosaicism, a phenomenon that has sparked much debate lately as it confounds results obtained through preimplantation genetic testing for aneuploidy (PGT-A). PGT-A in itself is still highly debated, not only on the modalities of its execution but also on whether it should be offered to patients at all. We will focus on post-zygotic chromosomal abnormalities leading to mosaicism. First, we will summarize what is known about the rates of chromosomal abnormalities at different developmental stages. Next, based on the current understanding of the origin and cellular consequences of chromosomal abnormalities, which is largely based on studies on cancer cells and model organisms, we will offer a number of hypotheses on which mechanisms may be at work in early human development. Finally, and very briefly, we will touch upon the impact our current knowledge has on the practice of PGT-A. What is the level of abnormal cells that an embryo can tolerate before it loses its potential for full development? And is blastocyst biopsy as harmless as it seems?

Cleavage-stage human embryo arrest, is it embryo genetic composition or others?

Embryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle.

We have reached a dead end for preimplantation genetic testing for aneuploidy.

The hypothesis of preimplantation genetic testing for aneuploidy (PGT-A) was first proposed 20 years ago, suggesting that during IVF elimination of aneuploid embryos prior to transfer will improve implantation rates of remaining embryos and, therefore, increase pregnancy and live birth rates, while also reducing miscarriages. Subsequently, unvalidated and increasingly unrestricted clinical utilization of PGT-A called for at least one properly randomized controlled trial (RCT) to assess cumulative live birth rates following a single oocyte retrieval, utilizing all fresh and frozen embryos of an IVF cycle. Only recently two such RCTs were published, however both, when properly analysed, not only failed to demonstrate significant advantages from utilization of PGT-A, but actually demonstrated outcome deficits in comparison to non-use of PGT-A, when patient selection biases in favour of PGT-A were reversed. Moreover, because of high embryo mosaicism at the blastocyst stage and, therefore, high false-positive rates from trophectoderm biopsies, large numbers of chromosomal-normal embryos with normal pregnancy potential are unnecessarily left unused or discarded, indisputably causing harm to affected couples. We, therefore, strongly call for restricting PGT-A to only research protocols and, as of this point in time, encourage professional societies in the field to follow suit with appropriate practice guidelines.

Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human.

Understanding new modulators of axon regeneration is central to neural repair. Our previous work demonstrated critical roles of atypical cadherin Celsr2 during neural development, including cilia organization, neuron migration and axon navigation. Here, we address its role in axon regeneration. We show that Celsr2 is highly expressed in both mouse and human spinal motor neurons. Celsr2 knockout promotes axon regeneration and fasciculation in mouse cultured spinal explants. Similarly, cultured Celsr2 mutant motor neurons extend longer neurites and larger growth cones, with increased expression of end-binding protein 3 and higher potassium-induced calcium influx. Mice with Celsr2 conditional knockout in spinal motor neurons do not exhibit any behavioural deficits; however, after branchial plexus injury, axon regeneration and functional forelimb locomotor recovery are significantly improved. Similarly, knockdown of CELSR2 using shRNA interference in cultured human spinal motor explants and motor neurons increases axonal fasciculation and growth. In mouse adult spinal cord after root avulsion, in mouse embryonic spinal cords, and in cultured human motor neurons, Celsr2 downregulation is accompanied by increased levels of GTP-bound Rac1 and Cdc42, and of JNK and c-Jun. In conclusion, Celsr2 negatively regulates motor axon regeneration and is a potential target to improve neural repair.

GDF9 concentration in embryo culture medium is linked to human embryo quality and viability.

PURPOSE: We aimed to evaluate the link between the GDF9 concentration in day 3 human embryo culture medium and embryo quality and viability. METHODS: Two independent, prospective, observational studies were conducted. In study 1, a total of 280 embryos from 70 patients who obtained at least 4 embryos with 6-10 blastomeres (2 transferable and 2 non-transferable embryos) at day 3 were enrolled. In study 2, a total of 119 embryos from 61 patients (29 fully implanted and 32 non-implanted patients) were enrolled. The corresponding GDF9 concentrations in spent culture medium of embryos were quantified by ELISA assay. The expression pattern of GDF9 in human embryos was investigated using Q-PCR and immunofluorescence. RESULTS: GDF9 mRNA and protein were detected from human oocytes to eight-cell embryos and displayed a slow decreasing trend. In study 1, GDF9 concentration in culture medium is lower for transferable embryos compared with non-transferable embryos (331 pg/mL (quartiles: 442, 664 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001), and increased commensurate with the diminution of the embryo quality (P < 0.001). In study 2, significantly lower GDF9 concentration was detected for implanted embryos than non-implanted embryos (331 pg/mL (quartiles: 156, 665 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001). The same trend was found between the embryos that led to live birth and those that failed. CONCLUSION: The GDF9 concentration in culture medium is linked to embryo quality and viability, and exhibited the potential to be a non-invasive biomarker for embryo selection.

Taking responsibility, making irresponsibility: Controversies in human gene editing.

In current discussions, human germline editing is often called 'irresponsible'. Looking at the international summits on human gene editing held in 2015 and in 2018 and the announcement by He Jiankui of the birth of two gene-edited babies in November 2018, this article analyses how 'irresponsible' research was the result of various (dis)qualifications and demarcations. Against a background of discussions of responsibility, an individual scientist was singled out, his experiments were scrutinized for their soundness, legality and safety and ethical and moral stances were questioned. These are features of a process that I call 'irresponsibilization'. This irresponsibilization of research is entangled with calls for further action: Irresponsible research like that of He Jiankui should be contained, the veracity of knowledge claims needs to be confirmed, and institutions and decision-makers are called to act. The controversy turned 'irresponsible' into an active category, and rendered explicit its political, institutional and practical ramifications.