H1FOO-DD promotes efficiency and uniformity in reprogramming to naive pluripotency.

Heterogeneity among both primed and naive pluripotent stem cell lines remains a major unresolved problem. Here we show that expressing the maternal-specific linker histone H1FOO fused to a destabilizing domain (H1FOO-DD), together with OCT4, SOX2, KLF4, and LMYC, in human somatic cells improves the quality of reprogramming to both primed and naive pluripotency. H1FOO-DD expression was associated with altered chromatin accessibility around pluripotency genes and with suppression of the innate immune response. Notably, H1FOO-DD generates naive induced pluripotent stem cells with lower variation in transcriptome and methylome among clones and a more uniform and superior differentiation potency. Furthermore, we elucidated that upregulation of FKBP1A, driven by these five factors, plays a key role in H1FOO-DD-mediated reprogramming.

Heparan Sulfate Chain-Conjugated Laminin-E8 Fragments Advance Paraxial Mesodermal Differentiation Followed by High Myogenic Induction from hiPSCs.

Human-induced pluripotent stem cells (hiPSCs) have great therapeutic potential. The cell source differentiated from hiPSCs requires xeno-free and robust methods for lineage-specific differentiation. Here, a system is described for differentiating hiPSCs on new generation laminin fragments (NGLFs), a recombinant form of a laminin E8 fragment conjugated to the heparan sulfate chains (HS) attachment domain of perlecan. Using NGLFs, hiPSCs are highly promoted to direct differentiation into a paraxial mesoderm state with high-efficiency muscle lineage generation. HS conjugation to the C-terminus of Laminin E8 fragments brings fibroblast growth factors (FGFs) bound to the HS close to the cell surface of hiPSCs, thereby facilitating stronger FGF signaling pathways stimulation and initiating HOX gene expression, which triggers the paraxial mesoderm differentiation of hiPSCs. This highly efficient differentiation system can provide a roadmap for paraxial mesoderm development and an infinite source of myocytes and muscle stem cells for disease modeling and regenerative medicine.

A let-7 microRNA-RALB axis links the immune properties of iPSC-derived megakaryocytes with platelet producibility.

We recently achieved the first-in-human transfusion of induced pluripotent stem cell-derived platelets (iPSC-PLTs) as an alternative to standard transfusions, which are dependent on donors and therefore variable in supply. However, heterogeneity characterized by thrombopoiesis-biased or immune-biased megakaryocytes (MKs) continues to pose a bottleneck against the standardization of iPSC-PLT manufacturing. To address this problem, here we employ microRNA (miRNA) switch biotechnology to distinguish subpopulations of imMKCLs, the MK cell lines producing iPSC-PLTs. Upon miRNA switch-based screening, we find imMKCLs with lower let-7 activity exhibit an immune-skewed transcriptional signature. Notably, the low activity of let-7a-5p results in the upregulation of RAS like proto-oncogene B (RALB) expression, which is crucial for the lineage determination of immune-biased imMKCL subpopulations and leads to the activation of interferon-dependent signaling. The dysregulation of immune properties/subpopulations, along with the secretion of inflammatory cytokines, contributes to a decline in the quality of the whole imMKCL population.

SARS-CoV-2-Specific Immune Responses in Vaccination and Infection during the Pandemic in 2020-2022.

To gain insight into how immunity develops against SARS-CoV-2 from 2020 to 2022, we analyzed the immune response of a small group of university staff and students who were either infected or vaccinated. We investigated the levels of receptor-binding domain (RBD)-specific and nucleocapsid (N)-specific IgG and IgA antibodies in serum and saliva samples taken early (around 10 days after infection or vaccination) and later (around 1 month later), as well as N-specific T-cell responses. One patient who had been infected in 2020 developed serum RBD and N-specific IgG antibodies, but declined eight months later, then mRNA vaccination in 2021 produced a higher level of anti-RBD IgG than natural infection. In the vaccination of naïve individuals, vaccines induced anti-RBD IgG, but it declined after six months. A third vaccination boosted the IgG level again, albeit to a lower level than after the second. In 2022, when the Omicron variant became dominant, familial transmission occurred among vaccinated people. In infected individuals, the levels of serum anti-RBD IgG antibodies increased later, while anti-N IgG peaked earlier. The N-specific activated T cells expressing IFN γ or CD107a were detected only early. Although SARS-CoV-2-specific salivary IgA was undetectable, two individuals showed a temporary peak in RBD- and N-specific IgA antibodies in their saliva on the second day after infection. Our study, despite having a small sample size, revealed that SARS-CoV-2 infection triggers the expected immune responses against acute viral infections. Moreover, our findings suggest that the temporary mucosal immune responses induced early during infection may provide better protection than the currently available intramuscular vaccines.

Early enteral nutrition and mobilization following head and neck surgery with free flap reconstruction.

BACKGROUND: Perioperative management methods that reduce surgery-associated invasiveness and improve the quality of postoperative recovery are being promoted as enhanced recovery after surgery programs in various areas. Early enteral nutrition and mobilization are essential elements for enhanced recovery after surgery; however, their safety and feasibility are unclear in head and neck surgery with free tissue transfer reconstruction. This study aimed to clarify these uncertainties. METHODS: This is a retrospective before-after study. From 2018 to 2022, 187 and 173 patients received conventional management on or before April 2020 and early management on or after May 2020, respectively. The conventional management and early management groups received enteral nutrition and mobilization on postoperative days 2 and 1, respectively. The primary outcome for safety assessment was the incidence of complications. The secondary outcome was the compliance rate of conventional management or early management for feasibility assessment and the length of hospital stay. RESULTS: The clinical tumour-node-metastasis stage and American Society of Anesthesiologists physical status showed significant differences between the groups. In multivariable analysis, the early management group demonstrated a significantly lower incidence of treatment-required complication classified Clavien-Dindo Grade 2 and above (odds ratio = 0.57; 95% confidence interval = 0.31-0.92) and lower wound infection (odds ratio = 0.53; 95% confidence interval = 0.31-0.92). The early management group had lower compliance rate than the conventional management group; however, no statistically significant difference was observed (79.8% vs. 85.0%, P = 0.21). CONCLUSION: Early management is safe and feasible following head and neck surgery with free tissue transfer reconstruction. It could reduce the complication rate and is considered a useful postoperative management method.

Comprehensive Immunophenotyping by Polychromatic Cytometry.

The human immune system comprises a diverse array of cells involved in innate and adaptive immunity, and these immune cells coordinate immune responses against pathogens through intricate interactions. Multicolor flow cytometry is a powerful technique for qualitatively and quantitatively measuring the characteristics of immune cells, offering advantages, such as high-dimensional analysis, elucidation of cellular heterogeneity, understanding of pathogenesis, development of therapeutic strategies, and platform flexibility. Here, we demonstrate a new immunophenotyping panel that allows simultaneous evaluation of the characteristics of T and B cells. This panel enables tracking of changes in the immune status due to aging, environmental factors, pathogen infections, and vaccine administration. Additionally, it includes co-stimulatory molecules for assessing the activation state of immune cells and inhibitory checkpoint molecules for evaluating exhaustion status, thereby providing valuable insights into the features of human immune responses. These analyses contribute to understanding the pathophysiology of diseases and developing therapeutic strategies while offering crucial information for assessing the correlation of symptoms with infections and evaluating the efficacy of vaccines.

Circulating tumor-associated antigen-specific IFNγ+4-1BB+ CD8+ T cells as peripheral biomarkers of treatment outcomes in patients with pancreatic cancer.

CD8+ T cells affect the outcomes of pancreatic ductal adenocarcinoma (PDAC). Using tissue samples at pre-treatment to monitor the immune response is challenging, while blood samples are beneficial in overcoming this limitation. In this study, we measured peripheral antigen-specific CD8+ T cell responses against four different tumor-associated antigens (TAAs) in PDAC using flow cytometry and investigated their relationships with clinical features. We analyzed the optimal timing within the treatment course for effective immune checkpoint inhibition in vitro. We demonstrated that the frequency of TAA-specific IFNγ+4-1BB+ CD8+ T cells was correlated with a fold reduction in CA19-9 before and after neoadjuvant therapy. Moreover, patients with TAA-specific IFNγ+4-1BB+ CD8+ T cells after surgery exhibited a significantly improved disease-free survival. Anti-PD-1 treatment in vitro increased the frequency of TAA-specific IFNγ+4-1BB+ CD8+ T cells before neoadjuvant therapy in patients, suggesting the importance of the timing of anti-PD-1 inhibition during the treatment regimen. Our results indicate that peripheral immunophenotyping, combined with highly sensitive identification of TAA-specific responses in vitro as well as detailed CD8+ T cell subset profiling via ex vivo analysis, may serve as peripheral biomarkers to predict treatment outcomes and therapeutic efficacy of immunotherapy plus neoadjuvant chemotherapy.

Micro-patterned culture of iPSC-derived alveolar and airway cells distinguishes SARS-CoV-2 variants.

The emergence of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants necessitated a rapid evaluation system for their pathogenesis. Lung epithelial cells are their entry points; however, in addition to their limited source, the culture of human alveolar epithelial cells is especially complicated. Induced pluripotent stem cells (iPSCs) are an alternative source of human primary stem cells. Here, we report a model for distinguishing SARS-CoV-2 variants at high resolution, using separately induced iPSC-derived alveolar and airway cells in micro-patterned culture plates. The position-specific signals induced the apical-out alveolar type 2 and multiciliated airway cells at the periphery and center of the colonies, respectively. The infection studies in each lineage enabled profiling of the pathogenesis of SARS-CoV-2 variants: infection efficiency, tropism to alveolar and airway lineages, and their responses. These results indicate that this culture system is suitable for predicting the pathogenesis of emergent SARS-CoV-2 variants.

Single-cell RNA sequencing of submandibular gland reveals collagen type XV-positive fibroblasts as a disease-characterizing cell population of IgG4-related disease.

OBJECTIVES: IgG4-related disease (IgG4-RD) is a systemic autoimmune disease with an unknown etiology, affecting single/multiple organ(s). Pathological findings include the infiltration of IgG4-producing plasma cells, obliterative phlebitis, and storiform fibrosis. Although immunological studies have shed light on the dysregulation of lymphocytes in IgG4-RD pathogenesis, the role of non-immune cells remains unclear. This study aimed to investigate the demographics and characteristics of non-immune cells in IgG4-RD and explore potential biomarkers derived from non-immune cells in the sera. METHODS: We conducted single-cell RNA sequence (scRNA-seq) on non-immune cells isolated from submandibular glands of IgG4-RD patients. We focused on fibroblasts expressing collagen type XV and confirmed the presence of those fibroblasts using immunohistochemistry. Additionally, we measured the levels of collagen type XV in the sera of IgG4-RD patients. RESULTS: The scRNA-seq analysis revealed several distinct clusters consisting of fibroblasts, endothelial cells, ductal cells, and muscle cells. Differential gene expression analysis showed upregulation of COL15A1 in IgG4-RD fibroblasts compared to control subjects. Notably, COL15A1-positive fibroblasts exhibited a distinct transcriptome compared to COL15A1-negative counterparts. Immunohistochemical analysis confirmed a significant presence of collagen type XV-positive fibroblasts in IgG4-RD patients. Furthermore, immune-suppressive therapy in active IgG4-RD patients resulted in decreased serum levels of collagen type XV. CONCLUSIONS: Our findings suggest that collagen type XV-producing fibroblasts may represent a disease-characterizing non-immune cell population in IgG4-RD and hold potential as a disease-monitoring marker.

Instrumentation failure following pediatric spine deformity growth-sparing surgery using traditional growing rods or vertical expandable prosthetic titanium ribs.

BACKGROUND: Instrumentation failure (IF) is a major complication associated with growth-sparing surgery for pediatric spinal deformities; however, studies focusing on IF following each surgical procedure are lacking. We aimed to evaluate the incidence, timing, and rates of unplanned return to the operating room (UPROR) associated with IF following each surgical procedure in growth-sparing surgeries using traditional growing rods (TGRs) and vertical expandable prosthetic titanium ribs (VEPTRs). METHODS: We reviewed 1,139 surgical procedures documented in a Japanese multicenter database from 2015 to 2017. Of these, 544 TGR and 455 VEPTR procedures were included for evaluation on a per-surgery basis. IF was defined as the occurrence of an implant-related complication requiring revision surgery. RESULTS: The surgery-based incidences of IF requiring revision surgery in the TGR and VEPTR groups were 4.3% and 4.0%, respectively, with no significant intergroup difference. Remarkably, there was a negative correlation between IF incidence per surgical procedure and the number of lengthening surgeries in both groups. In addition, rod breakage in the TGR group and anchor-related complications in the VEPTR group tended to occur relatively early in the treatment course. The surgery-based rates of UPROR due to IF in the TGR and VEPTR groups were 2.0% and 1.5%, respectively, showing no statistically significant difference. CONCLUSIONS: We found that IF, such as anchor related-complications and rod breakage, occurs more frequently earlier in the course of lengthening surgeries. This finding may help in patient counseling and highlights the importance of close postoperative follow-up to detect IF and improve outcomes.

Humoral and cellular immune responses to COVID-19 mRNA vaccines in immunosuppressed liver transplant recipients.

BACKGROUND: Liver transplant recipients (LTRs) are at a high risk of severe COVID-19 owing to immunosuppression and comorbidities. LTRs are less responsive to mRNA vaccines than healthy donors (HDs) or other immunosuppressed patients. However, the disruption mechanism in humoral and cellular immune memory responses is unclear. METHODS: We longitudinally collected peripheral blood mononuclear cells and plasma samples from HDs (n = 44) and LTRs (n = 54) who received BNT162b2 or mRNA-1273 vaccines. We measured the levels of anti-receptor-binding domain (RBD) antibodies and spike-specific CD4+ and CD8+ T-cell responses. RESULTS: Here, we show that the induction of anti-RBD IgG was weaker in LTRs than in HDs. The use of multiple immunosuppressive drugs is associated with lower antibody titers than only calcineurin inhibitor, and limits the induction of CD4+ T-cell responses. However, spike-specific CD4+ T-cell and antibody responses improved with a third vaccination. Furthermore, mRNA vaccine-induced spike-specific CD8+ T cells are quantitatively, but not qualitatively, limited to LTRs. Both CD4+ and CD8+ T cells react to omicron sublineages, regardless of the presence in HDs or LTRs. However, there is no boosting effect of spike-specific memory CD8+ T-cell responses after a third vaccination in HDs or LTRs. CONCLUSIONS: The third mRNA vaccination improves both humoral responses and spike-specific CD4+ T-cell responses in LTRs but provides no booster effect for spike-specific memory CD8+ T-cell responses. A third mRNA vaccination could be helpful in LTRs to prevent severe COVID-19, although further investigation is required to elicit CD8+ T-cell responses in LTRs and HDs.

People with a liver transplant don't have as strong an immune response to COVID-19 vaccines as healthy people. This study investigates how these individuals produce protective proteins, called antibodies, and CD4 and CD8 T cell immune responses. CD4 T cells are responsible for commanding the immune response and CD8 T cells for remembering and fighting the virus in future. We found that liver transplant recipients have a weaker ability to produce antibodies after vaccination, which is even more noticeable in those taking drugs to prevent transplant rejection. While a third vaccine dose improves their ability to produce antibodies, and to have a CD4 T cell response, it doesn't boost the CD8 T cell response. In summary, an extra vaccine dose can strengthen the immune response in liver transplant recipients but doesn't improve some aspects of their immune memory.

Oxidative phosphorylation is a pivotal therapeutic target of fibrodysplasia ossificans progressiva.

Heterotopic ossification (HO) is a non-physiological bone formation where soft tissue progenitor cells differentiate into chondrogenic cells. In fibrodysplasia ossificans progressiva (FOP), a rare genetic disease characterized by progressive and systemic HO, the Activin A/mutated ACVR1/mTORC1 cascade induces HO in progenitors in muscle tissues. The relevant biological processes aberrantly regulated by activated mTORC1 remain unclear, however. RNA-sequencing analyses revealed the enrichment of genes involved in oxidative phosphorylation (OXPHOS) during Activin A-induced chondrogenesis of mesenchymal stem cells derived from FOP patient-specific induced pluripotent stem cells. Functional analyses showed a metabolic transition from glycolysis to OXPHOS during chondrogenesis, along with increased mitochondrial biogenesis. mTORC1 inhibition by rapamycin suppressed OXPHOS, whereas OXPHOS inhibitor IACS-010759 inhibited cartilage matrix formation in vitro, indicating that OXPHOS is principally involved in mTORC1-induced chondrogenesis. Furthermore, IACS-010759 inhibited the muscle injury-induced enrichment of fibro/adipogenic progenitor genes and HO in transgenic mice carrying the mutated human ACVR1. These data indicated that OXPHOS is a critical downstream mediator of mTORC1 signaling in chondrogenesis and therefore is a potential FOP therapeutic target.

Chemically Recyclable Unnatural (1→6)-Polysaccharides from Cellulose-Derived Levoglucosenone and Dihydrolevoglucosenone.

Unnatural polysaccharide analogs and their biological activities and material properties have attracted considerable research interest. However, these efforts often encounter challenges, especially those related to synthetic complexity and scalability. Here, we report the chemical synthesis of unnatural (1→6)-polysaccharides using levoglucosenone (LGO) and dihydrolevoglucosenone (Cyrene), which are derived from cellulose. Using a versatile monomer synthesis from LGO and Cyrene and cationic ring-opening polymerization, (1→6)-polysaccharides with various tailored substituent patterns are obtained. Additionally, environmentally benign and easy-to-handle organic Brønsted acid catalysts are investigated. This study demonstrates well-controlled first-order polymerization kinetics for the reactive (1S,5R)-6,8-dioxabicyclo[3,2,1]octane (DBO) monomer. The synthesized (1→6)-polysaccharides exhibit high thermal stability and form amorphous solids under ambient conditions, which could be processed into highly transparent self-standing films. Additionally, these polymers exhibit excellent closed-loop chemical recyclability. This study provides an important approach to explore the chemical spaces of unnatural polysaccharides and contributes to the development of sustainable polymer materials from abundant biomass resources.

Safety and immunogenicity of VLPCOV-02, a SARS-CoV-2 self-amplifying RNA vaccine with a modified base, 5-methylcytosine.

Continuing emergence of variants of concern resulting in reduced SARS-CoV-2 vaccine efficacy necessitates additional prevention strategies. The structure of VLPCOV-01, a lipid nanoparticle-encapsulated, self-amplifying RNA COVID-19 vaccine with a comparable immune response to BNT162b2, was revised by incorporating a modified base, 5-methylcytosine, to reduce reactogenicity, and an updated receptor-binding domain derived from the Brazil (gamma) variant. Interim analyses of a phase 1 dose-escalation booster vaccination study with the resulting construct, VLPCOV-02, in healthy, previously vaccinated Japanese individuals (N = 96) are reported (jRCT2051230005). A dose-related increase in solicited local and systemic adverse events was observed, which were generally rated mild or moderate. The most commonly occurring events were tenderness, pain, fatigue, and myalgia. Serum SARS-CoV-2 immunoglobulin titers increased during the 4 weeks post-immunization. VLPCOV-02 demonstrated a favorable safety profile compared with VLPCOV-01, with reduced adverse events and fewer fever events at an equivalent dose. These findings support further study of VLPCOV-02.

Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells.

Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures in vitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages in vitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.

Single-cell and spatial transcriptomics in endocrine research.

Accumulating evidence suggests that cellular heterogeneity in organs and cell-cell and tissue-tissue interactions are crucial for maintaining physical homeostasis and disease progression. Endocrine organs also exhibit cellular heterogeneity and comprise multiple cell types. For instance, the pituitary gland comprises five types of pituitary hormone-producing cells as well as non-hormone-producing supporting cells, such as fibroblasts, endothelial cells, and folliculostellate cells. However, the functional roles of the interactions between hormone-producing and non-producing cells in the pituitary gland remain incompletely understood. Over the past decade, emerging technologies such as single-cell and spatial transcriptomics have provided excellent tools for studying cellular heterogeneity and their interactions; however, the application of these technologies in endocrine research remains limited. This review provides an overview of these technologies and discusses their strengths and limitations. Additionally, we also summarize the potential future applications of single-cell and spatial transcriptomics in the study of endocrine organs and their disorders.

Umbilical Vein Calcification Associated with Double-Lumen Catheter Malpositioning in an Extremely Low-Birth-Weight Infant.

Umbilical venous (UV) catheters (UVCs) are commonly used in severely ill neonates. Complications associated with UVC often result from an inappropriate UVC position. Calcification of the UV, a rare complication, was observed in an extremely low-birth-weight infant born at 23 weeks of gestation. After birth, the infant experienced respiratory and circulatory dysfunction, followed by disseminated intravascular coagulation (DIC). A UVC was inserted, and circulatory agonists and blood transfusions were administered, as well as a calcium gluconate infusion for hypocalcemia and hyperkalemia. Ten days after birth, calcification was detected in the UV, likely due to a tunica intima injury caused by UVC, a hypercoagulable state due to DIC, and a high-dose calcium gluconate infusion. Additionally, proximal port malpositioning of the double-lumen catheter might have contributed to calcification within the UV. To prevent such complications, real-time ultrasound confirmation with agitated saline contrast during UVC placement is recommended; in the absence of the facility or skills for ultrasonography, X-rays should be performed in the lateral and anteroposterior views. Furthermore, when using multi-lumen catheters, physicians should not only verify the tip position but also ensure proper placement of proximal ports and carefully select medications administered through the ports.

SARS-CoV-2-induced disruption of a vascular bed in a microphysiological system caused by type-I interferon from bronchial organoids.

Blood vessels show various COVID-19-related conditions including thrombosis and cytokine propagation. Existing in vitro blood vessel models cannot represent the consequent changes in the vascular structure or determine the initial infection site, making it difficult to evaluate how epithelial and endothelial tissues are damaged. Here, we developed a microphysiological system (MPS) that co-culture the bronchial organoids and the vascular bed to analyze infection site and interactions. In this system, virus-infected organoids caused damage in vascular structure. However, vasculature was not damaged or infected when the virus was directly introduced to vascular bed. The knockout of interferon-related genes and inhibition of the JAK/STAT pathway reduced the vascular damage, indicating the protective effect of interferon response suppression. The results demonstrate selective infection of bronchial epithelial cells and vascular damage by cytokines and also indicate the applicability of MPS to investigate how the infection influences vascular structure and functions.

Hypoblast from human pluripotent stem cells regulates epiblast development.

Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.