An update on human pre- and peri-implantation development: a blueprint for blastoids.

Despite over 40 years following the first birth from medically assisted reproduction (MAR) technologies, mechanisms underlying the key developmental events during the first 7 days of human development, such as signaling pathway contribution, are remaining a mystery. An in-depth mechanistic understanding of how the human preimplantation embryo develops would support the optimization of embryo quality assessment methods and culturing conditions, thereby increasing the success rate of MAR. However, the limited availability of human embryos, legitimate ethical concerns, and regulations still present an obstacle toward our advancement of knowledge. Stem cell-based embryonic models, including blastoids than model blastocysts, offer unprecedented opportunities to fill knowledge gaps and complement animal models. Blastoids' predictive power depends on how faithfully they recapitulate the blastocyst. Here, we review the state of the art of human pre- and peri-implantation development and outline the specificities of human embryo research to clarify the framework for blastoid research.

Discovery of potent 1,1-diarylthiogalactoside glycomimetic inhibitors of Pseudomonas aeruginosa LecA with antibiofilm properties.

In this work, ß-thiogalactoside mimetics bearing 1,1-diarylmethylene or benzophenone aglycons have been prepared and assayed for their affinity towards LecA, a lectin and virulence factor from Pseudomonas aeruginosa involved in bacterial adhesion and biofilm formation. The hit compound presents higher efficiency than previously described monovalent inhibitors and the crystal structure confirmed the occurrence of additional contacts between the aglycone and the protein surface. The highest affinity (160 nM) was obtained for a divalent ligand containing two galactosides. The monovalent high affinity compound (Kd = 1 µM) obtained through structure-activity relationship (SAR) showed efficient antibiofilm activity with no associated bactericidal activity.

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.

Synthesis and Biological Activity of 3-(Heteroaryl)quinolin-2(1H)-ones Bis-Heterocycles as Potential Inhibitors of the Protein Folding Machinery Hsp90.

In the context of our SAR study concerning 6BrCaQ analogues as C-terminal Hsp90 inhibitors, we designed and synthesized a novel series of 3-(heteroaryl)quinolin-2(1H), of types 3, 4, and 5, as a novel class of analogues. A Pd-catalyzed Liebeskind-Srogl cross-coupling was developed as a convenient approach for easy access to complex purine architectures. This series of analogues showed a promising biological effect against MDA-MB231 and PC-3 cancer cell lines. This study led to the identification of the best compounds, 3b (IC50 = 28 µM) and 4e, which induce a significant decrease of CDK-1 client protein and stabilize the levels of Hsp90 and Hsp70 without triggering the HSR response.

Human blastoids model blastocyst development and implantation.

One week after fertilization, human embryos implant into the uterus. This event requires the embryo to form a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Stem cells can form a blastocyst model that we called a blastoid1. Here we show that naive human pluripotent stem cells cultured in PXGL medium2 and triply inhibited for the Hippo, TGF-ß and ERK pathways efficiently (with more than 70% efficiency) form blastoids generating blastocyst-stage analogues of the three founding lineages (more than 97% trophectoderm, epiblast and primitive endoderm) according to the sequence and timing of blastocyst development. Blastoids spontaneously form the first axis, and we observe that the epiblast induces the local maturation of the polar trophectoderm, thereby endowing blastoids with the capacity to directionally attach to hormonally stimulated endometrial cells, as during implantation. Thus, we propose that such a human blastoid is a faithful, scalable and ethical model for investigating human implantation and development3,4.

Integrated pseudotime analysis of human pre-implantation embryo single-cell transcriptomes reveals the dynamics of lineage specification.

Understanding lineage specification during human pre-implantation development is a gateway to improving assisted reproductive technologies and stem cell research. Here we employ pseudotime analysis of single-cell RNA sequencing (scRNA-seq) data to reconstruct early mouse and human embryo development. Using time-lapse imaging of annotated embryos, we provide an integrated, ordered, and continuous analysis of transcriptomics changes throughout human development. We reveal that human trophectoderm/inner cell mass transcriptomes diverge at the transition from the B2 to the B3 blastocyst stage, just before blastocyst expansion. We explore the dynamics of the fate markers IFI16 and GATA4 and show that they gradually become mutually exclusive upon establishment of epiblast and primitive endoderm fates, respectively. We also provide evidence that NR2F2 marks trophectoderm maturation, initiating from the polar side, and subsequently spreads to all cells after implantation. Our study pinpoints the precise timing of lineage specification events in the human embryo and identifies transcriptomics hallmarks and cell fate markers.

Induction of Human Trophoblast Stem Cells from Somatic Cells and Pluripotent Stem Cells.

Human trophoblast stem cells (hTSCs) derived from blastocysts and first-trimester cytotrophoblasts offer an unprecedented opportunity to study the placenta. However, access to human embryos and first-trimester placentas is limited, thus preventing the establishment of hTSCs from diverse genetic backgrounds associated with placental disorders. Here, we show that hTSCs can be generated from numerous genetic backgrounds using post-natal cells via two alternative methods: (1) somatic cell reprogramming of adult fibroblasts with OCT4, SOX2, KLF4, MYC (OSKM) and (2) cell fate conversion of naive and extended pluripotent stem cells. The resulting induced/converted hTSCs recapitulated hallmarks of hTSCs including long-term self-renewal, expression of specific transcription factors, transcriptomic signature, and the potential to differentiate into syncytiotrophoblast and extravillous trophoblast cells. We also clarified the developmental stage of hTSCs and show that these cells resemble day 8 cytotrophoblasts. Altogether, hTSC lines of diverse genetic origins open the possibility to model both placental development and diseases in a dish.

Initiation of a conserved trophectoderm program in human, cow and mouse embryos.

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2-5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.

Synthesis of Benzofurans and Indoles from Terminal Alkynes and Iodoaromatics Catalyzed by Recyclable Palladium Nanoparticles Immobilized on Siliceous Mesocellular Foam.

Herein, we report on the utilization of a heterogeneous catalyst, consisting of Pd nanoparticles supported on a siliceous mesocellular foam (Pd0 -AmP-MCF), for the synthesis of heterocycles. Reaction of o-iodophenols and protected o-iodoanilines with acetylenes in the presence of a Pd nanocatalyst produced 2-substituted benzofurans and indoles, respectively. In general, the catalytic protocol afforded the desired products in good to excellent yields under mild reaction conditions without the addition of ligands. Moreover, the structure of the reported Pd nanocatalyst was further elucidated with extended X-ray absorption fine-structure spectroscopy, and it was proven that the catalyst could be recycled multiple times without significant loss of activity.

Stereoretentive Copper-Catalyzed Directed Thioglycosylation of C(sp2 )-H Bonds of Benzamides.

An efficient thioglycosylation of C(sp2 )-H bonds with thiosugars has been established for the first time. Using only Cu(OAc)2 ⋅H2 O as a catalyst and Ag2 CO3 as an additive in DMSO, the protocol proved to have broad scope, and a variety of complex thioglycosides have been prepared in good yields with exclusive ß-selectivity.

Formulation and in vitro efficacy of liposomes containing the Hsp90 inhibitor 6BrCaQ in prostate cancer cells.

6BrCaQ is a promising anti-cancer agent derived from novobiocin, which has been shown to inhibit Hsp90. 6BrCaQ was loaded into nanometer-scaled phospholipid vesicles (liposomes) suitable for drug delivery to solid tumors. The effective incorporation of the drug within the phospholipid bilayer was investigated by differential scanning calorimetry. Liposomal 6BrCaQ showed good activity on PC-3 cell lines in vitro in terms of apoptosis induction and cell growth arrest in G2/M. Liposomes containing 6BrCaQ were also shown to slow down migration of PC-3 cells in presence of chemokine ligand 2 and to synergize with doxorubicin. Several Hsp90 targeting molecules like geldanamycin induce accumulation of Hsp70, leading to cytoprotection and often correlated with poor prognosis. In this study, we did not report any Hsp70 induction after treatment with liposomal 6BrCaQ but a decrease in Hsp90 and CDK-4 protein expression, indicating an effect on the chaperon machinery. Liposomal encapsulation of 6BrCaQ revealed promising anti-cancer effects and a better understanding of its mechanism of action.

Stereoretentive palladium-catalyzed arylation, alkenylation, and alkynylation of 1-thiosugars and thiols using aminobiphenyl palladacycle precatalyst at room temperature.

A general and efficient protocol for the palladium-catalyzed functionalization of mono- and polyglycosyl thiols by using the palladacycle precatalyst G3-XantPhos was developed. The C-S bond-forming reaction was achieved rapidly at room temperature with various functionalized (hetero)aryl-, alkenyl-, and alkynyl halides. The functional group tolerance on the electrophilic partner is typically high and anomer selectivities of thioglycosides are high in all cases studied. New sulfur nucleophiles such as thiophenols, alkythiols, and thioaminoacids (cysteine) were also successfully coupled to lead to the most general and practical method yet reported for the functionalization of thiols.

A general Pd/Cu-catalyzed C-H heteroarylation of 3-bromoquinolin-2(1H)-ones.

3-(Heteroaryl)quinolin-2(1H)-ones were synthesized in good to excellent yields using a bimetallic catalytic system through the C-H heteroarylation strategy. Starting from 3-bromoquinolin-2(1H)-ones, various azoles have been successfully used. In all cases, the reactions take place rapidly in dioxane and efficiently proceed in the presence of a bimetallic Pd(OAc)2/CuI as the catalyst, PPh3 as the ligand and LiOtBu or KOAc as the base.

Stereoselective copper-catalyzed Chan-Lam-Evans N-arylation of glucosamines with arylboronic acids at room temperature.

An efficient and practical N-arylation of glycosylamines with substituted aryl boronic acids has been established. Using Cu(OAc)2 and pyridine at room temperature under air atmosphere, the protocol proved to be general, and a variety of aryl N-glycosides have been prepared in good to excellent yields with exclusive ß selectivity.