The ability to study human post-implantation development remains limited due to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (i.e., embryonic disk, bilaminar disk, yolk- and chorionic sacs, surrounding trophoblasts) remain lacking2. Mouse naïve embryonic stem cells (ESCs) have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation mouse Structured Stem cell-based Embryo Models with spatially organized morphogenesis (SEMs)3. Here, we extend these findings to humans, while using only genetically unmodified human naïve ESCs (in HENSM conditions)4. Such human fully integrated SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos including epiblast, hypoblast, extra-embryonic mesoderm, and trophoblast surrounding the latter layers. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days post-fertilization (dpf) (Carnegie stage 6a). This includes embryonic disk and bilaminar disk formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, PGC specification, polarized yolk sac with visceral and parietal endoderm, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, a trophoblast surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform may enable the experimental interrogation of previously inaccessible windows of human early post-implantation up to peri-gastrulation development.

The N6-methyladenosine (mA) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous mA-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers' expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers' role during early development that is gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.

The inherent limitations of fluorescence microscopy, notably the restricted number of color channels, have long constrained comprehensive spatial analysis in biological specimens. Here, we introduce cycleHCR technology that leverages multicycle DNA barcoding and Hybridization Chain Reaction (HCR) to surpass the conventional color barrier. cycleHCR facilitates high-specificity, single-shot imaging per target for RNA and protein species within thick specimens, mitigating the molecular crowding issues encountered with other imaging-based spatial omics techniques. We demonstrate whole-mount transcriptomics imaging of 254 genes within an E6.5\~7.0 mouse embryo, achieving precise three-dimensional gene expression and cell fate mapping across a specimen depth of \~ 310 µm. Utilizing expansion microscopy alongside protein cycleHCR, we unveil the complex network of 10 subcellular structures in primary mouse embryonic fibroblasts. Furthermore, in mouse hippocampal slice, we image 8 protein targets and profile the transcriptome of 120 genes, uncovering complex gene expression gradients and cell-type specific nuclear structural variances. cycleHCR provides a unifying framework for multiplex RNA and protein imaging, offering a quantitative solution for elucidating spatial regulations in deep tissue contexts for research and potentially diagnostic applications.

The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.

Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro, approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesis in mammals.

The recent derivation of human trophoblast stem cells (TSCs) from placental cytotrophoblasts and blastocysts opened opportunities for studying the development and function of the human placenta. Recent reports have suggested that human naïve, but not primed, pluripotent stem cells (PSCs) retain an exclusive potential to generate TSCs. Here we report that, in the absence of WNT stimulation, transforming growth factor β (TGF-β) pathway inhibition leads to direct and robust conversion of primed human PSCs into TSCs. The resulting primed PSC-derived TSC lines exhibit self-renewal, can differentiate into the main trophoblast lineages, and present RNA and epigenetic profiles that are indistinguishable from recently established TSC lines derived from human placenta, blastocysts, or isogenic human naïve PSCs expanded under human enhanced naïve stem cell medium (HENSM) conditions. Activation of nuclear Yes-associated protein (YAP) signaling is sufficient for this conversion and necessary for human TSC maintenance. Our findings underscore a residual plasticity in primed human PSCs that allows their in vitro conversion into extra-embryonic trophoblast lineages.

Neural progenitors (NP), found in fetal and adult brain, differentiate into neurons potentially able to be used in cell replacement therapies. This approach however, raises technical and ethical problems which limit their potential therapeutic use. Alternately, NPs can be obtained by transdifferentiation of non-neural somatic cells evading these difficulties. Human bone marrow mesenchymal stromal cells (MSCs) are suggested to transdifferentiate into NP-like cells, which however, have a low proliferation capacity. The present study demonstrates the requisite of cell adhesion for proliferation and survival of NP-like cells and re-evaluates some neuronal features after differentiation by standard procedures. Mature neuronal markers, though, were not detected by these procedures. A chemical differentiation approach was used in this study to convert MSCs-derived NP-like cells into neurons by using a cocktail of six molecules, CHIR99021, I-BET151, RepSox, DbcAMP, forskolin and Y-27632, defined after screening combinations of 22 small molecules. Direct transdifferentiation of MSCs into neuronal cells was obtained with the small molecule cocktail, without requiring the NP-like intermediate stage.

Human mesenchymal stem cells (MSCs) are good candidates for brain cell replacement strategies and have already been used as adjuvant treatments in neurological disorders. MSCs can be obtained from many different sources, and the present study compares the potential of neuronal transdifferentiation in MSCs from adult and neonatal sources (Wharton's jelly (WhJ), dental pulp (DP), periodontal ligament (PDL), gingival tissue (GT), dermis (SK), placenta (PLAC), and umbilical cord blood (UCB)) with a protocol previously tested in bone marrow- (BM-) MSCs consisting of a cocktail of six small molecules: I-BET151, CHIR99021, forskolin, RepSox, Y-27632, and dbcAMP (ICFRYA). Neuronal morphology and the presence of cells positive for neuronal markers (TUJ1 and MAP2) were considered attributes of neuronal induction. The ICFRYA cocktail did not induce neuronal features in WhJ-MSCs, and these features were only partial in the MSCs from dental tissues, SK-MSCs, and PLAC-MSCs. The best response was found in UCB-MSCs, which was comparable to the response of BM-MSCs. The addition of neurotrophic factors to the ICFRYA cocktail significantly increased the number of cells with complex neuron-like morphology and increased the number of cells positive for mature neuronal markers in BM- and UCB-MSCs. The neuronal cells generated from UCB-MSCs and BM-MSCs showed increased reactivity of the neuronal genes TUJ1, MAP2, NF-H, NCAM, ND1, TAU, ENO2, GABA, and NeuN as well as down- and upregulation of MSC and neuronal genes, respectively. The present study showed marked differences between the MSCs from different sources in response to the transdifferentiation protocol used here. These results may contribute to identifying the best source of MSCs for potential cell replacement therapies.