Generating human blastoids modeling blastocyst-stage embryos and implantation.

Human early development sets the stage for embryonic and adult life but remains difficult to investigate. A solution came from the ability of stem cells to organize into structures resembling preimplantation embryos-blastocysts-that we termed blastoids. This embryo model is available in unlimited numbers and could thus support scientific and medical advances. However, its predictive power depends on how faithfully it recapitulates the blastocyst. Here, we describe how we formed human blastoids that (1) efficiently achieve the morphology of the blastocyst and (2) form lineages according to the pace and sequence of blastocyst development, (3) ultimately forming cells that transcriptionally reflect the blastocyst (preimplantation stage). We employ three different commercially available 96- and 24-well microwell plates with results similar to our custom-made ones, and show that blastoids form in clinical in vitro fertilization medium and can be cryopreserved for shipping. Finally, we explain how blastoids replicate the directional process of implantation into endometrial organoids, specifically when these are hormonally stimulated. It takes 4 d for human blastoids to form and 10 d to prepare the endometrial implantation assay, and we have cultured blastoids up to 6 d (time-equivalent of day 13). On the basis of our experience, we anticipate that a person with ~1 year of human pluripotent stem cell culture experience and of organoid culture should be able to perform the protocol. Altogether, blastoids offer an opportunity to establish scientific and biomedical discovery programs for early pregnancy, and an ethical alternative to the use of embryos.

Protocol for Human Blastoids Modeling Blastocyst Development and Implantation.

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend on its ability to efficiently, timely, and faithfully recapitulate the sequences of blastocyst development (morphogenesis, specification, patterning), and to form cells reflecting the blastocyst stage. Here we show that naïve human pluripotent stem cells cultured in PXGL conditions and then triply inhibited for the Hippo, transforming growth factor- ß, and extracellular signal-regulated kinase pathways efficiently undergo morphogenesis to form blastoids (>70%). Matching with developmental timing (~4 days), blastoids unroll the blastocyst sequence of specification by producing analogs of the trophoblast and epiblast, followed by the formation of analogs of the primitive endoderm and the polar trophoblasts. This results in the formation of cells transcriptionally similar to the blastocyst (>96%) and a minority of post-implantation analogs. Blastoids efficiently pattern by forming the embryonic-abembryonic axis marked by the maturation of the polar region (NR2F2+), which acquires the specific potential to directionally attach to hormonally stimulated endometrial cells, as in utero. Such a human blastoid is a scalable, versatile, and ethical model to study human development and implantation in vitro.

Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction.

Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.

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.

Modulation of ß-catenin levels regulates cranial neural crest patterning and dispersal into first pharyngeal arch.

BACKGROUND: Vertebrate cranial neural crest cells (CNCCs) are multipotent, proximal to the source CNCC form the cranial ganglia. Distally, in the pharyngeal arches, they give rise to the craniofacial skeleton and connective tissues. Fate choices are made as CNCC pattern into distinct destination compartments. In spite of this importance, the mechanism patterning CNCC is poorly defined. RESULTS: Here, we report that a novel ß-catenin-dependent regulation of N-Cadherin levels may drive CNCC patterning. In mouse embryos, at the first pharyngeal arch axial level, membrane ß-catenin levels correlate with the extent of N-cadherin-mediated adhesion and thus suggest the presence of collective and dispersed states of CNCC. Using in vitro human neural crest model and chemical modulators of ß-catenin levels, we show a requirement for down-modulating ß-catenin for regulating N-cadherin levels and cell-cell adhesion. Similarly, in ß-catenin gain-of-function mutant mouse embryos, CNCC fail to lower N-cadherin levels. This indicates a failure to reduce cell-cell adhesion, which may underlie the failure of mutant CNCC to populate first pharyngeal arch. CONCLUSION: We suggest that ß-catenin-mediated regulation of CNCC adhesion, a previously underappreciated mechanism, underlies the patterning of CNCC into fate-specific compartments.

Infectivity of adeno-associated virus serotypes in mouse testis.

BACKGROUND: Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection. RESULTS: We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells. CONCLUSIONS: In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.

Divergent early mesoderm specification underlies distinct head and trunk muscle programmes in vertebrates.

Head and trunk muscles have discrete embryological origins and are governed by distinct regulatory programmes. Whereas the developmental route of trunk muscles from mesoderm is well studied, that of head muscles is ill defined. Here, we show that, unlike the myogenic trunk paraxial mesoderm, head mesoderm development is independent of the T/Tbx6 network in mouse. We reveal that, in contrast to Wnt and FGF-driven trunk mesoderm, dual inhibition of Wnt/ß-catenin and Nodal specifies head mesoderm. Remarkably, the progenitors derived from embryonic stem cells by dual inhibition efficiently differentiate into cardiac and skeletal muscle cells. This twin potential is the defining feature of cardiopharyngeal mesoderm: the head subtype giving rise to heart and branchiomeric head muscles. Therefore, our findings provide compelling evidence that dual inhibition specifies head mesoderm and unravel the mechanism that diversifies head and trunk muscle programmes during early mesoderm fate commitment. Significantly, this is the first report of directed differentiation of pluripotent stem cells, without transgenes, into progenitors with muscle/heart dual potential. Ability to generate branchiomeric muscle in vitro could catalyse efforts in modelling myopathies that selectively involve head muscles.

Co-expression of Tbx6 and Sox2 identifies a novel transient neuromesoderm progenitor cell state.

Elongation of the body axis is a key aspect of body plan development. Bipotential neuromesoderm progenitors (NMPs) ensure axial growth of embryos by contributing both to the spinal cord and mesoderm. The current model for the mechanism controlling NMP deployment invokes Tbx6, a T-box factor, to drive mesoderm differentiation of NMPs. Here, we identify a new population of Tbx6+ cells in a subdomain of the NMP niche in mouse embryos. Based on co-expression of a progenitor marker, Sox2, we identify this population as representing a transient cell state in the mesoderm-fated NMP lineage. Genetic lineage tracing confirms the presence of the Tbx6+ NMP cell state. Furthermore, we report a novel aspect of the documented Tbx6 mutant phenotype, namely an increase from two to four ectopic neural tubes, corresponding to the switch in NMP niche, thus highlighting the importance of Tbx6 function in NMP fate decision. This study emphasizes the function of Tbx6 as a bistable switch that turns mesoderm fate 'on' and progenitor state 'off', and thus has implications for the molecular mechanism driving NMP fate choice.