Evaluation of Stem-Cell Embryo Models by Integration with a Human Embryo Single-Cell Transcriptome Atlas.

Single-cell RNA sequencing (scRNA-seq) revolutionized our understanding of the molecular processes of early development and provided us with the means to capture biological heterogeneity and assess the cellular composition in early embryos. Comparative analysis of the transcriptional landscapes of embryos with single-cell resolution allows us to better understand and improve stem-cell-based embryo models. However, proper comparison between different single-cell datasets acquired by different laboratories and through different technologies is imperative for adequate analysis and findings. In this chapter, we focus on the analysis of human blastoids, which model the blastocyst, and their integrative analysis with human embryo datasets and a 2D in vitro early development model system dataset, which models epiblast, extraembryonic mesoderm, and trophoblast cells.

Spatially Self-Organized Three-Dimensional Neural Concentroid as a Novel Reductionist Humanized Model to Study Neurovascular Development.

Although human pluripotent stem cell (PSC)-derived brain organoids have enabled researchers to gain insight into human brain development and disease, these organoids contain solely ectodermal cells and are not vascularized as occurs during brain development. Here it is created less complex and more homogenous large neural constructs starting from PSC-derived neuroprogenitor cells (NPC), by fusing small NPC spheroids into so-called concentroids. Such concentroids consisted of a pro-angiogenic core, containing neuronal and outer radial glia cells, surrounded by an astroglia-dense outer layer. Incorporating PSC-derived endothelial cells (EC) around and/or in the concentroids promoted vascularization, accompanied by differential outgrowth and differentiation of neuronal and astroglia cells, as well as the development of ectodermal-derived pericyte-like mural cells co-localizing with EC networks. Single nucleus transcriptomic analysis revealed an enhanced neural cell subtype maturation and diversity in EC-containing concentroids, which better resemble the fetal human brain compared to classical organoids or NPC-only concentroids. This PSC-derived "vascularized" concentroid brain model will facilitate the study of neurovascular/blood-brain barrier development, neural cell migration, and the development of effective in vitro vascularization strategies of brain mimics.

A facile method to generate cerebral organoids from human pluripotent stem cells.

Human cerebral organoids (COs) are self-organizing three-dimensional (3D) neural structures that provide a human-specific platform to study the cellular and molecular processes that underlie different neurological events. The first step of CO generation from human pluripotent stem cells (hPSCs) is neural induction, which is an in vitro simulation of neural ectoderm development. Several signaling pathways cooperate during neural ectoderm development and in vitro differentiation of hPSCs toward neural cell lineages is also affected by them. In this study, we considered some of the known sources of these variable signaling cues arising from cell culture media components and sought to modulate their effects by applying a comprehensive combination of small molecules and growth factors for CO generation. Histological analysis demonstrated that these COs recapitulate the neural progenitor zone and early cortical layer organization, containing different types of neuronal and glial cells which was in accordance with single-nucleus transcriptome profiling results. Moreover, patch clamp and intracellular Ca2+ dynamic studies demonstrated that the COs behave as a functional neural network. Thus, this method serves as a facile protocol for generating hPSC-derived COs that faithfully mimic the features of their in vivo counterparts in the developing human brain. See also Figure 1(Fig. 1).

Inferring regulators of cell identity in the human adult pancreas.

Cellular identity during development is under the control of transcription factors that form gene regulatory networks. However, the transcription factors and gene regulatory networks underlying cellular identity in the human adult pancreas remain largely unexplored. Here, we integrate multiple single-cell RNA-sequencing datasets of the human adult pancreas, totaling 7393 cells, and comprehensively reconstruct gene regulatory networks. We show that a network of 142 transcription factors forms distinct regulatory modules that characterize pancreatic cell types. We present evidence that our approach identifies regulators of cell identity and cell states in the human adult pancreas. We predict that HEYL, BHLHE41 and JUND are active in acinar, beta and alpha cells, respectively, and show that these proteins are present in the human adult pancreas as well as in human induced pluripotent stem cell (hiPSC)-derived islet cells. Using single-cell transcriptomics, we found that JUND represses beta cell genes in hiPSC-alpha cells. BHLHE41 depletion induced apoptosis in primary pancreatic islets. The comprehensive gene regulatory network atlas can be explored interactively online. We anticipate our analysis to be the starting point for a more sophisticated dissection of how transcription factors regulate cell identity and cell states in the human adult pancreas.

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.

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.

Evaluating totipotency using criteria of increasing stringency.

Totipotency is the ability of a single cell to give rise to all of the differentiated cell types that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies on a variety of assays of variable stringency. Here, we describe criteria to define totipotency. We explain how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in mice, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbour increased totipotent potential relative to conventional embryonic stem cells under in vitro and in vivo conditions.

Dynamic reversal of random X-Chromosome inactivation during iPSC reprogramming.

Induction and reversal of chromatin silencing is critical for successful development, tissue homeostasis, and the derivation of induced pluripotent stem cells (iPSCs). X-Chromosome inactivation (XCI) and reactivation (XCR) in female cells represent chromosome-wide transitions between active and inactive chromatin states. Although XCI has long been studied, providing important insights into gene regulation, the dynamics and mechanisms underlying the reversal of stable chromatin silencing of X-linked genes are much less understood. Here, we use allele-specific transcriptomics to study XCR during mouse iPSC reprogramming in order to elucidate the timing and mechanisms of chromosome-wide reversal of gene silencing. We show that XCR is hierarchical, with subsets of genes reactivating early, late, and very late during reprogramming. Early genes are activated before the onset of late pluripotency genes activation. Early genes are located genomically closer to genes that escape XCI, unlike genes reactivating late. Early genes also show increased pluripotency transcription factor (TF) binding. We also reveal that histone deacetylases (HDACs) restrict XCR in reprogramming intermediates and that the severe hypoacetylation state of the inactive X Chromosome (Xi) persists until late reprogramming stages. Altogether, these results reveal the timing of transcriptional activation of monoallelically repressed genes during iPSC reprogramming, and suggest that allelic activation involves the combined action of chromatin topology, pluripotency TFs, and chromatin regulators. These findings are important for our understanding of gene silencing, maintenance of cell identity, reprogramming, and disease.

Lysosomal Pathways and Autophagy Distinctively Control Endothelial Cell Behavior to Affect Tumor Vasculature.

Cancer cell-stromal cell crosstalk is orchestrated by a plethora of ligand-receptor interactions generating a tumor microenvironment (TME) which favors tumor growth. The high pro-angiogenic nature of the TME perpetuates the chaotic network of structurally immature, low pericyte-covered vessels characteristic of the tumor vasculature. We previously demonstrated that chloroquine (CQ) -a lysosomotropic agent used as first-generation autophagy blocker in clinical trials- induced tumor vessel normalization and reduced tumor hypoxia. CQ improved both vessel structure and maturation, whereas the conditional knockout of the crucial autophagy gene Atg5 in endothelial cells (ECs) did not, thus highlighting a potential differential role for EC-associated autophagy and the lysosomes in pathological tumor angiogenesis. However, how CQ or ATG5-deficiency in ECs affect angiogenic signals regulating EC-pericyte interface and therefore vessel maturation, remains unknown. Here, we show that in ECs CQ constrained VEGF-A-mediated VEGF receptor (VEGFR)2 phosphorylation, a driver of angiogenic signaling. In the presence of CQ we observed increased expression of the decoy receptor VEGFR1 and of a lower molecular weight form of VEGFR2, suggesting receptor cleavage. Consequently, VEGF-A-driven EC spheroid sprouting was reduced by CQ treatment. Furthermore, CQ significantly affected the transcription and secretion of platelet-derived growth factor (PDGF)-AB/BB (upregulated) and Endothelin-1 (EDN1, downregulated), both modulators of perivascular cell (PC) behavior. In contrast, silencing of ATG5 in ECs had no effect on VEGFR2 to VEGFR1 ratio nor on PDGFB and EDN1 expression. Accordingly, mice harboring B16F10 melanoma tumors treated with CQ, displayed both an increased number of αSMA+ PCs covering tumor vessels and co-expressed PDGF receptor-ß, enabling PDGF ligand dependent recruitment. Moreover, upon CQ treatment the tumoral expression of angiopoietin-1 (Angpt1), which retains mural cells, and induces vessel stabilization by binding to the EC-localized cognate receptor (TIE2), was increased thus supporting the vessel normalization function of CQ. These features associated with improved tumor vasculature were not phenocopied by the specific deletion of Atg5 in ECs. In conclusion, this study further unravels endothelial cell autonomous and non-autonomous mechanisms by which CQ "normalizes" the intercellular communication in the tumor vasculature independent of autophagy.