The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues ordered with reference to three orthogonal axes 1 . While model organisms have provided much insight into this process, we know very little about gastrulation in humans due to the difficulty of obtaining embryos at such early stages of development, as well as to the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo 2 . Here we show that human embryonic stem cells can be used to generate gastruloids: three dimensional multicellular aggregates that differentiate to derivatives of the three germ layers organised spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis and, using spatial transcriptomics, we show that they exhibit patterned gene expression. This includes a somitogenesis signature that suggests that 72 hour human gastruloids exhibit features of Carnegie Stage 9 embryos 3 . Our study represents a new, experimentally tractable model system to reveal and probe human-specific regulatory processes occurring during axial organisation in early development.The body plan of mammalian embryos emerges through interactions of sequential cell fate decisions and morphogenetic events, which have hitherto been difficult to observe in humans.Human Embryonic Stem Cells (hESCs) 4 have opened up opportunities for studying early fate decisions, and have hinted at the existence of regulatory mechanisms specific to humans 5,6 . But, in contrast to the embryo, where proportionate populations interact with one another to generate tissues and organs, differentiation in adherent culture is heterogeneous and favours a limited number of cell types 7 . Seeding hESCs on micropatterned surfaces yields coordinated patterns of gene expression, but without the axial organization characteristic of embryos 8 . However, when mouse ESCs are aggregated in suspension under defined conditions, they generate 'gastruloids': a three-dimensional, in vitro model of mammalian development, which exhibits an embryo-like spatiotemporal organization of gene expression 9,10 . We hypothesised that similar human gastruloids could be derived from hESCs. Generation of human gastruloidsWhen hESCs in 2D culture were treated with Chiron, a Wnt agonist, for one day before seeding defined numbers in low-adherence plates in the presence of Chiron, they formed compact, spherical aggregates within a few hours (Fig. 1a, Extended Data Fig. 1a-c). These aggregates progressively broke symmetry and formed elongated structures, with maximal elongation at 72-96h (Fig. 1a-d). On average, ~66% of aggregates from each experiment displayed an elongated morphology at 72h. Although some of the structures remained TOP award (NWO-CW 714.016.001), and the Foundation for Fundamental Research on Matter, financially supported by NWO (FOM-14NOISE01) to S.C.v.d.B., A.A. and A.v.O.. This w...

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a na€ ıve pluripotent state. Efforts have been made to trigger na€ ıve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of na€ ıve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor 1 Ascorbic Acid 1 Forskolin) facilitating rapid induction of transgene-free na€ ıve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted na€ ıve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated na€ ıve pluripotency genes, and exhibited dependence on signaling pathways similar to na€ ıve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward na€ ıve pluripotency. Collectively, we demonstrate an alternate route to capture na€ ıve pluripotency in hESCs that is fast, reproducible, supports na€ ıve mESC derivation, and allows efficient differentiation. STEM CELLS 2015;33:2686-2698 SIGNIFICANCE STATEMENTNa€ ıve pluripotency, commonly displayed by mouse embryonic stem cells (ESCs), holds several advantages over stem cells exhibiting a primed pluripotent state such as human ESCs, which already show a bias towards certain lineages. We report the formulation of a novel culture condition with minimal components facilitating rapid, robust and efficient induction of na€ ıve pluripotency in primed human ESCs. These novel na€ ıve human ESCs were karyotypically normal, underwent efficient single cell passaging, exhibited a unique epigenetic and lncRNA profile and unbiased lineage-specific differentiation similar to mouse ESCs. This na€ ıve state of pluripotency is important for possible future regenerative cell applications including efficient genome engineering and targeted gene correction.

Mouse embryonic stem cells (mESCs) exist in a naive, primed and ground state of pluripotency. While comparative analyses of these pluripotency states have been reported, the mESCs utilized originated from various genetic backgrounds and were derived in different laboratories. mESC derivation in conventional LIF + serum culture conditions is strain dependent, with different genetic backgrounds potentially affecting subsequent stem cell characteristics. In the present study, we performed a comprehensive characterization of naive, primed and ground state mESCs originating from the same genetic background within our laboratory, by comparing their transcriptional profiles. We showed unique transcriptional profiles for naive, primed and ground state mESCs. While naive and ground state mESCs have more similar but not identical profiles, primed state mESCs show a very distinct profile. We further demonstrate that the differentiation propensity of mESCs to specific germ layers is highly dependent on their respective state of pluripotency.

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Objective Enteroendocrine cells (EECs) of the gastro-intestinal tract sense gut luminal factors and release peptide hormones or serotonin (5-HT) to coordinate energy uptake and storage. Our goal is to decipher the gene regulatory networks controlling EECs specification from enteroendocrine progenitors. In this context, we studied the role of the transcription factor Rfx6 which had been identified as the cause of Mitchell–Riley syndrome, characterized by neonatal diabetes and congenital malabsorptive diarrhea. We previously reported that Rfx6 was essential for pancreatic beta cell development and function; however, the role of Rfx6 in EECs differentiation remained to be elucidated. Methods We examined the molecular, cellular, and metabolic consequences of constitutive and conditional deletion of Rfx6 in the embryonic and adult mouse intestine. We performed single cell and bulk RNA-Seq to characterize EECs diversity and identify Rfx6-regulated genes. Results Rfx6 is expressed in the gut endoderm; later, it is turned on in, and restricted to, enteroendocrine progenitors and persists in hormone-positive EECs. In the embryonic intestine, the constitutive lack of Rfx6 leads to gastric heterotopia, suggesting a role in the maintenance of intestinal identity. In the absence of intestinal Rfx6, EECs differentiation is severely impaired both in the embryo and adult. However, the number of serotonin-producing enterochromaffin cells and mucosal 5-HT content are increased. Concomitantly, Neurog3-positive enteroendocrine progenitors accumulate. Combined analysis of single-cell and bulk RNA-Seq data revealed that enteroendocrine progenitors differentiate in two main cell trajectories, the enterochromaffin (EC) cells and the Peptidergic Enteroendocrine (PE) cells, the differentiation programs of which are differentially regulated by Rfx6. Rfx6 operates upstream of Arx , Pax6 and Isl1 to trigger the differentiation of peptidergic EECs such as GIP-, GLP-1-, or CCK-secreting cells. On the contrary, Rfx6 represses Lmx1a and Tph1 , two genes essential for serotonin biosynthesis. Finally, we identified transcriptional changes uncovering adaptive responses to the prolonged lack of enteroendocrine hormones and leading to malabsorption and lower food efficiency ratio in Rfx6-deficient mouse intestine. Conclusion These studies identify Rfx6 as an essential transcriptional regulator of EECs specification and shed light on the molecular mechanisms of intestinal failures in human RFX6-deficiencies such as Mitchell–Riley syndrome.