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...

Neutrophil granulocytes are the first and robust responders to the chemotactic molecules released from an inflamed acidic tissue. The aim of this study was to elucidate the role of microenvironmental pH in neutrophil chemotaxis. To this end, we used neutrophils from male C57BL/6J mice and combined live cell imaging chemotaxis assays with measurements of the intracellular pH (pHi) in varied extracellular pH (pHe). Observational studies were complemented by biochemical analyses of leukotriene B4 (LTB4) production and activation of the Cdc42 Rho GTPase. Our data show that pHi of neutrophils dose-dependently adapts to a given pH of the extracellular milieu. Neutrophil chemotaxis toward C5a has an optimum at pHi ∼7.1, and its pHi dependency is almost parallel to that of LTB4 production. Consequently, a shallow pHe gradient, resembling that encountered by neutrophils during extravasation from a blood vessel (pH ∼7.4) into the interstitium (pH ∼7.2), favors chemotaxis of stimulated neutrophils. Lowering pHe below pH 6.8, predominantly affects neutrophil chemotaxis, although the velocity is largely maintained. Inhibition of the Na+/H+ exchanger 1 (NHE1) with cariporide drastically attenuates neutrophil chemotaxis at the optimal pHi irrespective of the high LTB4 production. Neutrophil migration and chemotaxis are almost completely abrogated by inhibiting LTB4 production or blocking its receptor (BLT1). The abundance of the active GTP-bound form of Cdc42 is strongly reduced by NHE1 inhibition or pHe 6.5. In conclusion, we propose that the pH dependence of neutrophil chemotaxis toward C5a is caused by a pHi-dependent production of LTB4 and activation of Cdc42. Moreover, it requires the activity of NHE1.

The importance of the intracellular Ca 2+ concentration ([Ca 2+ ] i) in neutrophil function has been intensely studied. However, the role of the intracellular Na + concentration ([Na + ] i) which is closely linked to the intracellular Ca 2+ regulation has been largely overlooked. The [Na + ] i is regulated by Na + transport proteins such as the Na + /Ca 2+-exchanger (NCX1), Na + /K +-ATPase, and Na +-permeable, transient receptor potential melastatin 2 (TRPM2) channel. Stimulating with either N-formylmethionine-leucyl-phenylalanine (fMLF) or complement protein C5a causes distinct changes of the [Na + ] i. fMLF induces a sustained increase of [Na + ] i , surprisingly, reaching higher values in TRPM2 −/− neutrophils. This outcome is unexpected and remains unexplained. In both genotypes, C5a elicits only a transient rise of the [Na + ] i. The difference in [Na + ] i measured at t = 10 min after stimulation is inversely related to neutrophil chemotaxis. Neutrophil chemotaxis is more efficient in C5a than in an fMLF gradient. Moreover, lowering the extracellular Na + concentration from 140 to 72 mM improves chemotaxis of WT but not of TRPM2 −/− neutrophils. Increasing the [Na + ] i by inhibiting the Na + /K +-ATPase results in disrupted chemotaxis. This is most likely due to the impact of the altered Na + homeostasis and presumably NCX1 function whose expression was shown by means of qPCR and which critically relies on proper extra-to intracellular Na + concentration gradients. Increasing the [Na + ] i by a few mmol/l may suffice to switch its transport mode from forward (Ca 2+-efflux) to reverse (Ca 2+-influx) mode. The role of NCX1 in neutrophil chemotaxis is corroborated by its blocker, which also causes a complete inhibition of chemotaxis.

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