Co-development of central and peripheral neurons with trunk mesendoderm in human elongating multi-lineage organized gastruloids

Olmsted Z.T., Paluh J.L.

Abstract The inaccessibility of scientific discovery to human development at post-implantation, including specialization and differentiation, necessitates sophisticated heterogenous 3D in vitro models. This protocol describes the generation and self-organization of human elongating multi-lineage organized (EMLO) gastruloids in suspension without the need for supplied extracellular matrix (ECM). EMLO gastruloids can be matured to large organoid states comprised of interconnected tissue microstructures of the trunk. These include correlates of the neuromesodermal progenitor-derived central nervous system, the neural crest cell-derived peripheral nervous system, the endoderm-derived primitive gut tube, and the mesoderm-derived splanchnic mesenchyme. Herein, we provide critical details for the reproducible generation of human EMLOs in vitro.

Olmsted Z.T., Paluh J.L.

The ability to reliably repair spinal cord injuries (SCI) will be one of the greatest human achievements realized in regenerative medicine. Until recently, the cellular path to this goal has been challenging. However, as detailed developmental principles are revealed in mouse and human models, their application in the stem cell community brings trunk and spine embryology into efforts to advance human regenerative medicine. New models of posterior embryo development identify neuromesodermal progenitors (NMPs) as a major bifurcation point in generating the spinal cord and somites and is leading to production of cell types with the full range of axial identities critical for repair of trunk and spine disorders. This is coupled with organoid technologies including assembloids, circuitoids, and gastruloids. We describe a paradigm for applying developmental principles towards the goal of cell-based restorative therapies to enable reproducible and effective near-term clinical interventions.

Zalc A., Sinha R., Gulati G.S., Wesche D.J., Daszczuk P., Swigut T., Weissman I.L., Wysocka J.

Reactivating neural crest pluripotency Cranial neural crest cells (CNCCs) are a transient cell group with an extraordinary differentiation potential that extends beyond its ectodermal lineage to form the majority of facial mesenchyme. Zalc et al. identified a neuroepithelial precursor population that transiently reactivates pluripotency factors to generate CNCCs. The pluripotency factor Oct4 is required for the expansion of CNCC developmental potential to form facial mesenchyme. Analysis of the chromatin landscape of Oct4 + CNCC precursors showed that these cells resemble those of epiblast stem cells, with additional features suggestive of future priming for neural crest programs. Thus, to expand their cellular potency, CNCC precursors undergo a natural in vivo reprogramming event. Science , this issue p. eabb4776

Rossi G., Broguiere N., Miyamoto M., Boni A., Guiet R., Girgin M., Kelly R.G., Kwon C., Lutolf M.P.

Organoids are powerful models for studying tissue development, physiology, and disease. However, current culture systems disrupt the inductive tissue-tissue interactions needed for the complex morphogenetic processes of native organogenesis. Here, we show that mouse embryonic stem cells (mESCs) can be coaxed to robustly undergo fundamental steps of early heart organogenesis with an in-vivo-like spatiotemporal fidelity. These axially patterned embryonic organoids (gastruloids) mimic embryonic development and support the generation of cardiovascular progenitors, including first and second heart fields. The cardiac progenitors self-organize into an anterior domain reminiscent of a cardiac crescent before forming a beating cardiac tissue near a putative primitive gut-like tube, from which it is separated by an endocardial-like layer. These findings unveil the surprising morphogenetic potential of mESCs to execute key aspects of organogenesis through the coordinated development of multiple tissues. This platform could be an excellent tool for studying heart development in unprecedented detail and throughput.

Andersen J., Revah O., Miura Y., Thom N., Amin N.D., Kelley K.W., Singh M., Chen X., Thete M.V., Walczak E.M., Vogel H., Fan H.C., Paşca S.P.

Neurons in the cerebral cortex connect through descending pathways to hindbrain and spinal cord to activate muscle and generate movement. Although components of this pathway have been previously generated and studied in vitro, the assembly of this multi-synaptic circuit has not yet been achieved with human cells. Here, we derive organoids resembling the cerebral cortex or the hindbrain/spinal cord and assemble them with human skeletal muscle spheroids to generate 3D cortico-motor assembloids. Using rabies tracing, calcium imaging, and patch-clamp recordings, we show that corticofugal neurons project and connect with spinal spheroids, while spinal-derived motor neurons connect with muscle. Glutamate uncaging or optogenetic stimulation of cortical spheroids triggers robust contraction of 3D muscle, and assembloids are morphologically and functionally intact for up to 10 weeks post-fusion. Together, this system highlights the remarkable self-assembly capacity of 3D cultures to form functional circuits that could be used to understand development and disease.

Veenvliet J.V., Bolondi A., Kretzmer H., Haut L., Scholze-Wittler M., Schifferl D., Koch F., Guignard L., Kumar A.S., Pustet M., Heimann S., Buschow R., Wittler L., Timmermann B., Meissner A., et. al.

Trunk formation in a dish Building mammalian embryos from self-organizing stem cells in culture would accelerate the investigation of morphogenetic and differentiation processes that shape the body plan. Veenvliet et al. report a method for generating embryonic trunk-like structures (TLSs) with a neural tube, somites, and gut by embedding mouse embryonic stem cell aggregates in an extracellular matrix surrogate. Live imaging and comparative single-cell transcriptomics indicate that TLS formation is analogous to mouse development. TLSs therefore provide a scalable, tractable, and accessible high-throughput platform for decoding mammalian embryogenesis at a high level of resolution. Science , this issue p. eaba4937

Abdullah M.A., Amini N., Yang L., Paluh J.L., Wang J.

Multipotent neural stem cells (NSCs) are widely applied in pre-clinical and clinical trials as a cell source to promote tissue regeneration in neurodegenerative diseases. Frequently delivered as dissociated cells, aggregates or self-organized rosettes, it is unknown whether disruption of the NSC rosette morphology or method of formation affect signaling profiles of these cells that may impact uniformity of outcomes in cell therapies. Here we generate a neural cell-cell interaction microchip (NCCIM) as an in vitro platform to simultaneously track an informed panel of cytokines and co-evaluate cell morphology and biomarker expression coupled to a sandwich ELISA platform. We apply multiplex in situ tagging technology (MIST) to evaluate ten cytokines (PDGF-AA, GDNF, BDNF, IGF-1, FGF-2, IL-6, BMP-4, CNTF, β-NGF, NT-3) on microchips for EB-derived rosettes, single cell dissociated rosettes and reformed rosette neurospheres. Of the cytokines evaluated, EB-derived rosettes secrete PDGF-AA, GDNF and FGF-2 prominently, whereas this profile is temporarily lost upon dissociation to single cells and in reformed neurospheres two additional cytokines, BDNF and β-NGF, are also secreted. This study on NSC rosettes demonstrates the development, versatility and utility of the NCCIM as a sensitive multiplex detector of cytokine signaling in a high throughput and controlled microenvironment. The NCCIM is expected to provide important new information to refine cell source choices in therapies as well as to support development of informative 2D or 3D in vitro models including areas of neurodegeneration or neuroplasticity.

Frith T.J., Gogolou A., Hackland J.O., Hewitt Z.A., Moore H.D., Barbaric I., Thapar N., Burns A.J., Andrews P.W., Tsakiridis A., McCann C.J.

Summary The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1 and 7. Defects in the development and function of the ENS cause a range of enteric neuropathies, including Hirschsprung disease. Little is known about the signals that specify early ENS progenitors, limiting progress in the generation of enteric neurons from human pluripotent stem cells (hPSCs) to provide tools for disease modeling and regenerative medicine for enteric neuropathies. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and, following in vivo transplantation, achieved long-term colonization of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS.

Sahu S., Sharan S.K.

Summary The astounding capacity of pluripotent stem cells (PSCs) to differentiate and self-organize has revolutionized the development of 3D cell culture models. The major advantage is its ability to mimic in vivo microenvironments and cellular interactions when compared with the classical 2D cell culture models. Recent innovations in generating embryo-like structures (including blastoids and gastruloids) from PSCs have advanced the experimental accessibility to understand embryogenesis with immense potential to model human development. Taking cues on how embryonic development leads to organogenesis, PSCs can also be directly differentiated to form mini-organs or organoids of a particular lineage. Organoids have opened new avenues to augment our understanding of stem cell and regenerative biology, tissue homeostasis, and disease mechanisms. In this review, we provide insights from developmental biology with a comprehensive resource of signaling pathways that in a coordinated manner form embryo-like structures and organoids. Moreover, the advent of assembloids and multilineage organoids from PSCs opens a new dimension to study paracrine function and multi-tissue interactions in vitro. Although this led to an avalanche of enthusiasm to utilize organoids for organ transplantation studies, we examine the current limitations and provide perspectives to improve reproducibility, scalability, functional complexity, and cell-type characterization. Taken together, these 3D in vitro organ-specific and patient-specific models hold great promise for drug discovery, clinical management, and personalized medicine.

Han L., Chaturvedi P., Kishimoto K., Koike H., Nasr T., Iwasawa K., Giesbrecht K., Witcher P.C., Eicher A., Haines L., Lee Y., Shannon J.M., Morimoto M., Wells J.M., Takebe T., et. al.

Visceral organs, such as the lungs, stomach and liver, are derived from the fetal foregut through a series of inductive interactions between the definitive endoderm (DE) and the surrounding splanchnic mesoderm (SM). While DE patterning is fairly well studied, the paracrine signaling controlling SM regionalization and how this is coordinated with epithelial identity is obscure. Here, we use single cell transcriptomics to generate a high-resolution cell state map of the embryonic mouse foregut. This identifies a diversity of SM cell types that develop in close register with the organ-specific epithelium. We infer a spatiotemporal signaling network of endoderm-mesoderm interactions that orchestrate foregut organogenesis. We validate key predictions with mouse genetics, showing the importance of endoderm-derived signals in mesoderm patterning. Finally, leveraging these signaling interactions, we generate different SM subtypes from human pluripotent stem cells (hPSCs), which previously have been elusive. The single cell data can be explored at: https://research.cchmc.org/ZornLab-singlecell . The fetal murine foregut develops into visceral organs via interactions between the mesoderm and endoderm, but how is unclear. Here, the authors use single cell RNAseq to show a diversity in organ specific splanchnic mesoderm cell-types, infer a signalling network governing organogenesis and use this to differentiate human pluripotent stem cells.

Olmsted Z.T., Stigliano C., Badri A., Zhang F., Williams A., Koffas M.A., Xie Y., Linhardt R.J., Cibelli J., Horner P.J., Paluh J.L.

Cell therapy for the injured spinal cord will rely on combined advances in human stem cell technologies and delivery strategies. Here we encapsulate homotypic spinal cord neural stem cells (scNSCs) in an alginate-based neural ribbon delivery platform. We perform a comprehensive in vitro analysis and qualitatively demonstrate graft survival and injury site retention using a rat C4 hemi-contusion model. Pre-configured neural ribbons are transport-stable modules that enable site-ready injection, and can support scNSC survival and retention in vivo. Neural ribbons offer multifunctionality in vitro including co-encapsulation of the injury site extracellular matrix modifier chondroitinase ABC (chABC), tested here in glial scar models, and ability of cervically-patterned scNSCs to differentiate within neural ribbons and project axons for integration with 3-D external matrices. This is the first extensive in vitro characterization of neural ribbon technology, and constitutes a plausible method for reproducible delivery, placement, and retention of viable neural cells in vivo.

Moris N., Anlas K., van den Brink S.C., Alemany A., Schröder J., Ghimire S., Balayo T., van Oudenaarden A., Martinez Arias A.

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 that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids—three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development. Human gastruloids—three-dimensional aggregates derived from human embryonic stem cells—show features of human embryos at around 19–21 days, and provide a model for the study of early human development.

van den Brink S.C., Alemany A., van Batenburg V., Moris N., Blotenburg M., Vivié J., Baillie-Johnson P., Nichols J., Sonnen K.F., Martinez Arias A., van Oudenaarden A.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1–3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral–caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner. Single-cell RNA sequencing and spatial transcriptomics reveal that the somitogenesis clock is active in mouse gastruloids, which can be induced to generate somites with the correct rostral–caudal patterning.

Faustino Martins J., Fischer C., Urzi A., Vidal R., Kunz S., Ruffault P., Kabuss L., Hube I., Gazzerro E., Birchmeier C., Spuler S., Sauer S., Gouti M.

Neuromuscular networks assemble during early human embryonic development and are essential for the control of body movement. Previous neuromuscular junction modeling efforts using human pluripotent stem cells (hPSCs) generated either spinal cord neurons or skeletal muscles in monolayer culture. Here, we use hPSC-derived axial stem cells, the building blocks of the posterior body, to simultaneously generate spinal cord neurons and skeletal muscle cells that self-organize to generate human neuromuscular organoids (NMOs) that can be maintained in 3D for several months. Single-cell RNA-sequencing of individual organoids revealed reproducibility across experiments and enabled the tracking of the neural and mesodermal differentiation trajectories as organoids developed and matured. NMOs contain functional neuromuscular junctions supported by terminal Schwann cells. They contract and develop central pattern generator-like neuronal circuits. Finally, we successfully use NMOs to recapitulate key aspects of myasthenia gravis pathology, thus highlighting the significant potential of NMOs for modeling neuromuscular diseases in the future.

Sagner A., Briscoe J.

ABSTRACT The vertebrate spinal cord comprises multiple functionally distinct neuronal cell types arranged in characteristic positions. During development, these different types of neurons differentiate from transcriptionally distinct neural progenitors that are arrayed in discrete domains along the dorsal-ventral and anterior-posterior axes of the embryonic spinal cord. This organization arises in response to morphogen gradients acting upstream of a gene regulatory network, the architecture of which determines the spatial and temporal pattern of gene expression. In recent years, substantial progress has been made in deciphering the regulatory network that underlies the specification of distinct progenitor and neuronal cell identities. In this Review, we outline how distinct neuronal cell identities are established in response to spatial and temporal patterning systems, and outline novel experimental approaches to study the emergence and function of neuronal diversity in the spinal cord.

Sozen B., Tam P.P., Pera M.F.

ABSTRACT Pluripotency, the capacity to generate all cells of the body, is a defining property of a transient population of epiblast cells found in pre-, peri- and post-implantation mammalian embryos. As development progresses, the epiblast cells undergo dynamic transitions in pluripotency states, concurrent with the specification of extra-embryonic and embryonic lineages. Recently, stem cell-based models of pre- and post-implantation human embryonic development have been developed using stem cells that capture key properties of the epiblast at different developmental stages. Here, we review early primate development, comparing pluripotency states of the epiblast in vivo with cultured pluripotent cells representative of these states. We consider how the pluripotency status of the starting cells influences the development of human embryo models and, in turn, what we can learn about the human pluripotent epiblast. Finally, we discuss the limitations of these models and questions arising from the pioneering studies in this emerging field.

Braccioli L., van den Brand T., Alonso Saiz N., Fountas C., Celie P.H., Kazokaitė-Adomaitienė J., de Wit E.

Şenkal-Turhan S., Bulut-Okumuş E., Şahin F., Yavuz Y., Yılmaz B., Şişli H.B., Kalaycı S., Özgün H.B., Ömeroğlu Ulu Z., Akkuş Süt P., Doğan A.

Paredes-Espinosa M.B., Paluh J.L.

The evolution of stem cell-based heart models from cells and tissues to organoids and assembloids and recently synthetic embryology gastruloids, is poised to revolutionize our understanding of cardiac development, congenital to adult diseases, and patient customized therapies. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have already been integrated into transplantable patches and are in preclinical efforts to reverse fibrotic scarring from myocardial infarctions. To inform on the complexity of heart diseases, multi-tissue morphogenic heart models are needed that replicate fundamental components of heart function to heart organogenesis in vitro and which require a deep understanding of heart development. Organoid and assembloid models capture selected multicellular cardiac processes, such as chamber formation and priming events for vascularization. Gastruloid heart models offer deeper insights as synthetic embryology to mimic multi-staged developmental events of in vivo heart organogenesis including established heart fields, crescent formation and heart tube development along with vascular systemic foundation and even further steps. The human Elongating Multi-Lineage Organized Cardiac (EMLOC) gastruloid model captures these stages and additional events including chamber genesis, patterned vascularization, and extrinsic central and intrinsic cardiac nervous system (CNS-ICNS) integration guided by spatiotemporal and morphogenic processes with neural crest cells. Gastruloid synthetic embryology heart models offer new insights into previously hidden processes of development and provide powerful platforms for addressing heart disease that extends beyond cardiomyocytes, such as arrhythmogenic diseases, congenital defects, and systemic injury interactions, as in spinal cord injuries. The holistic view that is emerging will reveal heart development and disease in unprecedented detail to drive transformative state-of-the-art innovative applications for heart health.

Ascenzi B.M., Badner A., Vidal P.M.

Rito T., Libby A.R., Demuth M., Domart M., Cornwall-Scoones J., Briscoe J.

Abstract The formation of the vertebrate body involves the coordinated production of trunk tissues from progenitors located in the posterior of the embryo. Although in vitro models using pluripotent stem cells replicate aspects of this process1–10, they lack crucial components, most notably the notochord—a defining feature of chordates that patterns surrounding tissues11. Consequently, cell types dependent on notochord signals are absent from current models of human trunk formation. Here we performed single-cell transcriptomic analysis of chick embryos to map molecularly distinct progenitor populations and their spatial organization. Guided by this map, we investigated how differentiating human pluripotent stem cells develop a stereotypical spatial organization of trunk cell types. We found that YAP inactivation in conjunction with FGF-mediated MAPK signalling facilitated WNT pathway activation and induced expression of TBXT (also known as BRA). In addition, timely inhibition of WNT-induced NODAL and BMP signalling regulated the proportions of different tissue types, including notochordal cells. This enabled us to create a three-dimensional model of human trunk development that undergoes morphogenetic movements, producing elongated structures with a notochord and ventral neural and mesodermal tissues. Our findings provide insights into the mechanisms underlying vertebrate notochord formation and establish a more comprehensive in vitro model of human trunk development. This paves the way for future studies of tissue patterning in a physiologically relevant environment.

Robles-Garcia M., Thimonier C., Angoura K., Ozga E., MacPherson H., Blin G.

Notochord progenitors (NotoPs) represent a scarce yet crucial embryonic cell population, playing important roles in embryo patterning and eventually giving rise to the cells that form and maintain intervertebral discs. The mechanisms regulating NotoPs emergence are unclear. This knowledge gap persists due to the inherent complexity of cell fate patterning during gastrulation, particularly within the anterior primitive streak (APS), where NotoPs first arise alongside neuro-mesoderm and endoderm. To gain insights into this process, we use micropatterning together with FGF and the WNT pathway activator CHIR9901, to guide the development of human embryonic stem cells into reproducible patterns of APS cell fates. We show that CHIR9901 dosage dictates the downstream dynamics of endogenous TGFbeta signalling which in turn controls cell fate decisions. While sustained NODAL signalling defines endoderm and NODAL inhibition is imperative for neuro-mesoderm emergence, timely inhibition of NODAL signalling with spatial confinement potentiates WNT activity and enables us to generate NotoPs efficiently. Our work elucidates the signalling regimes underpinning NotoPs emergence and provides novel insights into the regulatory mechanisms controlling the balance of APS cell fates during gastrulation.

Sun P., Yuan Y., Lv Z., Yu X., Ma H., Liang S., Zhang J., Zhu J., Lu J., Wang C., Huan L., Jin C., Wang C., Li W.

Chen Z., Yao H., Yao X., Zheng R., Yang Y., Liu Z., Zhang R., Cheng Y.

Heart failure (HF) represents the terminal stage of cardiovascular diseases, with limited therapeutic options currently available. Calotropin (CAL), a cardenolide isolated from Calotropis gigantea, exhibits a similar chemical structure and inhibitory effect on Na+/K+-ATPase to digoxin, a positive inotropic drugs used in heart failure treatment. However, the specific effect of calotropin in ischemic HF (IHF) remains unknown. The objective of this study is to assess the anti-HF effect and clarify its underlying mechanisms. The left anterior descending (LAD) artery ligation on Male Sprague-Dawley (SD) rats was used to construct ischemic HF model. Daily administration of CAL at 0.05 mg/kg significantly enhanced ejection fraction (EF) and fractional shortening (FS), while inhibiting cardiac fibrosis in IHF rats. CAL reduced the OGD/R-induced H9c2 cell injury. Furthermore, CAL upregulated the expression of SERCA2a and SIRT1. The cardioprotective effect of CAL against IHF was abrogated in the presence of the SIRT1 inhibitor EX527. Notably, we identified FOXD3 as a pivotal transcription factor mediating CAL-induced SERCA2a regulation. CAL promoted the deacetylation and nuclear translocation of FOXD3 in a SIRT1-dependent manner. In conclusion, our study explores a novel mechanism of calotropin for improving cardiac dysfunction in ischemic heart failure by regulating SIRT1/FOXD3/SERCA2a pathway.

Kee N., Leboeuf M., Gómez S., Petipré C., Mei I., Benlefki S., Hagey D.W., Dias J., Lallemend F., EL Andaloussi S., Ericson J., Hedlund E.

ABSTRACTElongation of the posterior body axis is driven by multi-potent neuromesodermal progenitors (NMPs), which both self-renew and simultaneously generate neural tube, neural crest, and presomitc mesoderm lineages at successive anterior posterior (A-P) levels. The ensuing diversification of these three NMP lineages is remarkably extensive, and also essential for an immense range of clinically important adult posterior body tissues. Here, we describe a human pluripotent stem cell protocol that successfully specifies authentic NMPs using a cocktail of seven factors (7F). 7F-NMPs express requisite markers, exhibit co-linearHOXactivation, and can be purposely specified into each of the three NMP daughter lineages, demonstrating genuine multi-potency. 3D assembly of neural tube, neural crest, and presomitic mesoderm spheroids followed by long-term floating culture derives mature, multi-compartment Posterior Axial Assembloids, or PAXAs. PAXAs constitute a complex heterogeneous tissue containing spinal motor neurons and interneurons, central and peripheral glia, connective tissues, muscle satellite cells and contractile muscle fibres. Together, 7F-NMP and PAXA protocols establish a versatile in vitro platform to model mechanisms of human posterior body axis development, and for the study of a wide range of human diseases.

Turner D.A., Martinez Arias A.

AbstractGastrulation is a key milestone in the development of an organism. It is a period of cell proliferation and coordinated cellular rearrangement, that creates an outline of the body plan. Our current understanding of mammalian gastrulation has been improved by embryo culture, but there are still many open questions that are difficult to address because of the intrauterine development of the embryos and the low number of specimens. In the case of humans, there are additional difficulties associated with technical and ethical challenges. Over the last few years, pluripotent stem cell models are being developed that have the potential to become useful tools to understand the mammalian gastrulation. Here we review these models with a special emphasis on gastruloids and provide a survey of the methods to produce them robustly, their uses, relationship to embryos, and their prospects as well as their limitations.

Hamazaki N., Yang W., Kubo C.A., Qiu C., Martin B.K., Garge R.K., Regalado S.G., Nichols E.K., Pendyala S., Bradley N., Fowler D.M., Lee C., Daza R.M., Srivatsan S., Shendure J.

AbstractGastruloids are a powerful in vitro model of early human development. However, although elongated and composed of all three germ layers, human gastruloids do not morphologically resemble post-implantation human embryos. Here we show that an early pulse of retinoic acid (RA), together with later Matrigel, robustly induces human gastruloids with posterior embryo-like morphological structures, including a neural tube flanked by segmented somites and diverse cell types, including neural crest, neural progenitors, renal progenitors and myocytes. Through in silico staging based on single-cell RNA sequencing, we find that human RA-gastruloids progress further than other human or mouse embryo models, aligning to E9.5 mouse and CS11 cynomolgus monkey embryos. We leverage chemical and genetic perturbations of RA-gastruloids to confirm that WNT and BMP signalling regulate somite formation and neural tube length in the human context, while transcription factors TBX6 and PAX3 underpin presomitic mesoderm and neural crest, respectively. Looking forward, RA-gastruloids are a robust, scalable model for decoding early human embryogenesis.

Wu J., Fu J.

Developing functional organs from stem cells remains a challenging goal in regenerative medicine. Existing methodologies, such as tissue engineering, bioprinting, and organoids, only offer partial solutions. This perspective focuses on two promising approaches emerging for engineering human organs from stem cells: stem cell-based embryo models and interspecies organogenesis. Both approaches exploit the premise of guiding stem cells to mimic natural development. We begin by summarizing what is known about early human development as a blueprint for recapitulating organogenesis in both embryo models and interspecies chimeras. The latest advances in both fields are discussed before highlighting the technological and knowledge gaps to be addressed before the goal of developing human organs could be achieved using the two approaches. We conclude by discussing challenges facing embryo modeling and interspecies organogenesis and outlining future prospects for advancing both fields toward the generation of human tissues and organs for basic research and translational applications.

Mavrommatis L., Daya N., Volke L., Lu I., Zhuge H., Stehling M., Zeuschner D., Jeong H., Yang J.H., Meyer zu Horste G., Brand-Saberi B., Scholer H.R., Vorgerd M., Zaehres H.

SummarySpatiotemporal recapitulation of long-range trajectories for lineages that influence body patterning along the medio-lateral and proximal-distal axes during embryogenesis in anin vitrosystem remains elusive. Here we introduce a three-dimensional organoid approach, termed Gastruloids-Lateraloid-Musculoids (GLMs), to model human neural crest, lateral plate mesoderm and skeletal muscle lineage development at the forelimb level following gastrulation and during limb patterning. GLMs harvest neuro-mesodermal progenitors with the potential to establish neural and paraxial mesodermal populations, while single cell analyses and spatial transcriptomics demonstrate promotion of mesodermal lineage segregation during gastrulation and spatial recapitulation of migration events along the medio-lateral axis for vagal neural crest, hypaxial myogenesis and lateral plate mesodermal lineages. Comparative analyses to developmental atlases and adult muscle stem cell data confirm a pool of hypaxial migrating myogenic progenitors that in a niche dependent manner change their embryonic anatomical developmental program to a fetal myogenic program, thus enabling them to resist specification in a cell autonomous manner and facilitate long termin vitroexpansion. GLMs model human myogenesis at the forelimb level, establish fetal muscle stem cells equivalent to those that sustain the growth phase of the embryo and provide a 3Din vitrosystem for investigating neural crest, early fore-gut and lateral plate mesoderm development.

Rossant J.

Understanding the processes and mechanisms underlying early human embryo development has become an increasingly active and important area of research. It has potential for insights into important clinical issues such as early pregnancy loss, origins of congenital anomalies and developmental origins of adult disease, as well as fundamental insights into human biology. Improved culture systems for preimplantation embryos, combined with the new tools of single cell genomics and live imaging, are providing new insights into the similarities and differences between human and mouse development. However, access to human embryo material is still restricted and extended culture of early embryos has regulatory and ethical concerns. Stem cell-derived models of different phases of human development can potentially overcome these limitations and provide a scalable source of material to explore the early postimplantation stages of human development. To date, such models are clearly incomplete replicas of normal development but future technological improvements can be envisaged. The ethical and regulatory environment for such studies remains to be fully resolved.