Extended culture of 2D gastruloids to model human mesoderm development.

Micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 (2D gastruloids) are among the most widely used stem cell models for human gastrulation. Due to its simplicity and reproducibility, this system is ideal for high throughput quantitative studies of tissue patterning and has led to many insights into the mechanisms of mammalian gastrulation. However, 2D gastruloids have only been studied up to 48h. Here we extended this system to 96h. We discovered a phase of highly reproducible morphogenesis during which directed migration from the primitive streak-like region gives rise to a mesodermal layer beneath an epiblast-like layer. Multiple types of mesoderm arise with striking spatial organization including lateral mesoderm-like cells on the colony border and paraxial mesoderm-like further inside the colony. Single cell transcriptomics showed strong similarity of these cells to mesoderm in human and non-human primate embryos. However, our data suggest that the annotation of the reference human embryo may need to be revised. This illustrates that extended culture of 2D gastruloids provides a powerful model for human mesoderm differentiation and morphogenesis.

Time-integrated BMP signaling determines fate in a stem cell model for early human development.

How paracrine signals are interpreted to yield multiple cell fate decisions in a dynamic context during human development in vivo and in vitro remains poorly understood. Here we report an automated tracking method to follow signaling histories linked to cell fate in large numbers of human pluripotent stem cells (hPSCs). Using an unbiased statistical approach, we discover that measured BMP signaling history correlates strongly with fate in individual cells. We find that BMP response in hPSCs varies more strongly in the duration of signaling than the level. However, both the level and duration of signaling activity control cell fate choices only by changing the time integral. Therefore, signaling duration and level are interchangeable in this context. In a stem cell model for patterning of the human embryo, we show that signaling histories predict the fate pattern and that the integral model correctly predicts changes in cell fate domains when signaling is perturbed. Our data suggest that mechanistically, BMP signaling is integrated by SOX2.

The many dimensions of germline competence.

Primordial germ cell (PGC) specification is the first step in the development of the germline. Recent work has elucidated human-mouse differences in PGC differentiation and identified cell states with enhanced competency for PGC-like cell (PGCLC) differentiation in vitro in both species. However, it remains a subject of debate how different PGC competent states in vitro relate to each other, to embryonic development, and to the origin of PGCs in vivo. Here we review recent literature on human PGCLC differentiation in the context of mouse and non-human primate models. In contrast to what was previously thought, recent work suggests human pluripotent stem cells (hPSCs) are highly germline competent. We argue that paradoxical observations regarding the origin and signaling requirements of hPGCLCs may be due to local cell interactions. These confound assays of competence by generating endogenous signaling gradients and spatially modulating the ability to receive exogenous inductive signals. Furthermore, combinatorial signaling suggests that there is no unique germline competent state: rather than a one-dimensional spectrum of developmental progression, competence should be considered in a higher dimensional landscape of cell states.

Spatial Single Cell Analysis of Proteins in 2D Human Gastruloids Using Iterative Immunofluorescence.

During development, cell signaling instructs tissue patterning, the process by which initially identical cells give rise to spatially organized structures consisting of different cell types. How multiple signals combinatorially instruct fate in space and time remains poorly understood. Simultaneous measurement of signaling activity through multiple signaling pathways and of the cell fates they control is critical to addressing this problem. Here we describe an iterative immunofluorescence protocol and computational pipeline to interrogate pattern formation in a 2D model of human gastrulation with far greater multiplexing than is possible with standard immunofluorescence techniques. This protocol and computational pipeline together enable imaging followed by spatial and co-localization analysis of over 27 proteins in the same gastruloids. We demonstrate this by clustering single cell protein expression, using techniques familiar from scRNA-seq, and linking this to spatial position to calculate spatial distributions and cell signaling activity of different cell types. These methods are not limited to patterning in 2D gastruloids and can be easily extended to other contexts. In addition to the iterative immunofluorescence protocol and analysis pipeline, Support Protocols for 2D gastruloid differentiation and producing micropatterned multi-well slides are included. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Iterative immunofluorescence Basic Protocol 2: Computational analysis pipeline Support Protocol 1: Generating micropatterned multi-well slides Support Protocol 2: Differentiation of 2D gastruloids.

The time integral of BMP signaling determines fate in a stem cell model for early human development.

How paracrine signals are interpreted to yield multiple cell fate decisions in a dynamic context during human development in vivo and in vitro remains poorly understood. Here we report an automated tracking method to follow signaling histories linked to cell fate in large numbers of human pluripotent stem cells (hPSCs). Using an unbiased statistical approach, we discovered that measured BMP signaling history correlates strongly with fate in individual cells. We found that BMP response in hPSCs varies more strongly in the duration of signaling than the level. However, we discovered that both the level and duration of signaling activity control cell fate choices only by changing the time integral of signaling and that duration and level are therefore interchangeable in this context. In a stem cell model for patterning of the human embryo, we showed that signaling histories predict the fate pattern and that the integral model correctly predicts changes in cell fate domains when signaling is perturbed. Using an RNA-seq screen we then found that mechanistically, BMP signaling is integrated by SOX2.

Stable iPSC-derived NKX2-1+ lung bud tip progenitor organoids give rise to airway and alveolar cell types.

Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.

Efficient differentiation of human primordial germ cells through geometric control reveals a key role for Nodal signaling.

Human primordial germ cells (hPGCs) form around the time of implantation and are the precursors of eggs and sperm. Many aspects of hPGC specification remain poorly understood because of the inaccessibility of the early postimplantation human embryo for study. Here, we show that micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 give rise to hPGC-like cells (hPGCLC) and use these as a quantitatively reproducible and simple in vitro model to interrogate this important developmental event. We characterize micropatterned hPSCs up to 96 hr and show that hPGCLC populations are stable and continue to mature. By perturbing signaling during hPGCLC differentiation, we identify a previously unappreciated role for Nodal signaling and find that the relative timing and duration of BMP and Nodal signaling are critical parameters controlling the number of hPGCLCs. We formulate a mathematical model for a network of cross-repressive fates driven by Nodal and BMP signaling, which predicts the measured fate patterns after signaling perturbations. Finally, we show that hPSC colony size dictates the efficiency of hPGCLC specification, which led us to dramatically improve the efficiency of hPGCLC differentiation.

In humans and other animals, eggs and sperm are unique cells that pass on genetic material to the next generation. They originate from a small group of cells called primordial germ cells that form early in life in the developing embryo. Several different signal molecules including ones known as BMP4, Wnt, and Nodal, instruct certain cells in the embryo to become primordial germ cells. The process by which primordial germ cells are made in humans is very different to how primordial germ cells are made in mice and other so-called model animals that are commonly used in research. This has made it more challenging to uncover the details of the process in humans. Fortunately, new methods have recently been created that mimic aspects of how human embryos develop using human stem cells in a laboratory dish, providing an opportunity to gain a deeper understanding of how human germ cells form. Jo et al. used a technique called micropatterning to control the shape and size of groups of human stem cells growing in a laboratory dish. Treating these cells with a signal known as BMP4 gave rise to cells that resembled primordial germ cells. The team then used this system as a model to study how primordial germ cells form in humans. The experiments found that reducing Wnt signals in stem cells stopped primordial germ cells from forming in response to BMP4, confirming that Wnt signals made by the cells in response to BMP4 are essential. However, this block was overcome by providing the stem cells with another signal called Nodal. This suggests that the role of Wnt signaling in primordial germ cell formation is in part indirect by switching on Nodal in stem cells. Defects in eggs and sperm may lead to infertility, therefore, the findings of Jo et al. have the potential to help researchers develop new fertility treatments that use eggs or sperm grown in a laboratory from the patients' own stem cells. Such research would benefit from first developing a better understanding of how to make primordial germ cells.

Nodal is a short-range morphogen with activity that spreads through a relay mechanism in human gastruloids.

Morphogens are signaling molecules that convey positional information and dictate cell fates during development. Although ectopic expression in model organisms suggests that morphogen gradients form through diffusion, little is known about how morphogen gradients are created and interpreted during mammalian embryogenesis due to the combined difficulties of measuring endogenous morphogen levels and observing development in utero. Here we take advantage of a human gastruloid model to visualize endogenous Nodal protein in living cells, during specification of germ layers. We show that Nodal is extremely short range so that Nodal protein is limited to the immediate neighborhood of source cells. Nodal activity spreads through a relay mechanism in which Nodal production induces neighboring cells to transcribe Nodal. We further show that the Nodal inhibitor Lefty, while biochemically capable of long-range diffusion, also acts locally to control the timing of Nodal spread and therefore of mesoderm differentiation during patterning. Our study establishes a paradigm for tissue patterning by an activator-inhibitor pair.

Roadmap for the multiscale coupling of biochemical and mechanical signals during development.

The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.

Full of potential: Pluripotent stem cells for the systems biology of embryonic patterning.

Recently, it has become possible to recapitulate embryonic patterning in vitro using embryonic stem cells. The technical advantages and reduced complexity of these embryoids make them ideally suited to rigorously test long standing paradigms for pattern formation in the mammalian embryo. Here, we discuss insights into the underlying mechanisms that have been gained from stem cell models to date, focusing on models for gastrulation.

A novel self-organizing embryonic stem cell system reveals signaling logic underlying the patterning of human ectoderm.

During development, the ectoderm is patterned by a combination of BMP and WNT signaling. Research in model organisms has provided substantial insight into this process; however, there are currently no systems in which to study ectodermal patterning in humans. Further, the complexity of neural plate border specification has made it difficult to transition from discovering the genes involved to deeper mechanistic understanding. Here, we develop an in vitro model of human ectodermal patterning, in which human embryonic stem cells self-organize to form robust and quantitatively reproducible patterns corresponding to the complete medial-lateral axis of the embryonic ectoderm. Using this platform, we show that the duration of endogenous WNT signaling is a crucial control parameter, and that cells sense relative levels of BMP and WNT signaling in making fate decisions. These insights allowed us to develop an improved protocol for placodal differentiation. Thus, our platform is a powerful tool for studying human ectoderm patterning and for improving directed differentiation protocols.This article has an associated 'The people behind the papers' interview.

Rapid changes in morphogen concentration control self-organized patterning in human embryonic stem cells.

During embryonic development, diffusible signaling molecules called morphogens are thought to determine cell fates in a concentration-dependent way. Yet, in mammalian embryos, concentrations change rapidly compared to the time for making cell fate decisions. Here, we use human embryonic stem cells (hESCs) to address how changing morphogen levels influence differentiation, focusing on how BMP4 and Nodal signaling govern the cell-fate decisions associated with gastrulation. We show that BMP4 response is concentration dependent, but that expression of many Nodal targets depends on rate of concentration change. Moreover, in a self-organized stem cell model for human gastrulation, expression of these genes follows rapid changes in endogenous Nodal signaling. Our study shows a striking contrast between the specific ways ligand dynamics are interpreted by two closely related signaling pathways, highlighting both the subtlety and importance of morphogen dynamics for understanding mammalian embryogenesis and designing optimized protocols for directed stem cell differentiation. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Opening the black box: Stem cell-based modeling of human post-implantation development.

Proper development of the human embryo following its implantation into the uterine wall is critical for the successful continuation of pregnancy. However, the complex cellular and molecular changes that occur during this post-implantation period of human development are not amenable to study in vivo. Recently, several new embryo-like human pluripotent stem cell (hPSC)-based platforms have emerged, which are beginning to illuminate the current black box state of early human post-implantation biology. In this review, we will discuss how these experimental models are carving a way for understanding novel molecular and cellular mechanisms during early human development.

Morphogen and community effects determine cell fates in response to BMP4 signaling in human embryonic stem cells.

Paracrine signals maintain developmental states and create cell fate patterns in vivo and influence differentiation outcomes in human embryonic stem cells (hESCs) in vitro Systematic investigation of morphogen signaling is hampered by the difficulty of disentangling endogenous signaling from experimentally applied ligands. Here, we grow hESCs in micropatterned colonies of 1-8 cells ('µColonies') to quantitatively investigate paracrine signaling and the response to external stimuli. We examine BMP4-mediated differentiation in µColonies and standard culture conditions and find that in µColonies, above a threshold concentration, BMP4 gives rise to only a single cell fate, contrary to its role as a morphogen in other developmental systems. Under standard culture conditions BMP4 acts as a morphogen but this requires secondary signals and particular cell densities. We find that a 'community effect' enforces a common fate within µColonies, both in the state of pluripotency and when cells are differentiated, and that this effect allows a more precise response to external signals. Using live cell imaging to correlate signaling histories with cell fates, we demonstrate that interactions between neighbors result in sustained, homogenous signaling necessary for differentiation.

A synthetic planar cell polarity system reveals localized feedback on Fat4-Ds1 complexes.

The atypical cadherins Fat and Dachsous (Ds) have been found to underlie planar cell polarity (PCP) in many tissues. Theoretical models suggest that polarity can arise from localized feedbacks on Fat-Ds complexes at the cell boundary. However, there is currently no direct evidence for the existence or mechanism of such feedbacks. To directly test the localized feedback model, we developed a synthetic biology platform based on mammalian cells expressing the human Fat4 and Ds1. We show that Fat4-Ds1 complexes accumulate on cell boundaries in a threshold-like manner and exhibit dramatically slower dynamics than unbound Fat4 and Ds1. This suggests a localized feedback mechanism based on enhanced stability of Fat4-Ds1 complexes. We also show that co-expression of Fat4 and Ds1 in the same cells is sufficient to induce polarization of Fat4-Ds1 complexes. Together, these results provide direct evidence that localized feedbacks on Fat4-Ds1 complexes can give rise to PCP.

Active Tension Network model suggests an exotic mechanical state realized in epithelial tissues.

Mechanical interactions play a crucial role in epithelial morphogenesis, yet understanding the complex mechanisms through which stress and deformation affect cell behavior remains an open problem. Here we formulate and analyze the Active Tension Network (ATN) model, which assumes that the mechanical balance of cells within a tissue is dominated by cortical tension and introduces tension-dependent active remodeling of the cortex. We find that ATNs exhibit unusual mechanical properties. Specifically, an ATN behaves as a fluid at short times, but at long times supports external tension like a solid. Furthermore, an ATN has an extensively degenerate equilibrium mechanical state associated with a discrete conformal - "isogonal" - deformation of cells. The ATN model predicts a constraint on equilibrium cell geometries, which we demonstrate to approximately hold in certain epithelial tissues. We further show that isogonal modes are observed in the fruit y embryo, accounting for the striking variability of apical areas of ventral cells and helping understand the early phase of gastrulation. Living matter realizes new and exotic mechanical states, the study of which helps to understand biological phenomena.

Differential growth triggers mechanical feedback that elevates Hippo signaling.

Mechanical stress can influence cell proliferation in vitro, but whether it makes a significant contribution to growth control in vivo, and how it is modulated and experienced by cells within developing tissues, has remained unclear. Here we report that differential growth reduces cytoskeletal tension along cell junctions within faster-growing cells. We propose a theoretical model to explain the observed reduction of tension within faster-growing clones, supporting it by computer simulations based on a generalized vertex model. This reduced tension modulates a biomechanical Hippo pathway, decreasing recruitment of Ajuba LIM protein and the Hippo pathway kinase Warts, and decreasing the activity of the growth-promoting transcription factor Yorkie. These observations provide a specific mechanism for a mechanical feedback that contributes to evenly distributed growth, and we show that genetically suppressing mechanical feedback alters patterns of cell proliferation in the developing Drosophila wing. By providing experimental support for the induction of mechanical stress by differential growth, and a molecular mechanism linking this stress to the regulation of growth in developing organs, our results confirm and extend the mechanical feedback hypothesis.

Pluripotent stem cells as a model for embryonic patterning: From signaling dynamics to spatial organization in a dish.

In vivo studies have identified the signaling pathways and transcription factors involved in patterning the vertebrate embryo, but much remains unknown about how these are organized in space and time to orchestrate embryogenesis. Recently, embryonic stem cells have been established as a platform for studying spatial pattern formation and differentiation dynamics in the early mammalian embryo. The ease of observing and manipulating stem cell systems promises to fill gaps in our understanding of developmental dynamics and identify aspects that are uniquely human. Developmental Dynamics 245:976-990, 2016. © 2016 Wiley Periodicals, Inc.

Tissue cartography: compressing bio-image data by dimensional reduction.

The high volumes of data produced by state-of-the-art optical microscopes encumber research. We developed a method that reduces data size and processing time by orders of magnitude while disentangling signal by taking advantage of the laminar structure of many biological specimens. Our Image Surface Analysis Environment automatically constructs an atlas of 2D images for arbitrarily shaped, dynamic and possibly multilayered surfaces of interest. Built-in correction for cartographic distortion ensures that no information on the surface is lost, making the method suitable for quantitative analysis. We applied our approach to 4D imaging of a range of samples, including a Drosophila melanogaster embryo and a Danio rerio beating heart.