Neural tube morphogenesis in synthetic 3D microenvironments.

Three-dimensional organoid constructs serve as increasingly widespread in vitro models for development and disease modeling. Current approaches to recreate morphogenetic processes in vitro rely on poorly controllable and ill-defined matrices, thereby largely overlooking the contribution of biochemical and biophysical extracellular matrix (ECM) factors in promoting multicellular growth and reorganization. Here, we show how defined synthetic matrices can be used to explore the role of the ECM in the development of complex 3D neuroepithelial cysts that recapitulate key steps in early neurogenesis. We demonstrate how key ECM parameters are involved in specifying cytoskeleton-mediated symmetry-breaking events that ultimately lead to neural tube-like patterning along the dorsal-ventral (DV) axis. Such synthetic materials serve as valuable tools for studying the discrete action of extrinsic factors in organogenesis, and allow for the discovery of relationships between cytoskeletal mechanobiology and morphogenesis.

Bioengineering approaches to guide stem cell-based organogenesis.

During organogenesis, various molecular and physical signals are orchestrated in space and time to sculpt multiple cell types into functional tissues and organs. The complex and dynamic nature of the process has hindered studies aimed at delineating morphogenetic mechanisms in vivo, particularly in mammals. Recent demonstrations of stem cell-driven tissue assembly in culture offer a powerful new tool for modeling and dissecting organogenesis. However, despite the highly organotypic nature of stem cell-derived tissues, substantial differences set them apart from their in vivo counterparts, probably owing to the altered microenvironment in which they reside and the lack of mesenchymal influences. Advances in the biomaterials and microtechnology fields have, for example, afforded a high degree of spatiotemporal control over the cellular microenvironment, making it possible to interrogate the effects of individual microenvironmental components in a modular fashion and rapidly identify organ-specific synthetic culture models. Hence, bioengineering approaches promise to bridge the gap between stem cell-driven tissue formation in culture and morphogenesis in vivo, offering mechanistic insight into organogenesis and unveiling powerful new models for drug discovery, as well as strategies for tissue regeneration in the clinic. We draw on several examples of stem cell-derived organoids to illustrate how bioengineering can contribute to tissue formation ex vivo. We also discuss the challenges that lie ahead and potential ways to overcome them.

Defined three-dimensional microenvironments boost induction of pluripotency.

Since the discovery of induced pluripotent stem cells (iPSCs), numerous approaches have been explored to improve the original protocol, which is based on a two-dimensional (2D) cell-culture system. Surprisingly, nothing is known about the effect of a more biologically faithful 3D environment on somatic-cell reprogramming. Here, we report a systematic analysis of how reprogramming of somatic cells occurs within engineered 3D extracellular matrices. By modulating microenvironmental stiffness, degradability and biochemical composition, we have identified a previously unknown role for biophysical effectors in the promotion of iPSC generation. We find that the physical cell confinement imposed by the 3D microenvironment boosts reprogramming through an accelerated mesenchymal-to-epithelial transition and increased epigenetic remodelling. We conclude that 3D microenvironmental signals act synergistically with reprogramming transcription factors to increase somatic plasticity.

Artificial three-dimensional niches deconstruct pancreas development in vitro.

In the context of a cellular therapy for diabetes, methods for pancreatic progenitor expansion and subsequent differentiation into insulin-producing beta cells would be extremely valuable. Here we establish three-dimensional culture conditions in Matrigel that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the medium composition we generate either hollow spheres, which are mainly composed of pancreatic progenitors, or complex organoids that spontaneously undergo pancreatic morphogenesis and differentiation. The in vitro maintenance and expansion of pancreatic progenitors require active Notch and FGF signaling, thus recapitulating in vivo niche signaling interactions. Our experiments reveal new aspects of pancreas development, such as a community effect by which small groups of cells better maintain progenitor properties and expand more efficiently than isolated cells, as well as the requirement for three-dimensionality. Finally, growth conditions in chemically defined biomaterials pave the way for testing the biophysical and biochemical properties of the niche that sustains pancreatic progenitors.

Hyaluronic Acid Hydrogels Formed in Situ by Transglutaminase-Catalyzed Reaction.

Enzymatically cross-linked hydrogels can be formed in situ and permit highly versatile and selective tethering of bioactive molecules, thereby allowing for a wealth of applications in cell biology and tissue engineering. While a number of studies have reported the bioconjugation of extracellular matrix (ECM) proteins and peptides into such matrices, the site-specific incorporation of biologically highly relevant polysaccharides such as hyaluronic acid (HA) has thus far not been reported, limiting our ability to reconstruct this key feature of the in vivo ECM. Here we demonstrate a novel strategy for transglutaminase-mediated covalent linking of HA moieties to a synthetic poly(ethylene glycol) (PEG) macromer resulting in the formation of hybrid HA-PEG hydrogels. We characterize the ensuing matrix properties and demonstrate how these cytocompatible gels can serve to modulate the cellular phenotype of human mammary cancer epithelial cells as well as mouse myoblasts. The use of HA as a novel building block in the increasingly varied library of synthetic PEG-based artificial ECMs should have applications as a structural as well as a signaling component and offers significant potential as an injectable matrix for regenerative medicine.

Heparin-binding domain of fibrin(ogen) binds growth factors and promotes tissue repair when incorporated within a synthetic matrix.

By binding growth factors (GFs), the ECM tightly regulates their activity. We recently reported that the heparin-binding domain II of fibronectin acts as a promiscuous high-affinity GF-binding domain. Here we hypothesized that fibrin, the provisional ECM during tissue repair, also could be highly promiscuous in its GF-binding capacity. Using multiple affinity-based assays, we found that fibrin(ogen) and its heparin-binding domain bind several GFs from the PDGF/VEGF and FGF families and some GFs from the TGF-ß and neurotrophin families. Overall, we identified 15 unique binding interactions. The GF binding ability of fibrinogen caused prolonged retention of many of the identified GFs within fibrin. Thus, based on the promiscuous and high-affinity interactions in fibrin, GF binding may be one of fibrin's main physiological functions, and these interactions may potentially play an important and ubiquitous role during tissue repair. To prove this role in a gain-of-function model, we incorporated the heparin-binding domain of fibrin into a synthetic fibrin-mimetic matrix. In vivo, the multifunctional synthetic matrix could fully mimic the effect of fibrin in a diabetic mouse model of impaired wound healing, demonstrating the benefits of generating a hybrid biomaterial consisting of a synthetic polymeric scaffold and recombinant bioactive ECM domains. The reproduction of GF-ECM interactions with a fibrin-mimetic matrix could be clinically useful, and has the significant benefit of a more straightforward regulatory path associated with chemical synthesis rather than human sourcing.

Mechanical Actuation of Organoids in Synthetic Microenvironments.

Organoids are a powerful model system to explore the role of mechanical forces in sculpting emergent tissue cytoarchitecture. The modulation of the mechanical microenvironment is most readily performed using synthetic extracellular matrices (ECM); however, such materials provide passive, rather than active force modulation. Actuation technologies enable the active tuning of mechanical forces in both time and magnitude. Using such instruments, our group has shown that extrinsically imposed stretching on human neural tube organoids (hNTOs) enhanced patterning of the floor plate domain. Here, we provide a detailed protocol on the implementation of mechanical actuation of organoids embedded in synthetic 3D microenvironments, with additional details on methods to characterize organoid fate and behavior. Our protocol is easy to reproduce and is expected to be broadly applicable to investigate the role of active mechanics with in vitro model systems.

3D biomimetic environment enabling ex utero trophoblast invasion and co-culture of embryos and somatic cells.

The first direct contact between the embryo and the mother is established during implantation. This process is inaccessible for direct studies as the implanting embryo is concealed by the maternal tissues. Here, we present a protocol for establishing a 3D biomimetic environment based on synthetic hydrogels which harbor key biomechanical properties of the uterine stroma. We describe steps for isolating and culturing embryos in PEG/DexMA hydrogel. We then detail the co-culture of embryos and endothelial cells in a microfluidic device. For complete details on the use and execution of this protocol, please refer to Govindasamy et al. (2021)1 and Ozguldez et al. (2023).2.

Targeted mechanical stimulation via magnetic nanoparticles guides in vitro tissue development.

Tissues take shape through a series of morphogenetic movements guided by local cell-scale mechanical forces. Current in vitro approaches to recapitulate tissue mechanics rely on uncontrolled self-organization or on the imposition of extrinsic and homogenous forces using matrix or instrument-driven stimulation, thereby failing to recapitulate highly localized and spatially varying forces. Here we develop a method for targeted mechanical stimulation of organoids using embedded magnetic nanoparticles. We show that magnetic clusters within organoids can be produced by sequential aggregation of magnetically labeled and non-labeled human pluripotent stem cells. These clusters impose local mechanical forces on the surrounding cells in response to applied magnetic fields. We show that precise, spatially defined actuation provides short-term mechanical tissue perturbations as well as long-term cytoskeleton remodeling in these organoids, which we term "magnetoids". We demonstrate that targeted magnetic nanoparticle-driven actuation guides asymmetric tissue growth and proliferation, leading to enhanced patterning in human neural magnetoids. This approach, enabled by nanoparticle technology, allows for precise and locally controllable mechanical actuation in human neural tube organoids, and could be widely applicable to interrogate the role of local mechanotransduction in developmental and disease model systems.

Neuroepithelial organoid patterning is mediated by a neighborhood watch mechanism.

During epithelial tissue patterning, morphogens operate across multiple length scales to instruct cell identities. However, how cell fate changes are coordinated over these scales to establish spatial organization remains poorly understood. Here, we use human neural tube organoids as models of epithelial patterning and develop an in silico approach to define conditions permissive to patterning. By systematically varying morphogen position, diffusivity, and fate-inducing concentration levels, we show that cells follow a "neighborhood watch" (NW) mechanism that is deterministically dictated by initial morphogen source positions, reflecting scale-invariant in vitro phenotypes. We define how the frequency and local bias of morphogen sources stabilize pattern orientation. The model predicts enhanced patterning through floor plate inhibition, and receptor-ligand interaction analysis of single-cell RNA sequencing (scRNA-seq) data identifies wingless-related integration site (WNT) and bone morphogenic protein (BMP) as inhibition modulators, which we validate in vitro. These results suggest that developing neuroepithelia employ NW-based mechanisms to organize morphogen sources, define cellular identity, and establish patterns.

Large-scale perfused tissues via synthetic 3D soft microfluidics.

The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm3 tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing neural and liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.

Polarity inversion reorganizes the stem cell compartment of the trophoblast lineage.

The extra-embryonic tissues that form the placenta originate from a small population of trophectoderm cells with stem cell properties, positioned at the embryonic pole of the mouse blastocyst. During the implantation stages, the polar trophectoderm rapidly proliferates and transforms into extra-embryonic ectoderm. The current model of trophoblast morphogenesis suggests that tissue folding reshapes the trophoblast during the blastocyst to egg cylinder transition. Instead of through folding, here we found that the tissue scale architecture of the stem cell compartment of the trophoblast lineage is reorganized via inversion of the epithelial polarity axis. Our findings show the developmental significance of polarity inversion and provide a framework for the morphogenetic transitions in the peri-implantation trophoblast.

Paraxial mesoderm organoids model development of human somites.

During the development of the vertebrate embryo, segmented structures called somites are periodically formed from the presomitic mesoderm (PSM) and give rise to the vertebral column. While somite formation has been studied in several animal models, it is less clear how well this process is conserved in humans. Recent progress has made it possible to study aspects of human paraxial mesoderm (PM) development such as the human segmentation clock in vitro using human pluripotent stem cells (hPSCs); however, somite formation has not been observed in these monolayer cultures. Here, we describe the generation of human PM organoids from hPSCs (termed Somitoids), which recapitulate the molecular, morphological, and functional features of PM development, including formation of somite-like structures in vitro. Using a quantitative image-based screen, we identify critical parameters such as initial cell number and signaling modulations that reproducibly yielded formation of somite-like structures in our organoid system. In addition, using single-cell RNA-sequencing and 3D imaging, we show that PM organoids both transcriptionally and morphologically resemble their in vivo counterparts and can be differentiated into somite derivatives. Our organoid system is reproducible and scalable, allowing for the systematic and quantitative analysis of human spine development and disease in vitro.

Humans are part of a group of animals called vertebrates, which are all the animals with backbones. Broadly, all vertebrates have a similar body shape with a head at one end and a left and right side that are similar to each other. Although this is not very obvious in humans, vertebrate bodies are derived from pairs of segments arranged from the head to the tail. Each of these segments or somites originates early in embryonic development. Cells from each somite then divide, grow and specialize to form bones such as the vertebrae of the vertebral column, muscles, skin, and other tissues that make up each segment. Studying different animals during embryonic development has provided insights into how somites form and grow, but it is technically difficult to do and only provides an approximate model of how somites develop in humans. Being able to make and study somites using human cells in the lab would help scientists learn more about how somite formation in humans is regulated. Budjan et al. grew human stem cells in the lab as three-dimensional structures called organoids, and used chemical signals similar to the ones produced in the embryo during development to make the cells form somites. Various combinations of signals were tested to find the best way to trigger somite formation. Once the somites formed, Budjan et al. measured them and studied their structure and the genes they used. They found that these lab-grown somites have the same size and structure as natural somites and use many of the same genes. This new organoid model provides a way to study human somite formation and development in the lab for the first time. This can provide insights into the development and evolution of humans and other animals that could then help scientists understand diseases such as the development of abnormal spinal curvature that affects around 1 in 10,000 newborns.

Engineering neurovascular organoids with 3D printed microfluidic chips.

The generation of tissue and organs requires close interaction with vasculature from the earliest moments of embryonic development. Tissue-specific organoids derived from pluripotent stem cells allow for the in vitro recapitulation of elements of embryonic development. However, they are not intrinsically vascularized, which poses a major challenge for their sustained growth, and for understanding the role of vasculature in fate specification and morphogenesis. Current organoid vascularization strategies do not recapitulate the temporal synchronization and spatial orientation needed to ensure in vivo-like early co-development. Here, we developed a human pluripotent stem cell (hPSC)-based approach to generate organoids which interact with vascular cells in a spatially determined manner. The spatial interaction between organoid and vasculature is enabled by the use of a custom designed 3D printed microfluidic chip which allows for a sequential and developmentally matched co-culture system. We show that on-chip hPSC-derived pericytes and endothelial cells sprout and self-assemble into organized vascular networks, and use cerebral organoids as a model system to explore interactions with this de novo generated vasculature. Upon co-development, vascular cells physically interact with the cerebral organoid and form an integrated neurovascular organoid on chip. This 3D printing-based platform is designed to be compatible with any organoid system and is an easy and highly cost-effective way to vascularize organoids. The use of this platform, readily performed in any lab, could open new avenues for understanding and manipulating the co-development of tissue-specific organoids with vasculature.

Valproic acid stimulates myogenesis in pluripotent stem cell-derived mesodermal progenitors in a NOTCH-dependent manner.

Muscular dystrophies are debilitating neuromuscular disorders for which no cure exists. As this disorder affects both cardiac and skeletal muscle, patients would benefit from a cellular therapy that can simultaneously regenerate both tissues. The current protocol to derive bipotent mesodermal progenitors which can differentiate into cardiac and skeletal muscle relies on the spontaneous formation of embryoid bodies, thereby hampering further clinical translation. Additionally, as skeletal muscle is the largest organ in the human body, a high myogenic potential is necessary for successful regeneration. Here, we have optimized a protocol to generate chemically defined human induced pluripotent stem cell-derived mesodermal progenitors (cdMiPs). We demonstrate that these cells contribute to myotube formation and differentiate into cardiomyocytes, both in vitro and in vivo. Furthermore, the addition of valproic acid, a clinically approved small molecule, increases the potential of the cdMiPs to contribute to myotube formation that can be prevented by NOTCH signaling inhibitors. Moreover, valproic acid pre-treated cdMiPs injected in dystrophic muscles increase physical strength and ameliorate the functional performances of transplanted mice. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents.

3D biomimetic platform reveals the first interactions of the embryo and the maternal blood vessels.

The process of implantation and the cellular interactions at the embryo-maternal interface are intrinsically difficult to analyze, as the implanting embryo is concealed by the uterine tissues. Therefore, the mechanisms mediating the interconnection of the embryo and the mother are poorly understood. Here, we established a 3D biomimetic culture environment that harbors the key features of the murine implantation niche. This culture system enabled direct analysis of trophoblast invasion and revealed the first embryonic interactions with the maternal vasculature. We found that implantation is mediated by the collective migration of penetrating strands of trophoblast giant cells, which acquire the expression of vascular receptors, ligands, and adhesion molecules, assembling a network for communication with the maternal blood vessels. In particular, Pdgf signaling cues promote the establishment of the heterologous contacts. Together, the biomimetic platform and our findings thereof elucidate the hidden dynamics of the early interactions at the implantation site.

A fully defined matrix to support a pluripotent stem cell derived multi-cell-liver steatohepatitis and fibrosis model.

Chronic liver injury, as observed in non-alcoholic steatohepatitis (NASH), progressive fibrosis, and cirrhosis, remains poorly treatable. Steatohepatitis causes hepatocyte loss in part by a direct lipotoxic insult, which is amplified by derangements in the non-parenchymal cellular (NPC) interactive network wherein hepatocytes reside, including, hepatic stellate cells, liver sinusoidal endothelial cells and liver macrophages. To create an in vitro culture model encompassing all these cells, that allows studying liver steatosis, inflammation and fibrosis caused by NASH, we here developed a fully defined hydrogel microenvironment, termed hepatocyte maturation (HepMat) gel, that supports maturation and maintenance of pluripotent stem cell (PSC) derived hepatocyte- and NPC-like cells for at least one month. The HepMat-based co-culture system modeled key molecular and functional features of TGFß-induced liver fibrosis and fatty-acid induced inflammation and fibrosis better than monocultures of its constituent cell populations. The novel co-culture system should open new avenues for studying mechanisms underlying liver steatosis, inflammation and fibrosis as well as for assessing drugs counteracting these effects.

Actuation enhances patterning in human neural tube organoids.

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.

Nanoparticles as Versatile Tools for Mechanotransduction in Tissues and Organoids.

Organoids are 3D multicellular constructs that rely on self-organized cell differentiation, patterning and morphogenesis to recapitulate key features of the form and function of tissues and organs of interest. Dynamic changes in these systems are orchestrated by biochemical and mechanical microenvironments, which can be engineered and manipulated to probe their role in developmental and disease mechanisms. In particular, the in vitro investigation of mechanical cues has been the focus of recent research, where mechanical manipulations imparting local as well as large-scale mechanical stresses aim to mimic in vivo tissue deformations which occur through proliferation, folding, invagination, and elongation. However, current in vitro approaches largely impose homogeneous mechanical changes via a host matrix and lack the required positional and directional specificity to mimic the diversity of in vivo scenarios. Thus, while organoids exhibit limited aspects of in vivo morphogenetic events, how local forces are coordinated to enable large-scale changes in tissue architecture remains a difficult question to address using current techniques. Nanoparticles, through their efficient internalization by cells and dispersion through extracellular matrices, have the ability to provide local or global, as well as passive or active modulation of mechanical stresses on organoids and tissues. In this review, we explore how nanoparticles can be used to manipulate matrix and tissue mechanics, and highlight their potential as tools for fate regulation through mechanotransduction in multicellular model systems.

Mammary epithelial morphogenesis in 3D combinatorial microenvironments.

Human mammary epithelial cells can proliferate and reorganize into polarized multi-cellular constructs in-vitro, thereby functioning as an important model system in recapitulating key steps of in-vivo morphogenesis. Current approaches to constructing such three-dimensional mimics of the in-vivo microenvironment have involved the use of complex and ill-defined naturally derived matrices, whose properties are difficult to manipulate independently, and which have therefore limited our ability to understand the extrinsic regulation of morphogenesis. Here, we employ an automated, high-throughput approach to array modular building blocks of synthetic components, and develop a systematic approach to analyze colonies resulting from these varied microenvironmental combinations. This methodology allows us to systematically map the relationship between microenvironmental properties and ensuing morphogenetic phenotypes. Our analysis reveals that apico-basal polarity of mammary epithelial cells occurs within a narrow range of matrix stiffness, and that phenotypic homogeneity is favored in matrices which are insensitive to MMP-mediated degradation. Furthermore, combinations of extracellular proteins in the matrix finely tune the morphology of the mammary colonies, suggesting that subtle disregulations of the microenvironment may play a significant role in pathological disease states. This approach, which leverages the combinatorial possibilities of modular synthetic artificial extracellular matrices with an automated technology platform, demonstrates how morphogenesis can be assessed systematically in 3D, and provides new insights into mammary epithelial multicellularity.