Spatiotemporally resolved colorectal oncogenesis in mini-colons ex vivo.

Three-dimensional organoid culture technologies have revolutionized cancer research by allowing for more realistic and scalable reproductions of both tumour and microenvironmental structures1-3. This has enabled better modelling of low-complexity cancer cell behaviours that occur over relatively short periods of time4. However, available organoid systems do not capture the intricate evolutionary process of cancer development in terms of tissue architecture, cell diversity, homeostasis and lifespan. As a consequence, oncogenesis and tumour formation studies are not possible in vitro and instead require the extensive use of animal models, which provide limited spatiotemporal resolution of cellular dynamics and come at a considerable cost in terms of resources and animal lives. Here we developed topobiologically complex mini-colons that are able to undergo tumorigenesis ex vivo by integrating microfabrication, optogenetic and tissue engineering approaches. With this system, tumorigenic transformation can be spatiotemporally controlled by directing oncogenic activation through blue-light exposure, and emergent colon tumours can be tracked in real-time at the single-cell resolution for several weeks without breaking the culture. These induced mini-colons display rich intratumoural and intertumoural diversity and recapitulate key pathophysiological hallmarks displayed by colorectal tumours in vivo. By fine-tuning cell-intrinsic and cell-extrinsic parameters, mini-colons can be used to identify tumorigenic determinants and pharmacological opportunities. As a whole, our study paves the way for cancer initiation research outside living organisms.

A New Approach to Generate Gastruloids to Develop Anterior Neural Tissues.

Embryonic development is a complex process integrating cell fate decisions and morphogenesis in a spatiotemporally controlled manner. Previous studies with model organisms laid the foundation of our knowledge on post-implantation development; however, studying mammalian embryos at this stage is a difficult and laborious process. Early attempts to recapitulate mammalian development in vitro begun with embryoid bodies (EBs), in which aggregates of mouse embryonic stem cells (mESCs) were shown to differentiate into spatially arranged germ layers. A more revised version of EBs, gastruloids, improved the germ layer differentiation efficiency and demonstrated cell fate patterning on multiple axes. However, gastruloids lack anterior neural progenitors that give rise to brain tissues in the embryo. Here, we report a novel culture protocol to coax mESCs into post-implantation epiblast-like (EPI) aggregates in high throughput on bioengineered microwell arrays. We show that upon inhibition of the Wnt signaling pathway, EPI aggregates establish an extended axial patterning, leading to co-derivation of anterior neural progenitors and posterior tissues. Our approach is amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing embryoid models. This work should contribute to the advancement of the nascent field of synthetic embryology, opening up exciting perspectives for various applications of pluripotent stem cells in disease modeling and tissue engineering. Key features A new gastruloid culture system to model post-implantation mouse embryonic development in vitro High-throughput formation of epiblast-like aggregates on hydrogel microwells Builds upon conventional gastruloid cultures and provides insight into the role of Wnt signaling for the formation of anterior neural tissues Graphical overview.

Dissecting mechanisms of chamber-specific cardiac differentiation and its perturbation following retinoic acid exposure.

The specification of distinct cardiac lineages occurs before chamber formation and acquisition of bona fide atrial or ventricular identity. However, the mechanisms underlying these early specification events remain poorly understood. Here, we performed single cell analysis at the murine cardiac crescent, primitive heart tube and heart tube stages to uncover the transcriptional mechanisms underlying formation of atrial and ventricular cells. We find that progression towards differentiated cardiomyocytes occurs primarily based on heart field progenitor identity, and that progenitors contribute to ventricular or atrial identity through distinct differentiation mechanisms. We identify new candidate markers that define such differentiation processes and examine their expression dynamics using computational lineage trajectory methods. We further show that exposure to exogenous retinoic acid causes defects in ventricular chamber size, dysregulation in FGF signaling and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit resulting in formation of hypomorphic ventricles. Collectively, our data identify, at a single cell level, distinct lineage trajectories during cardiac specification and differentiation, and the precise effects of manipulating cardiac progenitor patterning via retinoic acid signaling.

Next-generation cancer organoids.

Organotypic models of patient-specific tumours are revolutionizing our understanding of cancer heterogeneity and its implications for personalized medicine. These advancements are, in part, attributed to the ability of organoid models to stably preserve genetic, proteomic, morphological and pharmacotypic features of the parent tumour in vitro, while also offering unprecedented genomic and environmental manipulation. Despite recent innovations in organoid protocols, current techniques for cancer organoid culture are inherently uncontrolled and irreproducible, owing to several non-standardized facets including cancer tissue sources and subsequent processing, medium formulations, and animal-derived three-dimensional matrices. Given the potential for cancer organoids to accurately recapitulate the intra- and intertumoral biological heterogeneity associated with patient-specific cancers, eliminating the undesirable technical variability accompanying cancer organoid culture is necessary to establish reproducible platforms that accelerate translatable insights into patient care. Here we describe the current challenges and recent multidisciplinary advancements and opportunities for standardizing next-generation cancer organoid systems.

Synthetic dynamic hydrogels promote degradation-independent in vitro organogenesis.

Epithelial organoids are most efficiently grown from mouse-tumour-derived, reconstituted extracellular matrix hydrogels, whose poorly defined composition, batch-to-batch variability and immunogenicity limit clinical applications. Efforts to replace such ill-defined matrices for organoid culture have largely focused on non-adaptable hydrogels composed of covalently crosslinked hydrophilic macromolecules. However, the excessive forces caused by tissue expansion in such elastic gels severely restrict organoid growth and morphogenesis. Chemical or enzymatic degradation schemes can partially alleviate this problem, but due to their irreversibility, long-term applicability is limited. Here we report a family of synthetic hydrogels that promote extensive organoid morphogenesis through dynamic rearrangements mediated by reversible hydrogen bonding. These tunable matrices are stress relaxing and thus promote efficient crypt budding in intestinal stem-cell epithelia through increased symmetry breaking and Paneth cell formation dependent on yes-associated protein 1. As such, these well-defined gels provide promising versatile matrices for fostering elaborate in vitro morphogenesis.

Bioengineered embryoids mimic post-implantation development in vitro.

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro.

Factor XIII Cross-Linked Adhesive Chitosan Hydrogels.

Biomedical adhesives have been found to be an attractive alternative to suturing in several circumstances. However, to date most of the clinically approved formulations are based on synthetic and highly reactive toxic chemicals. In this work, we aimed to combine for the first time the bioactive properties of the cationic polysaccharide chitosan and its intrinsic electrostatic binding to negatively charged tissues with the biocompatible and clinically compliant enzymatic cross-linking scheme of fibrin glue. This synergistic activity led to the generation of a transglutaminase Factor XIII cross-linkable chitosan formulation with fast gelation kinetics, tunable mechanical properties, antibacterial activity, and strong adhesion to cartilage.

Gastruloids generated without exogenous Wnt activation develop anterior neural tissues.

When stimulated with a pulse from an exogenous WNT pathway activator, small aggregates of mouse embryonic stem cells (ESCs) can undergo embryo-like axial morphogenesis and patterning along the three major body axes. However, these structures, called gastruloids, currently lack the anterior embryonic regions, such as those belonging to the brain. Here, we describe an approach to generate gastruloids that have a more complete antero-posterior development. We used hydrogel microwell arrays to promote the robust derivation of mouse ESCs into post-implantation epiblast-like (EPI) aggregates in a reproducible and scalable manner. These EPI aggregates break symmetry and axially elongate without external chemical stimulation. Inhibition of WNT signaling in early stages of development leads to the formation of gastruloids with anterior neural tissues. Thus, we provide a new tool to study the development of the mouse after implantation in vitro, especially the formation of anterior neural regions.

Capturing Cardiogenesis in Gastruloids.

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.

Homeostatic mini-intestines through scaffold-guided organoid morphogenesis.

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1-4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.

Axon Growth of CNS Neurons in Three Dimensions Is Amoeboid and Independent of Adhesions.

During development of the central nervous system (CNS), neurons polarize and rapidly extend their axons to assemble neuronal circuits. The growth cone leads the axon to its target and drives axon growth. Here, we explored the mechanisms underlying axon growth in three dimensions. Live in situ imaging and super-resolution microscopy combined with pharmacological and molecular manipulations as well as biophysical force measurements revealed that growth cones extend CNS axons independent of pulling forces on their substrates and without the need for adhesions in three-dimensional (3D) environments. In 3D, microtubules grow unrestrained from the actomyosin cytoskeleton into the growth cone leading edge to enable rapid axon extension. Axons extend and polarize even in adhesion-inert matrices. Thus, CNS neurons use amoeboid mechanisms to drive axon growth. Together with our understanding that adult CNS axons regenerate by reactivating developmental processes, our findings illuminate how cytoskeletal manipulations enable axon regeneration in the adult CNS.

Morphogenesis Guided by 3D Patterning of Growth Factors in Biological Matrices.

Three-dimensional (3D) control over the placement of bioactive cues is fundamental to understand cell guidance and develop engineered tissues. Two-photon patterning (2PP) provides such placement at micro- to millimeter scale, but nonspecific interactions between proteins and functionalized extracellular matrices (ECMs) restrict its use. Here, a 2PP system based on nonfouling hydrophilic photocages and Sortase A (SA)-based enzymatic coupling is presented, which offers unprecedented orthogonality and signal-to-noise ratio in both inert hydrogels and complex mammalian matrices. Improved photocaged peptide synthesis and protein functionalization protocols with broad applicability are introduced. Importantly, the method enables 2PP in a single step in the presence of fragile biomolecules and cells, and is compatible with time-controlled growth factor presentation. As a corollary, the guidance of axons through 3D-patterned nerve growth factor (NGF) within brain-mimetic ECMs is demonstrated. The approach allows for the interrogation of the role of complex signaling molecules in 3D matrices, thus helping to better understand biological guidance in tissue development and regeneration.

Characterization of polydactyly chondrocytes and their use in cartilage engineering.

Treating cartilage injuries and degenerations represents an open surgical challenge. The recent advances in cell therapies have raised the need for a potent off-the-shelf cell source. Intra-articular injections of TGF-ß transduced polydactyly chondrocytes have been proposed as a chronic osteoarthritis treatment but despite promising results, the use of gene therapy still raises safety concerns. In this study, we characterized infant, polydactyly chondrocytes during in vitro expansion and chondrogenic re-differentiation. Polydactyly chondrocytes have a steady proliferative rate and re-differentiate in 3D pellet culture after up to five passages. Additionally, we demonstrated that polydactyly chondrocytes produce cartilage-like matrix in a hyaluronan-based hydrogel, namely transglutaminase cross-linked hyaluronic acid (HA-TG). We utilized the versatility of TG cross-linking to augment the hydrogels with heparin moieties. The heparin chains allowed us to load the scaffolds with TGF-ß1, which induced cartilage-like matrix deposition both in vitro and in vivo in a subcutaneous mouse model. This strategy introduces the possibility to use infant, polydactyly chondrocytes for the clinical treatment of joint diseases.

Macroporous hydrogels derived from aqueous dynamic phase separation.

A method to generate injectable macroporous hydrogels based on partitioning of polyethylene glycol (PEG) and high viscous polysaccharides is presented. Step growth polymerization of PEG was used to initiate a phase separation and the formation of a connected macroporous network with tunable dimensions. The possibilities and physical properties of this new category of materials were examined, and then applied to address some challenges in neural engineering. First, non-degradable macroporous gels were shown to support rapid neurite extension from encapsulated dorsal root ganglia (DRGs) with unprecedented long-term stability. Then, dissociated primary rat cortical neurons could be encapsulated with >95% viability, and extended neurites at the fast rate of ≈100 µm/day and formed synapses, resulting in functional, highly viable and long-term stable 3D neural networks in the synthetic extracellular matrix (ECM). Adhesion cues were found unnecessary provided the gels have optimal physical properties. Normal electrophysiological properties were confirmed on 3D cultured mouse hippocampal neurons. Finally, the macroporous gels supported axonal growth in a rat sciatic nerve injury model when used as a conduit filling. The combination of injectability, tunable pore size, stability, connectivity, transparency, cytocompatibility and biocompatibility, makes this new class of materials attractive for a wide range of applications.

Growth of Epithelial Organoids in a Defined Hydrogel.

Epithelial organoids are simplified models of organs grown in vitro from embryonic and adult stem cells. They are widely used to study organ development and disease, and enable drug screening in patient-derived primary tissues. Current protocols, however, rely on animal- and tumor-derived basement membrane extract (BME) as a 3D scaffold, which limits possible applications in regenerative medicine. This prompted us to study how organoids interact with their matrix, and to develop a well-defined hydrogel that supports organoid generation and growth. It is found that soft fibrin matrices provide suitable physical support, and that naturally occurring Arg-Gly-Asp (RGD) adhesion domains on the scaffold, as well as supplementation with laminin-111, are key parameters required for robust organoid formation and expansion. The possibility to functionalize fibrin via factor XIII-mediated anchoring also allows to covalently link fluorescent nanoparticles to the matrix for 3D traction force microscopy. These measurements suggest that the morphogenesis of budding intestinal organoids results from internal pressure combined with higher cell contractility in the regions containing differentiated cells compared to the regions containing stem cells. Since the fibrin/laminin matrix supports long-term expansion of all tested murine and human epithelial organoids, this hydrogel can be widely used as a defined equivalent to BME.

Cell-Instructive Alginate Hydrogels Targeting RhoA.

Cellular processes involve dynamic rearrangement of the cytoskeleton. The GTPase RhoA plays a fundamental role in controlling cytoskeletal architecture. The phenotypic stability of chondrocytes is enhanced through inhibition of RhoA, whereas RhoA activation leads to dedifferentiation. We hypothesized that local inhibition of this pathway could induce chondrogenesis and cartilage regeneration. In this study, a novel alginate-derived hydrogel system was developed for sustained RhoA targeting. Specifically, an engineered variant of C. botulinum C3 transferase, a potent RhoA inhibitor, was immobilized onto a hydrogel to achieve sustained release and enzymatic activity. Chondrocytes encapsulated within this fully biocompatible, mechanically stable scaffold produced a stable collagen type II-rich matrix in vitro which matured over a six-week period. Samples were implanted subcutaneously in mice, and similar production of a collagen type II-rich matrix was observed. The intrinsically versatile system has the potential to treat a number of clinical disorders, including osteoarthritis, linked with RhoA dysregulation.

Sortase A as a cross-linking enzyme in tissue engineering.

The bacterial ligase Sortase A (SA) and its mutated variants have become increasingly popular over the last years for post-translational protein modifications due to their unparalleled specificity and efficiency. The aim of this work was to study SA as a cross-linking enzyme for hydrogel-based tissue engineering. For this, we optimized SA pentamutant production and purification from E. coli to achieve high yields and purity. Then using hyaluronan (HA) as a model biopolymer and modifying it with SA-substrate peptides, we studied the cross-linking kinetics obtained with SA, the enzyme stability, cytocompatibility, and immunogenicity, and compared those to state-of-the-art standards. The transglutaminase activated factor XIII (FXIIIa) was used as the reference cross-linking enzyme, and the clinical collagen scaffold Chondro-Gide (CG) was used as a reference biocompatible material for in vivo studies. We found SA could be produced in large amounts in the lab without special equipment, whereas the only viable source of FXIIIa is currently a prescription medicine purified from donated blood. SA was also remarkably more stable in solution than FXIIIa, and it could provide even much faster gelation, making it possible to achieve nearly-instantaneous gel formation upon delivery with a double-barrel syringe. This is an interesting improvement for in vivo work, to allow in situ gel formation in a wet environment, and could also be useful for applications like bioprinting where very fast gelation is needed. The cytocompatibility and lack of immunogenicity were still uncompromised. These results support the use of SA as a versatile enzymatic cross-linking strategy for 3D culture and tissue engineering applications. STATEMENT OF SIGNIFICANCE: Enzymatic crosslinking has immense appeal for tissue engineers as one of the most biocompatible methods of hydrogel crosslinking. Sortase A has a number of unique advantages over previous systems. We show an impressive and tunable range of crosslinking kinetics, from almost instantaneous gelation to several minutes. We also demonstrate that Sortase A crosslinked hydrogels have good cytocompatibility and cause no immune reaction when implanted in vivo. With its additional benefits of excellent stability in solution and easy large-scale synthesis available to any lab, we believe this novel crosslinking modality will find multiple applications in high throughput screening, tissue engineering, and biofabrication.

Enzymatically crosslinked poly(2-alkyl-2-oxazoline) networks for 3D cell culture.

Molecularly designed, random copolymer-bioconjugates based on poly(2-methyl-2-oxazoline) (PMOXA) and poly(2-ethyl-2-oxazoline) (PEOXA) are crosslinked in the presence of sortase A (SA) and human articular chondrocytes (hACs), to yield cellularized polymer networks. Their gelation kinetics and mechanical properties are finely tuned by varying the concentration of SA, while a cell viability >90% is achieved after several weeks of culture, even in the absence of any cell-adhesive cue.

Novel enzymatically cross-linked hyaluronan hydrogels support the formation of 3D neuronal networks.

Hyaluronan (HA) is an essential component of the central nervous system's extracellular matrix and its high molecular weight (MW) form has anti-inflammatory and anti-fibrotic properties relevant for regenerative medicine. Here, we introduce a new hydrogel based on high MW HA which is cross-linked using the transglutaminase (TG) activity of the activated blood coagulation factor XIII (FXIIIa). These HA-TG gels have significant advantages for neural tissue engineering compared to previous HA gels. Due to their chemical inertness in the absence of FXIIIa, the material can be stored long-term, is stable in solution, and shows no cytotoxicity. The gelation is completely cell-friendly due to the specificity of the enzyme and the gelation rate can be tuned from seconds to hours at physiological pH and independently of stiffness. The gels are injectable, and attach covalently to fibrinogen and fibrin, two common bioactive components in in vitro tissue engineering, as well as proteins present in vivo, allowing the gels to covalently bind to brain or spinal cord defects. These optimal chemical and bioactive properties of HA-TG gels enabled the formation of 3D neuronal cultures of unprecedented performance, showing fast neurite outgrowth, axonal and dendritic speciation, strong synaptic connectivity in 3D networks, and rapidly-occurring and long-lasting coordinated electrical activity.

Factor XIII Cross-Linked Hyaluronan Hydrogels for Cartilage Tissue Engineering.

In this study, transglutaminase-cross-linked hyaluronan (HA-TG) hydrogels are investigated for their potential to treat cartilage lesions. We show the hydrogels fulfill key requirements: they are simultaneously injectable, fast-gelling, biocompatible with encapsulated cells, mitogenic, chondroinductive, and form a stable and strongly adhesive bond to native cartilage. Human chondroprogenitors encapsulated in HA-TG gels simultaneously show good growth and chondrogenesis. Strikingly, within soft gels (∼1 kPa), chondroprogenitors proliferate and deposit extracellular matrix to the extent that the hydrogels reach a modulus (∼0.3 MPa) approaching that of native cartilage (∼1 MPa) within 3 weeks. The combination of such an off-the-shelf human chondroprogenitor cell source with HA-TG hydrogels lays the foundation for a cell-based treatment for cartilage lesions which is based on a minimally invasive one-step procedure, with improved reproducibility due to the defined cells and with improved integration with the surrounding tissue due to the new hydrogel chemistry.