Toward developing human organs via embryo models and chimeras.

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.

A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling.

Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

Reverse Engineering Human Pathophysiology with Organs-on-Chips.

While studies of cultured cells have led to new insights into biological control, greater understanding of human pathophysiology requires the development of experimental systems that permit analysis of intercellular communications and tissue-tissue interactions in a more relevant organ context. Human organs-on-chips offer a potentially powerful new approach to confront this long-standing problem.

Mesenchymal condensation in tooth development and regeneration: a focus on translational aspects of organogenesis.

The teeth are vertebrate-specific, highly specialized organs performing fundamental functions of mastication and speech, the maintenance of which is crucial for orofacial homeostasis and is further linked to systemic health and human psychosocial well-being. However, with limited ability for self-repair, the teeth can often be impaired by traumatic, inflammatory, and progressive insults, leading to high prevalence of tooth loss and defects worldwide. Regenerative medicine holds the promise to achieve physiological restoration of lost or damaged organs, and in particular an evolving framework of developmental engineering has pioneered functional tooth regeneration by harnessing the odontogenic program. As a key event of tooth morphogenesis, mesenchymal condensation dictates dental tissue formation and patterning through cellular self-organization and signaling interaction with the epithelium, which provides a representative to decipher organogenetic mechanisms and can be leveraged for regenerative purposes. In this review, we summarize how mesenchymal condensation spatiotemporally assembles from dental stem cells (DSCs) and sequentially mediates tooth development. We highlight condensation-mimetic engineering efforts and mechanisms based on ex vivo aggregation of DSCs, which have achieved functionally robust and physiologically relevant tooth regeneration after implantation in animals and in humans. The discussion of this aspect will add to the knowledge of development-inspired tissue engineering strategies and will offer benefits to propel clinical organ regeneration.

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.

Cartilage-like protein hydrogels engineered via entanglement.

Load-bearing tissues, such as muscle and cartilage, exhibit high elasticity, high toughness and fast recovery, but have different stiffness (with cartilage being significantly stiffer than muscle)1-8. Muscle achieves its toughness through finely controlled forced domain unfolding-refolding in the muscle protein titin, whereas articular cartilage achieves its high stiffness and toughness through an entangled network comprising collagen and proteoglycans. Advancements in protein mechanics and engineering have made it possible to engineer titin-mimetic elastomeric proteins and soft protein biomaterials thereof to mimic the passive elasticity of muscle9-11. However, it is more challenging to engineer highly stiff and tough protein biomaterials to mimic stiff tissues such as cartilage, or develop stiff synthetic matrices for cartilage stem and progenitor cell differentiation12. Here we report the use of chain entanglements to significantly stiffen protein-based hydrogels without compromising their toughness. By introducing chain entanglements13 into the hydrogel network made of folded elastomeric proteins, we are able to engineer highly stiff and tough protein hydrogels, which seamlessly combine mutually incompatible mechanical properties, including high stiffness, high toughness, fast recovery and ultrahigh compressive strength, effectively converting soft protein biomaterials into stiff and tough materials exhibiting mechanical properties close to those of cartilage. Our study provides a general route towards engineering protein-based, stiff and tough biomaterials, which will find applications in biomedical engineering, such as osteochondral defect repair, and material sciences and engineering.

Cellular and molecular mechanisms of development and regeneration.

Regeneration involves a highly coordinated interplay of intricate cellular processes, enabling living organisms to renew and repair themselves, from individual cells to entire ecosystems. Further, regeneration offers profound insights into developmental biology, tissue engineering and regenerative medicine. The Cellular and Molecular Mechanisms of Development and Regeneration (CMMDR) 2024 conference, which took place at the Shiv Nadar Institute of Eminence and University (India), gathered together an international array of researchers studying a wide variety of organisms across both plant and animal kingdoms. In this short Meeting Review, we highlight some of the outstanding research presented at this conference and draw together some of the common themes that emerged.

Spatial cell fate manipulation of human pluripotent stem cells by controlling the microenvironment using photocurable hydrogel.

Human pluripotent stem cells (hPSCs) dynamically respond to their chemical and physical microenvironment, dictating their behavior. However, conventional in vitro studies predominantly employ plastic culture wares, which offer a simplified representation of the in vivo microenvironment. Emerging evidence underscores the pivotal role of mechanical and topological cues in hPSC differentiation and maintenance. In this study, we cultured hPSCs on hydrogel substrates with spatially controlled stiffness. The use of culture substrates that enable precise manipulation of spatial mechanical properties holds promise for better mimicking in vivo conditions and advancing tissue engineering techniques. We designed a photocurable polyethylene glycol-polyvinyl alcohol (PVA-PEG) hydrogel, allowing the spatial control of surface stiffness and geometry at a micrometer scale. This versatile hydrogel can be functionalized with various extracellular matrix proteins. Laminin 511-functionalized PVA-PEG gel effectively supports the growth and differentiation of hPSCs. Moreover, by spatially modulating the stiffness of the patterned gel, we achieved spatially selective cell differentiation, resulting in the generation of intricate patterned structures.

Multiscale embedded printing of engineered human tissue and organ equivalents.

Creating tissue and organ equivalents with intricate architectures and multiscale functional feature sizes is the first step toward the reconstruction of transplantable human tissues and organs. Existing embedded ink writing approaches are limited by achievable feature sizes ranging from hundreds of microns to tens of millimeters, which hinders their ability to accurately duplicate structures found in various human tissues and organs. In this study, a multiscale embedded printing (MSEP) strategy is developed, in which a stimuli-responsive yield-stress fluid is applied to facilitate the printing process. A dynamic layer height control method is developed to print the cornea with a smooth surface on the order of microns, which can effectively overcome the layered morphology in conventional extrusion-based three-dimensional bioprinting methods. Since the support bath is sensitive to temperature change, it can be easily removed after printing by tuning the ambient temperature, which facilitates the fabrication of human eyeballs with optic nerves and aortic heart valves with overhanging leaflets on the order of a few millimeters. The thermosensitivity of the support bath also enables the reconstruction of the full-scale human heart on the order of tens of centimeters by on-demand adding support bath materials during printing. The proposed MSEP demonstrates broader printable functional feature sizes ranging from microns to centimeters, providing a viable and reliable technical solution for tissue and organ printing in the future.

An engineered model of metastatic colonization of human bone marrow reveals breast cancer cell remodeling of the hematopoietic niche.

Incomplete understanding of metastatic disease mechanisms continues to hinder effective treatment of cancer. Despite remarkable advancements toward the identification of druggable targets, treatment options for patients in remission following primary tumor resection remain limited. Bioengineered human tissue models of metastatic sites capable of recreating the physiologically relevant milieu of metastatic colonization may strengthen our grasp of cancer progression and contribute to the development of effective therapeutic strategies. We report the use of an engineered tissue model of human bone marrow (eBM) to identify microenvironmental cues regulating cancer cell proliferation and to investigate how triple-negative breast cancer (TNBC) cell lines influence hematopoiesis. Notably, individual stromal components of the bone marrow niche (osteoblasts, endothelial cells, and mesenchymal stem/stromal cells) were each critical for regulating tumor cell quiescence and proliferation in the three-dimensional eBM niche. We found that hematopoietic stem and progenitor cells (HSPCs) impacted TNBC cell growth and responded to cancer cell presence with a shift of HSPCs (CD34+CD38-) to downstream myeloid lineages (CD11b+CD14+). To account for tumor heterogeneity and show proof-of-concept ability for patient-specific studies, we demonstrate that patient-derived tumor organoids survive and proliferate in the eBM, resulting in distinct shifts in myelopoiesis that are similar to those observed for aggressively metastatic cell lines. We envision that this human tissue model will facilitate studies of niche-specific metastatic progression and individualized responses to treatment.

Development of porcine skeletal muscle extracellular matrix-derived hydrogels with improved properties and low immunogenicity.

Hydrogels derived from decellularized extracellular matrices (ECM) of animal origin show immense potential for regenerative applications due to their excellent cytocompatibility and biomimetic properties. Despite these benefits, the impact of decellularization protocols on the properties and immunogenicity of these hydrogels remains relatively unexplored. In this study, porcine skeletal muscle ECM (smECM) underwent decellularization using mechanical disruption (MD) and two commonly employed decellularization detergents, sodium deoxycholate (SDC) or Triton X-100. To mitigate immunogenicity associated with animal-derived ECM, all decellularized tissues were enzymatically treated with α-galactosidase to cleave the primary xenoantigen-the α-Gal antigen. Subsequently, the impact of the different decellularization protocols on the resultant hydrogels was thoroughly investigated. All methods significantly reduced total DNA content in hydrogels. Moreover, α-galactosidase treatment was crucial for cleaving α-Gal antigens, suggesting that conventional decellularization methods alone are insufficient. MD preserved total protein, collagen, sulfated glycosaminoglycan, laminin, fibronectin, and growth factors more efficiently than other protocols. The decellularization method impacted hydrogel gelation kinetics and ultrastructure, as confirmed by turbidimetric and scanning electron microscopy analyses. MD hydrogels demonstrated high cytocompatibility, supporting satellite stem cell recruitment, growth, and differentiation into multinucleated myofibers. In contrast, the SDC and Triton X-100 protocols exhibited cytotoxicity. Comprehensive in vivo immunogenicity assessments in a subcutaneous xenotransplantation model revealed MD hydrogels' biocompatibility and low immunogenicity. These findings highlight the significant influence of the decellularization protocol on hydrogel properties. Our results suggest that combining MD with α-galactosidase treatment is an efficient method for preparing low-immunogenic smECM-derived hydrogels with enhanced properties for skeletal muscle regenerative engineering and clinical applications.

A bioactive supramolecular and covalent polymer scaffold for cartilage repair in a sheep model.

Regeneration of hyaline cartilage in human-sized joints remains a clinical challenge, and it is a critical unmet need that would contribute to longer healthspans. Injectable scaffolds for cartilage repair that integrate both bioactivity and sufficiently robust physical properties to withstand joint stresses offer a promising strategy. We report here on a hybrid biomaterial that combines a bioactive peptide amphiphile supramolecular polymer that specifically binds the chondrogenic cytokine transforming growth factor ß-1 (TGFß-1) and crosslinked hyaluronic acid microgels that drive formation of filament bundles, a hierarchical motif common in natural musculoskeletal tissues. The scaffold is an injectable slurry that generates a porous rubbery material when exposed to calcium ions once placed in cartilage defects. The hybrid material was found to support in vitro chondrogenic differentiation of encapsulated stem cells in response to sustained delivery of TGFß-1. Using a sheep model, we implanted the scaffold in shallow osteochondral defects and found it can remain localized in mechanically active joints. Evaluation of resected joints showed significantly improved repair of hyaline cartilage in osteochondral defects injected with the scaffold relative to defects injected with the growth factor alone, including implantation in the load-bearing femoral condyle. These results demonstrate the potential of the hybrid biomimetic scaffold as a niche to favor cartilage repair in mechanically active joints using a clinically relevant large-animal model.

Liquid-embedded (bio)printing of alginate-free, standalone, ultrafine, and ultrathin-walled cannular structures.

While there has been considerable success in the three-dimensional bioprinting of relatively large standalone filamentous tissues, the fabrication of solid fibers with ultrafine diameters or those cannular featuring ultrathin walls remains a particular challenge. Here, an enabling strategy for (bio)printing of solid and hollow fibers whose size ranges could be facilely adjusted across a broad spectrum, is reported, using an aqueous two-phase embedded (bio)printing approach combined with specially designed cross-linking and extrusion methods. The generation of standalone, alginate-free aqueous architectures using this aqueous two-phase strategy allowed freeform patterning of aqueous bioinks, such as those composed of gelatin methacryloyl, within the immiscible aqueous support bath of poly(ethylene oxide). Our (bio)printing strategy revealed the fabrication of standalone solid or cannular structures with diameters as small as approximately 3 or 40 µm, respectively, and wall thicknesses of hollow conduits down to as thin as <5 µm. With cellular functions also demonstrated, we anticipate the methodology to serve as a platform that may satisfy the needs for the different types of potential biomedical and other applications in the future, especially those pertaining to cannular tissues of ultrasmall diameters and ultrathin walls used toward regenerative medicine and tissue model engineering.

Regenerative engineering of long bones using the small molecule forskolin.

Bone grafting procedures have become increasingly common in the United States, with approximately 500,000 cases occurring each year at a societal cost exceeding $2.4 billion. Recombinant human bone morphogenetic proteins (rhBMPs) are therapeutic agents that have been widely used by orthopedic surgeons to stimulate bone tissue formation alone and when paired with biomaterials. However, significant limitations such as immunogenicity, high production cost, and ectopic bone growth from these therapies remain. Therefore, efforts have been made to discover and repurpose osteoinductive small-molecule therapeutics to promote bone regeneration. Previously, we have demonstrated that a single-dose treatment with the small-molecule forskolin for just 24 h induces osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro, while mitigating adverse side effects attributed with prolonged small-molecule treatment schemes. In this study, we engineered a composite fibrin-PLGA [poly(lactide-co-glycolide)]-sintered microsphere scaffold for the localized, short-term delivery of the osteoinductive small molecule, forskolin. In vitro characterization studies showed that forskolin released out of the fibrin gel within the first 24 h and retained its bioactivity toward osteogenic differentiation of bone marrow-derived stem cells. The forskolin-loaded fibrin-PLGA scaffold was also able to guide bone formation in a 3-mo rabbit radial critical-sized defect model comparable to recombinant human bone morphogenetic protein-2 (rhBMP-2) treatment, as demonstrated through histological and mechanical evaluation, with minimal systemic off-target side effects. Together, these results demonstrate the successful application of an innovative small-molecule treatment approach within long bone critical-sized defects.

Application of stem cells in engineered vascular graft and vascularized organs.

Recent studies report that stem cell therapies have been applied successfully to patients, This has increased anticipations that this regeneration strategy could be a potential method to treat a wide range of intractable diseases some day. Stem cells offer new prospects for the treatment of incurable diseases and for tissue regeneration and repairation because of their unique biological properties. Angiogenesis a key process in tissue regeneration and repairation. Vascularization of organs is one of the main challenges hindering the clinical application of engineered tissues. Efficient production of engineered vascular grafts and vascularized organs is of critical importance for regenerative medicine. In this review, we focus on the types of stem cells that are widely used in tissue engineering and regeneration, as well as their application of these stem cells in the construction of tissue-engineered vascular grafts and vascularization of tissue-engineered organs.

Physical and biological advances in endothelial cell-based engineered co-culture model systems.

Scientific knowledge in the field of cell biology and mechanobiology heavily leans on cell-based in vitro experiments and models that favor the examination and comprehension of certain biological processes and occurrences across a variety of environments. Cell culture assays are an invaluable instrument for a vast spectrum of biomedical and biophysical investigations. The quality of experimental models in terms of simplicity, reproducibility, and combinability with other methods, and in particular the scale at which they depict cell fate in native tissues, is critical to advancing the knowledge of the comprehension of cell-cell and cell-matrix interactions in tissues and organs. Typically, in vitro models are centered on the experimental tinkering of mammalian cells, most often cultured as monolayers on planar, two-dimensional (2D) materials. Notwithstanding the significant advances and numerous findings that have been accomplished with flat biology models, their usefulness for generating further new biological understanding is constrained because the simple 2D setting does not reproduce the physiological response of cells in natural living tissues. In addition, the co-culture systems in a 2D stetting weakly mirror their natural environment of tissues and organs. Significant advances in 3D cell biology and matrix engineering have resulted in the creation and establishment of a new type of cell culture shapes that more accurately represents the in vivo microenvironment and allows cells and their interactions to be analyzed in a biomimetic approach. Contemporary biomedical and biophysical science has novel advances in technology that permit the design of more challenging and resilient in vitro models for tissue engineering, with a particular focus on scaffold- or hydrogel-based formats, organotypic cultures, and organs-on-chips, which cover the purposes of co-cultures. Even these complex systems must be kept as simplified as possible in order to grasp a particular section of physiology too very precisely. In particular, it is highly appreciated that they bridge the space between conventional animal research and human (patho)physiology. In this review, the recent progress in 3D biomimetic culturation is presented with a special focus on co-cultures, with an emphasis on the technological building blocks and endothelium-based co-culture models in cancer research that are available for the development of more physiologically relevant in vitro models of human tissues under normal and diseased conditions. Through applications and samples of various physiological and disease models, it is possible to identify the frontiers and future engagement issues that will have to be tackled to integrate synthetic biomimetic culture systems far more successfully into biomedical and biophysical investigations.

'Chip'-ing away at morphogenesis - application of organ-on-chip technologies to study tissue morphogenesis.

Emergent cell behaviors that drive tissue morphogenesis are the integrated product of instructions from gene regulatory networks, mechanics and signals from the local tissue microenvironment. How these discrete inputs intersect to coordinate diverse morphogenic events is a critical area of interest. Organ-on-chip technology has revolutionized the ability to construct and manipulate miniaturized human tissues with organotypic three-dimensional architectures in vitro. Applications of organ-on-chip platforms have increasingly transitioned from proof-of-concept tissue engineering to discovery biology, furthering our understanding of molecular and mechanical mechanisms that operate across biological scales to orchestrate tissue morphogenesis. Here, we provide the biological framework to harness organ-on-chip systems to study tissue morphogenesis, and we highlight recent examples where organ-on-chips and associated microphysiological systems have enabled new mechanistic insight in diverse morphogenic settings. We further highlight the use of organ-on-chip platforms as emerging test beds for cell and developmental biology.

Hyaline cartilage differentiation of fibroblasts in regeneration and regenerative medicine.

Amputation injuries in mammals are typically non-regenerative; however, joint regeneration is stimulated by BMP9 treatment, indicating the presence of latent articular chondrocyte progenitor cells. BMP9 induces a battery of chondrogenic genes in vivo, and a similar response is observed in cultures of amputation wound cells. Extended cultures of BMP9-treated cells results in differentiation of hyaline cartilage, and single cell RNAseq analysis identified wound fibroblasts as BMP9 responsive. This culture model was used to identify a BMP9-responsive adult fibroblast cell line and a culture strategy was developed to engineer hyaline cartilage for engraftment into an acutely damaged joint. Transplanted hyaline cartilage survived engraftment and maintained a hyaline cartilage phenotype, but did not form mature articular cartilage. In addition, individual hypertrophic chondrocytes were identified in some samples, indicating that the acute joint injury site can promote osteogenic progression of engrafted hyaline cartilage. The findings identify fibroblasts as a cell source for engineering articular cartilage and establish a novel experimental strategy that bridges the gap between regeneration biology and regenerative medicine.

Challenges and opportunities for the next generation of cardiovascular tissue engineering.

Engineered cardiac tissues derived from human induced pluripotent stem cells offer unique opportunities for patient-specific disease modeling, drug discovery and cardiac repair. Since the first engineered hearts were introduced over two decades ago, human induced pluripotent stem cell-based three-dimensional cardiac organoids and heart-on-a-chip systems have now become mainstays in basic cardiovascular research as valuable platforms for investigating fundamental human pathophysiology and development. However, major obstacles remain to be addressed before the field can truly advance toward commercial and clinical translation. Here we provide a snapshot of the state-of-the-art methods in cardiac tissue engineering, with a focus on in vitro models of the human heart. Looking ahead, we discuss major challenges and opportunities in the field and suggest strategies for enabling broad acceptance of engineered cardiac tissues as models of cardiac pathophysiology and testbeds for the development of therapies.

Kidney Disease Modeling with Organoids and Organs-on-Chips.

Kidney disease is a global health crisis affecting more than 850 million people worldwide. In the United States, annual Medicare expenditures for kidney disease and organ failure exceed $81 billion. Efforts to develop targeted therapeutics are limited by a poor understanding of the molecular mechanisms underlying human kidney disease onset and progression. Additionally, 90% of drug candidates fail in human clinical trials, often due to toxicity and efficacy not accurately predicted in animal models. The advent of ex vivo kidney models, such as those engineered from induced pluripotent stem (iPS) cells and organ-on-a-chip (organ-chip) systems, has garnered considerable interest owing to their ability to more accurately model tissue development and patient-specific responses and drug toxicity. This review describes recent advances in developing kidney organoids and organ-chips by harnessing iPS cell biology to model human-specific kidney functions and disease states. We also discuss challenges that must be overcome to realize the potential of organoids and organ-chips as dynamic and functional conduits of the human kidney. Achieving these technological advances could revolutionize personalized medicine applications and therapeutic discovery for kidney disease.