
For several decades, developmental biologists have studied aspects that can control stem cell behavior, such as differentiation and self-renewal, along with certain tissue lineages. To understand human biology, researchers have attempted to create models of the human developmental stages
As one of the promising hPSCs for organ formation, disease modeling, and applications
Organoids have been acknowledged as an important platform for drug screening (21) and have the potential to be used for studying the effect of long-term alternative therapies in regenerative medicine (22). Additionally, organoid technology can be used for numerous purposes by coupling it with the following: genome editing with clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) tools to obtain important insights into genetic disorders (23); co-culturing with pathogens for studying infectious diseases (24); and cancer modeling to understand cancer pathogenesis, development, and progression (25, 26). Principally, organoids serve as exploration tools for understanding the processes underlying human development and diseases (13). However, despite the advances made, most of the human iPSC-derived organoids remain immature in cultures owing to the absence of important features found in adult tissues (27). Therefore, robust
This review will explain the recent efforts at increasing the complexity of organoids and describe the current potential of using mature organoids in applications as diverse as bioengineering, disease modeling, drug discovery and regenerative medicine. We discuss the approaches to build the next-generation organoid, platform and highlight the challenges that need to be addressed for the organoid technology to reach its full potential in the field of regenerative medicine (28).
Organoids are generated by leveraging the self-organizing and self-patterning properties of homogeneous cell populations (3). They are
Adult stem cells (ASCs) (24, 29) and tissue-specific stem cells derived from hPSCs (9, 10, 12, 13) differ in their developmental potential, and thus follow their respective differentiation pathways
Most organoids are derived from a population of starting cells exposed to a particular morphogen at a defined point of time, resulting in the activation of the desired developmental signaling pathway. Notably, iPSC-derived organoids require exposure to specific growth factors at a precise time for differentiation into the target organs. Each organ is derived from three germ layers formed during embryonic development that undergo particular differentiation pathways. Diverse organs are formed from the same germ layer depending on the inductive signal. Precisely using these pathways to form organoids, along with the various combinations and concentrations of factors, and timing can cause different outcomes. Typically with intestinal organoids, the hindgut is formed from the posterior endoderm in response to induction by fibroblast growth factor (FGF)/Wnt (9). Gastric organoids induce foregut formation by inhibiting bone morphogenetic protein (BMP) signaling and generate the posterior foregut via retinoic acid signaling (13). Subsequently, the epidermal growth factor promotes the development of antral gastric organoids. Organoids can differentiate into fundic gastric organoids in response to continuous exposure to these conditions–activation of Wnt signaling; mitogen-ac-tivated protein kinase inhibition; BMP activation (34). Human colonic organoids (hCOs) can be produced by regulating BMP signaling after the formation of hindgut spheroids (35). iPSC-derived retinal organoids can be driven to differentiate into retinal tissue
Currently, organoids are cultured using an ECM to build 3D culture environments. In many reports, the ECM from Engelbreth-Holm-Swarm murine sarcoma, Matrigel, has been mainly used for culturing cells (11, 17). Matrigel has been widely used in epithelial cell culture, but batch- to-batch variability impedes reproducible
Since 2010, numerous papers have described the generation of various iPSC-derived organoids that represent human tissues. These organoids can be divided into ectoderm, mesoderm, and endoderm according to the lineage-specific differentiation process to describe organoid formation. Brain, eye, inner ear, and skin organoid represent the ectoderm. The Lancaster group first developed the iPSC-derived cerebral organoid culture system, which has shown it can reproduce brain development (12). Models in which cerebral organoids containing neurons and glial cell types, including oligodendrocytes, have been created offer new opportunities to examine processes associated with early neuronal development and diseases (46-48). Even more, to predict the central nervous system permeability of drug compounds, a human choroidal plexus organoid with cerebrospinal fluid secretion was established and could be used in brain homeostasis studies (49). Similarly, retinal and corneal organoids have been generated that contain photoreceptor cells that respond to light stimulus; positive results have been reported following their use in cell therapy (10, 36, 50). Ear organoids that contain sensory neurons and cochlear hair cells have recently been developed (17). Further, skin organoids that simulate the complex structure of human skin have been created (19). In mesoderm, one of the three germ layers, kidney, heart and recently blood vessels were developed. Renal organoids containing nephrons have been established which have recently been vascularized using ECM and suspension culture methods (15, 23, 40, 45, 51). Blood vessel organoids, including epithelial cells and pericytes have been generated; the organoids exhibit self-assembly and vascular tree formation when implanted into mice (20). Importantly, cardiac organoids were developed by forced fusion
By stimulating cells with human developmental signals, it is possible to generate a variety of cell types present
In many studies, organoids have been transplanted into immunodeficient mice to enable maturation
Organoids have great potential for drug development and cell-based therapies, which are further combined with various applications, such as genome editing tools and organs-on-a-chip technologies. Moreover, new applications allow creating advanced organoids, which could have tremendous potential in translational and regenerative medicine.
In principle, the generation of iPSC-derived organoids needs to strictly mimic human organ development. Therefore, the process should be required precise spatiotemporal signals and correct concentrations essential for cell differentiation and tissue assembly (14, 35, 57). Organoids as 3D models have contributed to an in-depth understanding of human tissue/organ biology; this approach is more realistic than two-dimensional (2D) cultures and mammalian models (65). Moreover, organoids have provided an easily accessible system for identifying organ formation and have opened new avenues for studying human developmental biology (Fig. 2).
Compared to the conventional 2D model, the organoid systems can better simulate histopathological characteristics by assembling various cells. In addition, genetic disorders can be recapitulated using genome editing technology, and in the case of infectious diseases, organoids can be directly co-cultured with pathogens, providing models for disease mechanisms and pathophysiology. Based on these advantages of organoid systems, certain diseases have been studied, such as genetic disorders (12, 21), host- pathogen diseases (13, 24), and cancers (25, 26). Organoids generated from patient-derived iPSCs should clearly recapitulate human pathophysiology to better predict the efficacy and toxicity of drugs at a tissue/organ level by reflecting unique clinical responses to drugs in individual patients. Disease-specific biobanks can be used as a source of samples to test powerful alternative tools for drug screening and precision medicine approaches (66) (Fig. 2). In particular, organoids have been used in a system to test drugs to treat of Zika virus infection (46), and to develop personalized medicine for cystic fibrosis (21) and colorectal cancer (67).
Human iPSC-derived organoids are generated by simulating a human developmental process, but their structure differs from that of real organs. In addition, since it is difficult to deliver growth factors that induce the
Organ-like bioengineered scaffolds are required for the cellular differentiation, organization, and activity, and they can also enhance the continuous culture process of organoids, to simulate the size and shape of actual organs by providing their physiological environment. For example, ISC cultures have maintained the shape of native small intestine with a crypt-villus structure when grown on a scaffold (69, 70). Besides, several studies have reported the use of chemically defined hydrogels (37) and collagen I (38) instead of Matrigel, to improve the growth of organoids and demonstrate clinical use. Applying hydrogels has led to developing photodegradable systems, whereby poly (ethylene glycol) hydrogels can also encapsulate embryonic stem cell-derived motor neurons and use infrared radiation for three-dimensional control. For physical micropatterning on the 3D tissue engineering scaffold, the structure within the hydrogel can be created using pulsed lasers and precisely controlled to enable the development of 3D cultured neural networks as well (71, 72). Alternatively, 3D bioprinting approaches that uniformly assemble cells in structures similar to those of adult organs, may decrease the variability of the organoid phenotypes and may be highly reproducible (73). The generation of organoids can be improved by growing microtubules on chips that mimic the scaffold and blood vessels, reproducing the required gradient of signal molecules (74). Organ-on-a-chip manufactured for organ-specific cell types can simulate a circulatory system with a microfluidic channel and has high reproducibility as an automated system. Organ-on-a-chip based on microfluidic technology allows cells to adapt to the culture environment of the chip system in which the medium is circulated within hollow microchannels (75). These devices can culture and maintain various types of cells simultaneously and predict drug response and toxicity at the organ- or body-levels, even as a multi-organ-on-a-chip. A multi-organ-on-a-chip, also known as a human-on-a-chip, contains various cell lines, including liver, lung, kidney, and adipose tissues, and a multi-channel 3D microfluidic system (76). Furthermore, an organ-on-a-chip is suitable for high-throughput systems including the drug or toxin screening, growth factor and/or signal identification, and may additionally study organ-organ and organ-vessel interaction (77).
The human intestinal mucosal barrier and immune system definitively develop during late gestation and infancy periods, which are associated with the first exposure of commensal and pathogenic microorganisms
Organoids are an important component of cell therapy in regenerative medicine, and the implantation of organoids in animal models has been demonstrated with various approaches. For example, hIOs were transplanted into colonic injuries in mice, following which the colonic mucosal damage was regenerated after 4 weeks (28). In addition, human iPSC-derived liver (11), kidney (87), and lung bud tips (14) were transplanted into each of the chemically induced damaged organs. An optic-cup was also transplanted in a mouse model with retinal degeneration to induce the photoreceptors and restore the synaptic connection to recover function (88). Human iPSC-derived brain organoids have been successfully implanted into the brains of adult mice, increasing the production of mature neuronal cells and the formation of synapses with host neurons (63). Overall, using orthotopic transplantation, it is possible to study the environment during the engraftment of organoids
Several studies have demonstrated rapid progress in organoid application, suggesting that organoids are a promising source for disease modeling, tissue engineering, and cell-based therapy in regenerative medicine. The production of mature organoids combined with challengeable applications can aid in the clinical treatment of tissues. Despite the aforementioned potential of organoids, numerous hurdles remain for achieving successful drug discovery and cell-based therapy. Ideally, protocols for differentiation of iPSC-derived organoids should be standardized for reproducibility and mass-production. Presently, iPSC-derived organoid systems are difficult to homogenize and lack scalability for high-throughput screens and large-scale cell therapy. Improving culture methods, including defined ECMs and multiscale micropatterning, may facilitate the production of more reproducible organoids. Additionally, for cell transplantation therapy, a defined ECM with excellent biocompatibility should be developed to replace Matrigel, and a scaffold that mimics real organ structure could be established. Conventional organoid differentiation protocols can generate a population of diverse cell types that occur spontaneously, but reducing their variability is a major challenge. Organoids with different characteristics are produced depending on the culture protocol, and specific mutations have been reported in patient-specific iPSC-derived organoids according to genetic background (12, 89). The introduction of gene editing technology can help generate new isogenic organoids that reduce background-related variability.
Based on the development methods of organoids mentioned in this review, organoids with stable scale-up capabilities and reproducibility should undergo three examinations before they are industrially produced as therapeutics. The first is to ensure a pathogen-free state that is directly linked to safety throughout the entire process from cell production to validation. Second, the use of advanced organoids in the industrial pharmaceutical pipeline requires the development of a storage or delivery method for organoids to optimize manufacturing costs. Finally, objective validation techniques for measuring the safety and efficacy of therapeutics
Human iPSC-derived organoids are an accessible and physiologically relevant model system that can mimic the functions of human organs without ethical concerns regarding human embryos and interspecies differences. Notably, organoids have tremendous potential in tissue biology research, disease modeling and alternative cell-based therapy. Moreover, in recent years, rapid progress has been made on the bioengineering aspects of ECM scaffolds, genome editing and organ-on-a-chip approaches to improve organoid functionality. Therefore, advanced organoid systems combined with various applications will undoubtedly expand the scope of regenerative medicine.
2D: two-dimensional; 3D: three-dimensional; ASCs: adult stem cells; BMP: bone morphogenetic protein; CRISPR: clustered regularly interspaced short palindromic repeats; ECM: extracellular matrix; ENS: enteric nervous system; FGF: fibroblast growth factor; hCOs: human colonic organoids; hESCs: human embryonic stem cells; hIOs: human intestinal organoids; hPSCs: human pluripotent stem cells; iPSCs: induced pluripotent stem cells; ISCs: intestinal stem cells
This work was supported by a grant from the Technology Innovation Program (No. 20008777) and 3D-TissueChip Based Drug Discovery Platform Technology Development Program (No. 20009774 and No. 20009209) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea), grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2018M3A9H3023077), Center for Agricul-tural Microorganism and Enzyme (Project No. PJ015049) of Rural Development Administration, and the KRIBB Research Initiative Program. The funders had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript. Figures were created using the site BioRender.com.
The authors have no conflicting financial interest.
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