Research Progress in Endoderm-Derived Cell Organoids

1. Introduction

Ectoderm, mesoderm and endoderm are important concepts in embryology. They refer to the three cell germ layers formed by the migration and transformation of blastula cells in the early stage of embryo development, which together constitute the posterior gastrula. The appearance of the three germ layers is a sign that the embryo smoothly enters the organ development stage from the cell division stage. Subsequently, ectodermal cells eventually differentiate into body surface structures, nervous system and some sensory organs. Mesoderm can differentiate into muscle, bone, cardiovascular, genitourinary and other tissues. Endoderm is located in the innermost layer of embryo through cell migration and rearrangement during gastrulation. In humans, endoderm-derived cells form most internal organs, such as the digestive tract (including esophagus, stomach, small intestine, large intestine, liver, pancreas, etc.), respiratory system (trachea, bronchus, alveoli, etc.), endocrine glands (thyroid, parathyroid, etc.) and urinary system (renal tubular epithelium, bladder epithelium, etc.), which are the most important and complex cell populations with the most difficult treatment of lesions and the highest medical value (Figure 1). In a word, endodermal cells have important values in the formation of major tissues and organs due to their complex functions and diverse differentiation potential. Through the in-depth study of endodermal cell behavior, development mechanism and cell function, readers can better understand its key tasks in the formation of digestive system, respiratory system, endocrine system and urinary system. It can more comprehensively provide new ideas and technical routes for the perfect function of organoids composed of endoderm-derived cells and provide new perspectives and methods for the further development of regenerative medicine [1, 2, 3, 4, 5].

In this chapter, we focus on the research progress of endoderm-derived cell organoids and introduce the theoretical system, application prospects and two-sided thinking of endoderm-derived cell organoid technology.

2. Development of endoderm-derived cell organoid technology

Endoderm-derived cell organoid refers to a micro tissue or an organ model that uses stem cells or tissue progenitor cells to spontaneously form through adult tissues derived from endoderm under three-dimensional culture conditions in vitro, or that is derived from pluripotent stem cells to directionally induce differentiation through the defined endoderm (DE) pathway and has the unique structure and function of endoderm cells. The study of endoderm-derived cell organoids is an important development direction in the fields of cell biology, developmental biology and tissue engineering in recent years. Its ultimate goal is to artificially reproduce its in vivo development process in vitro so as to obtain organs with infinite adult functions. We always believe that the solution of this problem will carry the good wish of mankind: to provide unlimited sources of biological materials for diseases, such as organ failure, degenerative diseases, cancer, genetic diseases and aging, which are difficult to obtain etiological treatment, so as to obtain complete cure of the disease. Endodermal organoids are the first organoid group studied by people. The embryonic form of the study can be traced back to 1907. Wilson first observed that sponge cells can self-assemble into a complete organism in vitro. This indicates that the cells have the ability of self-organization. Fortunately, in the 1950s–1970s, organoid technology made breakthroughs and researchers began to try organ culture in this period. However, limited by two-dimensional culture technology, it is difficult to simulate the real organ structure. By 2009, Hans Clevers’ research group had successfully constructed intestinal organoids with crypt-villus structure using lgr5+ intestinal stem cells, which marked the birth of endoderm-derived organoids and modern organoid construction technology in the true sense [6]. Organoid technology has advanced swiftly, producing various endoderm-derived organoids, such as liver, pancreas, lung and stomach. These organoids not only mimic the structure and cellular makeup of their corresponding organs but also replicate some of their functions. For instance, liver organoids can metabolize drugs and detoxify, while pancreatic organoids can secrete hormones such as insulin. These organoids are invaluable for studying embryonic development, disease modeling, drug testing and regenerative medicine. As the technology matures, a theoretical framework for researching these organoids has emerged, enhancing our understanding of organ development, disease mechanisms and treatment strategies. As a new biological model, organoids with stem cells as starting cells show great potential in the fields of drug screening and regenerative medicine with their unlimited proliferation, high homogeneity and multi-directional differentiation ability. The research theory of endoderm-derived cell organoids focuses on the differentiation of DE, the construction of organoids, functional evaluation and diverse applications. The formation of endoderm-derived cell organoids is not only dependent on the pluripotency of stem cells as starting cells but also restricted by complex microenvironments, such as intercellular signals, matrix support and physical environment. People usually use small molecule compounds or specific growth factors to induce these cells to differentiate toward the endodermal lineage. For example, molecules such as Nodal and Activin A can promote the formation of DE by activating SMAD2/3 signaling pathway [1]. Furthermore, DE cells obtained by induction can induce the formation of functional cell precursors of target organs and then obtain mature functional cells through the regulation of WNT, FGF and other signals under specific conditions [7]. Compared with the monolayer planar induction culture system, the three-dimensional culture system provides the space required for cell-cell interaction, simulates the cell polarity and hierarchical structure in organ development in vivo and enables the culture to obtain the core characteristics of self-organization ability and spatial structure. Organoids from different sources can usually simulate the differentiation pattern of original organs and form morphological features with characteristic structures. For example, during the formation of liver organoids, they will show a lobular structure of the liver, containing the expression of identity markers unique to functional hepatocytes, such as ALB and AAT [8]. Lung organoids display alveolar-like gas exchange units and express specific markers of mature alveoli, such as Nkx2.1 [9]. In addition to morphological features and identity markers, cells in organoids should also have functional markers specific to the organ of interest, such as insulin secretion by islet organoids and digestive enzymes secretion by intestinal organoids. Although the induction of stem cells as a source material is very expensive and the conditions are extremely harsh, it has irreplaceable advantages in terms of the yield of functional target cells, reducing immune rejection and avoiding ethical disputes at the current technical level. In addition, endodermal organoids are not simple three-dimensional cell aggregates. Their formation is regulated by many factors, such as the composition of extracellular matrix, physical properties, gas composition, matrix stiffness and changes in adhesive materials, which can significantly affect the morphological structure and stability of organoids. These elements together constitute a micro organ niche (i.e., culture microenvironment). In addition, the interaction between cells and the establishment of cell polarity are also important factors that determine the morphology and function of organoids.

3. Culture system and differentiation strategy of endoderm-derived organoids

The core idea of establishing the culture system of endoderm-derived organoids is to simulate the in vivo development process of functional organs of the digestive and respiratory systems. The construction method of its corresponding organoids needs to integrate multidisciplinary cross technologies, such as embryonic developmental biology, material science, bioengineering and bioinformatics. In terms of methodology design, it can be carried out from three aspects: stem cell species selection, medium optimization and three-dimensional construction strategy and functional verification. The construction of endodermal organoids begins with the differentiation and regulation of stem cells. Stem cell systems from different sources can be selected according to research needs. Among pluripotent stem cells, human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are ideal models for studying the early differentiation of endoderm because of their totipotent differentiation characteristics. Among them, iPSC has more advantages as starting material compared with ESC due to more perfect ethical disputes avoided and more extensive sources. Through the gradient induction and synergistic effect of Activin A, FGF4 and WNT3a signaling pathways, people can directionally differentiate ESC/iPSC into DE cell population within 5–7 days. Such cells need to be maintained in RPMI-1640 medium containing B-27 supplement and noggin to inhibit BMP signaling and promote the expression of endoderm-specific genes (such as SOX17 and FOXA2).

Fetal intestinal stem cells (FSCs) and adult stem cells (ASCs) have also been successfully used as starting materials for obtaining organoids. FSC retains epigenetic memory in the middle of embryonic development and is suitable for simulating the transition state of organ development. Studies have shown that FSCs can self-assemble into villus-crypt structures in the medium containing R-spondin 1, EGF and prostaglandin E2, and their gene expression profiles are highly similar to human fetal intestinal tissues [10]. Intestinal crypt stem cells (lgr5⁺) in ASCs are a classic model for studying the function of mature intestinal epithelium [11]. So far, organoids expanded by ENR (EGF/NOGGIN/R-spondin) medium can achieve long-term culture in Matrigel. Some new culture systems have been able to achieve cell lineages, with functions similar to those of primary tissues [12, 13, 14]. The differentiation of endodermal organoids requires precise regulation of the spatiotemporal expression of growth factors. The current typical culture protocol takes iPSC as the starting material to induce colon organoids as an example, which can be roughly divided into three stages: The first is the differentiation step of DE. When differentiating organoids from hESCs or iPSCs, the process of embryonic development should be simulated using growth factors or small molecules. In the mature protocol, the DE of cells was treated with CHI99021 within the first 24 h, and the cells were cultured with Activin a⁺ medium for at least 72 h. The second step of colon organoid formation involves hindgut differentiation, in which FGF signaling pathway plays an important role. Studies have shown that FGF signaling activation promotes the formation of the posterior endoderm, which in turn initiates the development of the midgut and the hindgut. The combination of FGF4 and WNT3a can maintain the stability of hindgut morphology, but high concentration of FGF4, combined with stem cell differentiation supporting factors such as B27 and CHIR99021, can transform monolayer endodermal cells into three-dimensional structures within a week, and spontaneously form early colon spheroids. CHIR99021 cooperates with FGF4 to activate WNT and FGF signaling to complete hindgut differentiation and induce colon spheroid formation. At the final stage, early colonic spheroids spontaneously separate from endodermal tubular structures. After monolayer cells are collected and embedded in Matrigel, they transition to the maturation process. After evaluation, it was determined that 4 days were the standard time for hindgut differentiation, but some iPSC lines appeared self-serve sprouting on Days 5–6. At the end of differentiation, spheres were collected and embedded in Matrigel to efficiently acquire colon organoids. Subsequently, EGF is added to the culture medium to maintain self-renewal, and small molecules such as CHIR99021 and IDN193189 can help promote the continuous and healthy growth of organoids (Figure 2) [15].

In the traditional organoid culture system, hanging drop method and Matrigel are commonly used. However, the hanging drop method has large batch differences and relies heavily on the experience of operators. In addition to large batch differences, Matrigel also has risks of animal-derived ingredients and ethical issues. In the three-dimensional culture system of organoids, these two methods are gradually replaced by new substrates.

Synthetic hydrogel belongs to a new type of matrix that is more selected by people. Through its engineering transformation, taking VitroGel organoid matrix as an example, it can simulate the mechanical strength of different tissues. This kind of matrix has the advantages of no animal source and clear composition. It can be directly cultured from frozen cells and can immediately form spheroids, which avoid culturing monolayer cells and can further promote the growth of organoids. It can effectively simplify the process of organoid culture, and has high uniformity, which offers great advantages [16]. Out of concern about the difficulty in standardizing the defect of manually controlling micro organ niches, the precise regulation of microfluidic chips has become indispensable in organoid technology. Based on organ chip technology combined with microfluidic system, it has been able to help people build a dual channel system containing intestinal epithelial mesenchymal interaction. Microfluidic chips can also improve the barrier function of organoids by applying stable and controllable fluid shear force [17]. Mature endodermal organoids need to verify their biological characteristics through a multi-dimensional evaluation system, such as morphological phenotype, lineage composition, functional verification, molecular markers and drug screening ability. In terms of molecular marker evaluation objectives, at present, people can use single-cell RNA sequencing technology to compare the transcriptome similarities between organoids and fetal tissues, and then establish a marker gene library to achieve dynamic high-throughput monitoring. However, people still lack sufficient experience in verifying maturity and functional integrity. The reason is that although many organoids are similar to real organs in morphology, there is still a big gap in function. This defect makes their application in disease research and drug testing subject to many and long-term limitations.

In terms of the diversity of lineage composition, especially in adult mature organs, cell types are abundant and diverse, and there are complex interactions between different cells. However, it is often difficult for existing organoid culture technology to fully reproduce this cell diversity and interaction. For example, in liver organoids, there may be a lack of some key non-parenchymal cells, such as hepatic stellate cells and Kupffer cells, which will affect the comprehensive simulation of liver physiological functions and disease processes by organoids. The size and long-term stability of organoids is also one of the technical difficulties. At present, most organoids can only be called micro-organs, which limits their use in some research and applications. In addition, in the long-term culture process, organoids may have problems such as abnormal differentiation and genetic drift, resulting in changes in their characteristics and functions, which are difficult to meet the needs of long-term research and clinical application.

The establishment of endoderm-derived organoid culture system is a model of cross-innovation between developmental biology and bioengineering. With the emergence of new biomaterials and intelligent culture equipment, organoid models will more accurately simulate the spatiotemporal development process of human organs, providing a revolutionary tool for personalized medicine and regenerative medicine. Future research needs to further break through technical bottlenecks, such as heterogeneous regulation and vascularization construction, and promote the transformation of this technology from laboratory to clinic.

4. Endoderm-derived digestive and respiratory organoids

As expected, endoderm-derived organoids have been widely used to mimic genetic diseases (such as cystic fibrosis), infectious diseases (such as severe acute respiratory syndrome) and a variety of cancers originating from the endoderm lineage. Moreover, organoid technology has also brought effective solutions for personalized medicine, especially in patient-specific drug sensitivity testing. So far, among endoderm-derived organoids, digestive system organoids and respiratory system organoids have made great breakthroughs. In the field of basic research, among digestive system organoids, taking intestinal organoids as an example, effective intestinal organoids should be mainly composed of intestinal endocrine cells (EEC), which are hormone-secreting cells that exist in the epithelium of the stomach, small intestine and colon. The combination of RNA sequencing and organoid technology can help clarify the temporal hierarchy of gene expression profiles during the development of EEC. As a time-resolved reporter gene, NEUROG3 (the main regulator of endocrine development) can construct a real-time and lineage-specific map of EEC differentiation in mice at the single-cell level. At present, the difficulty is that the regulation mechanism of EEC differentiation in mice and humans is not completely consistent. The regulatory mechanism upstream of NEUROG3 and the endogenous inhibitory factors regulating NEUROG3 expression are not yet clear [18], and new discoveries in human-derived intestinal organoids are urgently needed.

In terms of new drug development, Merus N.V. of the Netherlands cooperated with the Barcelona Institute of Science and Technology of Spain to screen more than 500 bispecific antibodies using the organoid bank from cancer patients and obtained the EGFR, the EG bispecific antibody named mcla-158, which can effectively inhibit the growth of colorectal cancer organoids and inhibit cancer metastasis. The relevant research results were published in the journal Nature in April 2022 [19]. In 2023, based on previous research, Hans Clevers team established an organoid gene-screening platform, found that ZNF800 is a brand-new transcription factor that inhibits EEC differentiation and confirmed that PAX4 is a downstream target of ZNF800.The findings in this study can provide key clues for the research of gastrointestinal diseases and endocrine diseases and lay the foundation for the development of new treatment schemes and drugs. At present, people have been able to support the differentiation of iPSCs into hepatic sinusoidal endothelial precursor cells through the inverted multi-layer air-liquid interface culture system and further self-organize to form liver organoids with functional sinusoidal vessels. This innovative result, as a functional cellular drug, not only brings new hope for the treatment of hemophilia and other coagulation disorders, but also provides new possibilities for the tissue repair of patients with liver damage [20].

In the respiratory system organoids, taking alveolar organoids as an example, in the field of basic research, Wilkinson et al. conducted a systematic study, including but not limited to developing complex cellular lung organoids from iPSC and modeling lung diseases using them. Furthermore, the team also established an in vitro pulmonary fibrosis model using lung organoids containing alveolar epithelial cells for the first time and constructed iPSC-derived lung organoids around functional alginate beads, which included mesenchymal cells, alveolar epithelial cells and other cells. After treatment with TGF-GF and other cells, as well as after treatment with lung mesenchymal cells, alveolar epithelial cells and epithelial cell clonal alginate beads, including those with mesenchymal cell fibrosis, further analyses were performed [21]. If researchers can further screen or modify existing matrix materials to achieve higher histocompatibility and stability in the body, it will have broader clinical application prospects. Different from the iPSC pathway, Surolia et al. extracted cell suspensions from partial lung tissues of normal people and patients with pulmonary fibrosis through surgical biopsy to prepare lung spheroid-like organoids, including type II alveolar epithelial cells, myofibroblasts and other cells. Obvious invasive myofibroblast regions were observed in organoids derived from patients with pulmonary fibrosis. Compared with the normal group, the expression of extracellular matrix proteins was different. This construction method is more conducive to the design of personalized treatment schemes for different patients [22]. In the field of application research, Tsutsumi et al. took the COPD mouse model simulated by long-term exposure to cigarette smoke as the research object. Researchers used the ex vivo alveolar organoid model to explore the role of FAM13a. Alveolar organoids were generated from fumigated Fam13a^+/+ and Fam13a^−/− mice. The results showed that the loss of FAM13a was related to the proliferation and differentiation of type II alveolar epithelial cells and the growth trend of alveolar organoids. When exposed to cigarette smoke, FAM13a deletion would upregulate the activation of WNT in type II alveolar epithelial cells and develop more alveolar organoids than Fam13a^+/+ mice [23]. Yu et al. also tried to study the passive influence of harmful particles in the air through alveolar organoids. By adding PM2.5 to the growth medium and differentiation medium of alveolar organoids, they found that PM2.5 treatment caused damage to alveolar organoids, the proliferation level of type II alveolar epithelial cells increased but the repair function was impaired, and the ratio of type I to type II alveolar epithelial cells decreased significantly, indicating that the process of type II alveolar epithelial cells entering transdifferentiation was inhibited [24].

In the applied research of coronavirus infection, alveolar organoids play a huge role. Han et al. cultured iPSCs into lung organoids containing type II alveolar epithelial cells and used their characteristics of expressing ACE2 to make transcriptome sequencing analysis after the culture was infected with coronavirus. It was found that a variety of chemokines and cytokines have high expression levels, and there is almost no expression of type I/III interferon signal, which is similar to the situation observed in patients with coronavirus lung infection. The use of imatinib and mycophenolic acid can slow down the virus infection of organoids [25]. Katsura et al. determined the conditions for long-term expansion and differentiation of adult alveolar stem cells, constructed alveolar organoids that can be directly infected by neocoronavirus, and found that the infected organoids had similar characteristics to the lungs after neocoronavirus infection, such as the appearance of interferon-mediated inflammatory response, the loss of surfactant proteins and apoptosis. In addition, the reduction of virus replication was observed in alveolar organoids pretreated with low-dose interferon, indicating its preventive effect on neocoronavirus [26]. In regenerative medicine research, researchers cultured adult primary bronchial epithelial cells, lung fibroblasts and lung microvascular endothelial cells for 3D to generate airway organoids. The mixed cell population will undergo rapid condensation and self-organization to form discrete epithelial and endothelial structures that are mechanically robust and stable in long-term culture. This organoid also showed a complex multicellular response to typical fibrotic stimuli. After transplantation into the renal capsule of immunodeficient mice, it had limited maturation and implantation ability. It could obtain nutrients needed for growth from the blood vessels of mice near the transplantation area and achieve in vivo survival for more than 6 weeks. At the same time, cell-specific markers were detected for this organoid, and it was found that during its growth, new cell types could be detected, such as CC10⁺ airway secretory cells and AQP5, SPC+ alveolar epithelial cells. This study shows that lung organoids are expected to achieve in vivo transplantation and maintain growth capacity, which to a certain extent supports the use of lung organoids for lung regeneration [27].

Using these lung organoid models, researchers can study lung pathology in more detail, such as cystic fibrosis, chronic obstructive pulmonary disease or asthma, and viral infection. These models lay the foundation for further improvement of organs, for example, by combining multiple immune cell types or other organs in microfluidic organ chip devices, standardizing and coordinating these devices to reliably and reproducibly conduct high-throughput drug and vaccine testing. Therefore, the development of organoid models provides a more real and effective tool for the study of respiratory and lung diseases, and provides important support for in-depth understanding of disease mechanisms, developing new therapeutic strategies and reducing the dependence on animal experiments.

5. Challenges and prospects of endoderm-derived organoids

Organoid technology has brought great hope for personalized medicine, especially in patient-specific drug sensitivity testing. In the future, the integration of gene editing and bioprinting may further enhance the role of endoderm-derived organoids in organ transplantation and precision medicine. However, there are still many challenges in standardizing the culture program, achieving vascularization and expanding clinical application and production.

First of all, we need to think about the ethical and regulatory issues, which involve our original intention and mission of carrying out organoid research, and always need researchers to explore in depth and respond cautiously. Organoid construction relies on human tissue such as stem cells, requiring thorough informed consent to ensure donor awareness of research purposes and risks. Emotional connections to organoids mean donor preferences and privacy must be respected. However, organoid technology could worsen medical resource inequality, as its high cost and complexity limit accessibility to wealthier, developed areas. Efforts should focus on cost reduction and simplified processes, alongside policies for broader access. Future complex organoids, particularly brain organoids, raise ethical concerns about potential consciousness, necessitating clear ethical guidelines to manage emerging risks.

With ethical issues as the starting point, the regulatory demand for organoid technology arises at the historic moment. At present, the regulatory framework for organoid research and application is not perfect. There is an urgent need for clear laws and regulations to regulate the source, production, use and commercialization of organoids to ensure their safety and effectiveness. The regulatory framework should fully consider the ethical principles and timely adjust according to the technological development, so as to improve the supervision of organoid clinical trials, ensure that the trial design is scientific and reasonable, and the rights and interests of subjects are fully protected.

Although organoids can replicate the structure and function of some organs in vivo, compared with real organs, endoderm-derived organoids still have a large gap in structural complexity and functional maturity in the future. For example, many organoids lack complete vasculature, innervation and immune cells, which limit their application in simulating complex physiological and pathological processes. In addition, in vitro culture environment is difficult to fully simulate the complex cell-cell interactions and microenvironmental signals in vivo, resulting in the limited functional maturity of organoids.

On the other hand, the preparation process of organoids is affected by many factors, including cell source, medium composition, growth factor combination and culture method. Differences in these factors will lead to differences in the structure, function and cell composition of organoids prepared in different batches or laboratories, affecting the reproducibility and reliability of experimental results. Therefore, the establishment of standardized organoid preparation process and quality control system is the current focus.

Historically, manual endoderm-derived organoid preparation hindered large-scale production, limiting high-throughput applications. Future automated systems must reduce costs and boost efficiency to enable clinical translation.

The application of 3D bioprinting technology in organoids has begun to take shape, achieving exciting breakthroughs in the engineering production of endoderm-derived organoids, such as liver, intestine and pancreas. By adjusting parameters, precise spatial deposition of bioink can be achieved, enhancing repeatability and high-throughput production of multiple printed objects. However, further improvements are needed as this technology faces numerous technical limitations, such as low printing resolution and speed, as well as compatibility issues between bioink and cells themselves. Additionally, the structural integrity and functional simulation of printed products are inadequate, and the high cost remains an unresolved issue.

6. Conclusion and prospect

Endoderm-derived organoid technology has brought revolutionary opportunities for biomedical research, but it is also accompanied by complex ethical and regulatory challenges. Only through full ethical consideration, perfect regulatory framework and extensive social participation can we ensure that this technology can develop healthily on the ethical track and truly benefit mankind. In the face of the double-edged sword effect of science and technology, we must uphold a prudent and responsible attitude, pursue advantages and avoid disadvantages, in order to realize the maximum value of organoid technology and the good expectations of society.