The science and engineering of stem cell-derived organoids-examples from hepatic,biliary, and pancreatic tissues

The field of organoid engineering promises to revolutionize medicine with wide-ranging applications of scientific, engineering, and clinical interest, including precision and personalized medicine, gene editing, drug development, disease modelling, cellular therapy, and human development. Organoids are a three-dimensional (3D) miniature representation of a target organ, are initiated with stem/progenitor cells, and are extremely promising tools with which to model organ function. The biological basis for organoids is that they foster stem cell self-renewal, differentiation, and self-organization, recapitulating 3D tissue structure or function better than two-dimensional (2D) systems. In this review, we first discuss the importance of epithelial organs and the general properties of epithelial cells to provide a context and rationale for organoids of the liver, pancreas, and gall bladder. Next, we develop a general framework to understand self-organization, tissue hierarchy, and organoid cultivation. For each of these areas, we provide a historical context, and review a wide range of both biological and mathematical perspectives that enhance understanding of organoids. Next, we review existing techniques and progress in hepatobiliary and pancreatic organoid engineering. To do this, we review organoids from primary tissues, cell lines, and stem cells, and introduce engineering studies when applicable. We discuss non-invasive assessment of organoids, which can reveal the underlying biological mechanisms and enable improved assays for growth, metabolism, and function. Applications of organoids in cell therapy are also discussed. Taken together, we establish a broad scientific foundation for organoids and provide an in-depth review of hepatic, biliary and pancreatic organoids.     

The Emergence of Stem Cell‐Based Brain Organoids: Trends and Challenges

Recent developments in 3D cultures exploiting the self‐organization ability of pluripotent stem cells have enabled the generation of powerful in vitro systems termed brain organoids. These 3D tissues recapitulate many aspects of human brain development and disorders occurring in vivo. When combined with improved differentiation methods, these in vitro systems allow the generation of more complex “assembloids,” which are able to reveal cell diversities, microcircuits, and cell–cell interactions within their 3D organization. Here, the ways in which human brain organoids have contributed to demystifying the complexities of brain development and modeling of developmental disorders is reviewed and discussed. Furthermore, challenging questions that are yet to be addressed by emerging brain organoid research are discussed.     

Patterning of brain organoids derived from human pluripotent stem cells

The emerging technology of brain organoids deriving from human pluripotent stem cells provides unprecedented opportunities to study human brain development and associated disorders. Various brain organoid protocols have been developed that can recapitulate some key features of cell type diversity, cytoarchitectural organization, developmental processes, functions, and pathologies of the developing human brain. In this review, we focus on patterning of human stem cell-derived brain organoids. We start with an overview of general procedures to generate brain organoids. We then highlight some recently developed brain organoid protocols and chemical cues involved in modeling development of specific human brain regions, subregions, and multiple regions together. We also discuss limitations and potential future improvements of human brain organoid technology.     

Modelling neurodegenerative diseases with 3D brain organoids

Neurodegenerative diseases are incurable and debilitating conditions characterized by the deterioration of brain function. Most brain disease models rely on human post‐mortem brain tissue, non‐human primate tissue, or in vitro two‐dimensional (2D) experiments. Resource limitations and the complexity of the human brain are some of the reasons that make suitable human neurodegenerative disease models inaccessible. However, recently developed three‐dimensional (3D) brain organoids derived from pluripotent stem cells (PSCs), including embryonic stem cells and induced PSCs, may provide suitable models for the study of the pathological features of neurodegenerative diseases. In this review, we provide an overview of existing 3D brain organoid models and discuss recent advances in organoid technology that have increased our understanding of brain development. Moreover, we explain how 3D organoid models recapitulate aspects of specific neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, and explore the utility of these models, for therapeutic applications.     

Organoid technologies meet genome engineering

Three‐dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two‐dimensional (2D) cell culture models. Organoids, the 3D self‐organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease‐associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.     

Brain organoids: an ensemble of bioassays to investigate human neurodevelopment and disease

AbstractUnderstanding etiology of human neurological and psychiatric diseases is challenging. Genomic changes, protracted development, and histological features unique to human brain development limit the disease aspects that can be investigated using model organisms. Hence, in order to study phenotypes associated with human brain development, function, and disease, it is necessary to use alternative experimental systems that are accessible, ethically justified, and replicate human context. Human pluripotent stem cell (hPSC)-derived brain organoids offer such a system, which recapitulates features of early human neurodevelopment in vitro, including the generation, proliferation, and differentiation of neural progenitors into neurons and glial cells and the complex interactions among the diverse, emergent cell types of the developing brain in three-dimensions (3-D). In recent years, numerous brain organoid protocols and related techniques have been developed to recapitulate aspects of embryonic and fetal brain development in a reproducible and predictable manner. Altogether, these different organoid technologies provide distinct bioassays to unravel novel, disease-associated phenotypes and mechanisms. In this review, we summarize how the diverse brain organoid methods can be utilized to enhance our understanding of brain disorders.Facts

• Brain organoids offer an in vitro approach to study aspects of human brain development and disease.
• Diverse brain organoid techniques offer bioassays to investigate new phenotypes associated with human brain disorders that are difficult to study in monolayer cultures.
• Brain organoids have been particularly useful to study phenomena and diseases associated with neural progenitor morphology, survival, proliferation, and differentiation.

Open question

• Future brain organoid research needs to aim at later stages of neurodevelopment, linked with neuronal activity and connections, to unravel further disease-associated phenotypes.
• Continued improvement of existing organoid protocols is required to generate standardized methods that recapitulate in vivo-like spatial diversity and complexity.

Subject terms: Neuroscience, Neurological disorders

     

Retinal organoids derived from rhesus macaque iPSCs undergo accelerated differentiation compared to human stem cells

PurposeTo compare the timing and efficiency of the development of Macaca mulatta, a nonhuman primate (NHP), induced pluripotent stem cell (rhiPSC) derived retinal organoids to those derived from human embryonic stem cells (hESCs).ResultsGeneration of retinal organoids was achieved from both human and several NHP pluripotent stem cell lines. All rhiPSC lines resulted in retinal differentiation with the formation of optic vesicle‐like structures similar to what has been observed in hESC retinal organoids. NHP retinal organoids had laminated structure and were composed of mature retinal cell types including cone and rod photoreceptors. Single‐cell RNA sequencing was conducted at two time points; this allowed identification of cell types and developmental trajectory characterization of the developing organoids. Important differences between rhesus and human cells were measured regarding the timing and efficiency of retinal organoid differentiation. While the culture of NHP‐derived iPSCs is relatively difficult compared to that of human stem cells, the generation of retinal organoids from NHP iPSCs is feasible and may be less time‐consuming due to an intrinsically faster timing of retinal differentiation.ConclusionsRetinal organoids produced from rhesus monkey iPSCs using established protocols differentiate through the stages of organoid development faster than those derived from human stem cells. The production of NHP retinal organoids may be advantageous to reduce experimental time for basic biology studies in retinogenesis as well as for preclinical trials in NHPs studying retinal allograft transplantation.     

Revealing the inner workings of organoids

The mechanisms by which human stem cells self‐organise into brain‐like tissues in 3D organoid culture are poorly understood. In this issue of The EMBO Journal, Renner and Lancaster et al demonstrate that in the absence of external stimuli, human cerebral organoids develop large forebrain structures that display specific landmarks of spatial and temporal patterning, including signalling centres producing known morphogens. The generation of cerebral organoids is therefore likely to reflect normal brain development.     

Modelling of primary ciliary dyskinesia using patient‐derived airway organoids

Patient‐derived human organoids can be used to model a variety of diseases. Recently, we described conditions for long‐term expansion of human airway organoids (AOs) directly from healthy individuals and patients. Here, we first optimize differentiation of AOs towards ciliated cells. After differentiation of the AOs towards ciliated cells, these can be studied for weeks. When returned to expansion conditions, the organoids readily resume their growth. We apply this condition to AOs established from nasal inferior turbinate brush samples of patients suffering from primary ciliary dyskinesia (PCD), a pulmonary disease caused by dysfunction of the motile cilia in the airways. Patient‐specific differences in ciliary beating are observed and are in agreement with the patients'' genetic mutations. More detailed organoid ciliary phenotypes can thus be documented in addition to the standard diagnostic procedure. Additionally, using genetic editing tools, we show that a patient‐specific mutation can be repaired. This study demonstrates the utility of organoid technology for investigating hereditary airway diseases such as PCD.     

Dissolved oxygen concentration regulates human hepatic organoid formation from pluripotent stem cells in a fully controlled bioreactor

Developing technologies for scalable production of human organoids has gained increased attention for “organoid medicine” and drug discovery. We developed a scalable and integrated differentiation process for generation of hepatic organoid from human pluripotent stem cells (hPSCs) in a fully controlled stirred tank bioreactor with 150 ml working volume by application of physiological oxygen concentrations in different liver tissue zones. We found that the 20–40% dissolved oxygen concentration [DO] (corresponded to 30–60 mmHg pO₂ within the liver tissue) significantly influences the process outcome via regulating the differentiation fate of hPSC aggregates by enhancing mesoderm induction. Regulation of the [DO] at 30% DO resulted in efficient generation of human fetal-like hepatic organoids that had a uniform size distribution and were comprised of red blood cells and functional hepatocytes, which exhibited improved liver-specific marker gene expressions, key liver metabolic functions, and, more important, higher inducible cytochrome P450 activity compared to the other trials. These hepatic organoids were successfully engrafted in an acute liver injury mouse model and produced albumin after implantation. These results demonstrated the significant impact of the dissolved oxygen concentration on hPSC hepatic differentiation fate and differentiation efficacy that should be considered ascritical translational aspect of established scalable liver organoid generation protocols for potential clinical and drug discovery applications.     

Linking stem cell function and growth pattern of intestinal organoids

Intestinal stem cells (ISCs) require well-defined signals from their environment in order to carry out their specific functions. Most of these signals are provided by neighboring cells that form a stem cell niche, whose shape and cellular composition self-organize. Major features of this self-organization can be studied in ISC-derived organoid culture. In this system, manipulation of essential pathways of stem cell maintenance and differentiation results in well-described growth phenotypes.We here provide an individual cell-based model of intestinal organoids that enables a mechanistic explanation of the observed growth phenotypes. In simulation studies of the 3D structure of expanding organoids, we investigate interdependences between Wnt- and Notch-signaling which control the shape of the stem cell niche and, thus, the growth pattern of the organoids. Similar to in vitro experiments, changes of pathway activities alter the cellular composition of the organoids and, thereby, affect their shape. Exogenous Wnt enforces transitions from branched into a cyst-like growth pattern; known to occur spontaneously during long term organoid expansion. Based on our simulation results, we predict that the cyst-like pattern is associated with biomechanical changes of the cells which assign them a growth advantage. The results suggest ongoing stem cell adaptation to in vitro conditions during long term expansion by stabilizing Wnt-activity.Our study exemplifies the potential of individual cell-based modeling in unraveling links between molecular stem cell regulation and 3D growth of tissues. This kind of modeling combines experimental results in the fields of stem cell biology and cell biomechanics constituting a prerequisite for a better understanding of tissue regeneration as well as developmental processes.     

Organoid Transplantation Can Improve Reproductive Prognosis by Promoting Endometrial Repair in Mice

Objective: Intrauterine adhesion (IUA) is one of the major causes of refractory secondary infertility, especially in regions and countries with high abortion rates. In this study, we used the mouse IUA model to evaluate the feasibility of the organoids, a 3D cell structure derived from endometrial tissue, as grafts for the treatment of post-traumatic endometrial regeneration disorders. Methods: The isolated and cultured endometrial organoid was transplanted into the model IUA uterus by the hydrogel scaffold method. Results: The cultured endometrial organoids were transplanted into the basal layer of the damaged endometrium for 28 days. They were completely implanted and grew normally. They not only reconstructed the structural integrity of the endometrial epithelium but also realized the functional repair of the endometrium through differentiation cultures and secretory functions. Conclusion: For severe IUA, this method may be better than stem cell transplantation. These findings provide useful insights into the use of endometrial organoid regeneration in the treatment of injury repair.     

The development of an in vitro cerebral organoid model for investigating the pathomolecular mechanisms associated with the central nervous system involvement in Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)

Abstract

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disorder caused by mutations in the thymidine phosphorylase gene (TYMP), leading to secondary aberrations to the mitochondrial genome. The disease is characterised by gastrointestinal dysmotility, sensorimotor peripheral neuropathy and leukoencephalopathy. The understanding of the molecular mechanisms that underlie the central nervous system (CNS) is hindered by the lack of a representative disease model; to address this we have developed an in vitro 3-D cerebral organoid of MNGIE. Induced pluripotent stem cells (iPSCs) generated from peripheral blood mononuclear cells (PBMCs) of a healthy control and a patient with MNGIE were characterised to ascertain bona fide pluripotency through the evaluation of pluripotency markers and the differentiation to the germ layers. iPSC lines were differentiated into cerebral organoids. Thymidine phosphorylase expression in PBMCs, iPSCs and Day 92 organoids was evaluated by immunoblotting and intact organoids were sampled for histological evaluation of neural markers. iPSCs demonstrated the expression of pluripotency markers SOX2 and TRA1-60 and the plasticity to differentiate into the germ layers. Cerebral organoids stained positive for the neural markers GFAP, O4, Tuj1, Nestin, SOX2 and MBP. Consistent with the disease phenotypes, MNGIE cells did not display thymidine phosphorylase expression whereas control PBMCs and Day 92 organoids did. Remarkably, control iPSCs did not stain positive for thymidine phosphorylase. We have established for the first time a MNGIE iPSC line and cerebral organoid model, which exhibited the expression of cells relevant to the study of the disease, such as neural stem cells, astrocytes and myelinating oligodendrocytes.     

Human iNSC-derived brain organoid model of lysosomal storage disorder in Niemann–Pick disease type C

Recent studies on developing three-dimensional (3D) brain organoids from stem cells have allowed the generation of in vitro models of neural disease and have enabled the screening of drugs because these organoids mimic the complexity of neural tissue. Niemann-Pick disease, type C (NPC) is a neurodegenerative lysosomal storage disorder caused by mutations in the NPC1 or NPC2. The pathological features underlying NPC are characterized by the abnormal accumulation of cholesterol in acidic compartments, including late endosomes and lysosomes. Due to the inaccessibility of brain tissues from human NPC patients, we developed NPC brain organoids with induced neural stem cells from NPC patient-derived fibroblasts. NPC organoids exhibit significantly reduced size and proliferative ability, which are accompanied by accumulation of cholesterol, impairment in neuronal differentiation, and autophagic flux and dysfunction of lysosomes; therefore, NPC organoids can recapitulate the main phenotypes of NPC patients. Furthermore, these pathological phenotypes observed in NPC organoids were reversed by treatment with valproic acid and HPBCD, which are known to be an effective treatment for several neurodegenerative diseases. Our data present patient-specific phenotypes in 3D organoid-based models of NPC and highlight the application of this model to drug screening in vitro.Subject terms: Disease model, Lipid-storage diseases, Neural stem cells     

Dawn of the organoid era

Novel and updated approaches of culturing cells in 3D are rapidly advancing our understanding of development, health, and disease. As tissues have been found to behave more realistically in 3D than in 2D cultures, organoid technology in combination with recent advances in the isolation and generation of stem cells, has rapidly become a promising concept in developmental and regenerative research. The development of all kinds of tissues can now be studied “in a dish,” allowing more detailed observations of stem cell maintenance, morphogens, and differentiation. This review explores how organoids have revolutionized academic research over the last 4 decades, and how they may continue to do so. It also addresses remaining hurdles in 3D cell culturing, and how they may be overcome.     

Convergence of human pluripotent stem cell,organoid, and genome editing technologies

The last decade has seen many exciting technological breakthroughs that greatly expanded the toolboxes for biological and biomedical research, yet few have had more impact than induced pluripotent stem cells and modern-day genome editing. These technologies are providing unprecedented opportunities to improve physiological relevance of experimental models, further our understanding of developmental processes, and develop novel therapies. One of the research areas that benefit greatly from these technological advances is the three-dimensional human organoid culture systems that resemble human tissues morphologically and physiologically. Here we summarize the development of human pluripotent stem cells and their differentiation through organoid formation. We further discuss how genetic modifications, genome editing in particular, were applied to answer basic biological and biomedical questions using organoid cultures of both somatic and pluripotent stem cell origins. Finally, we discuss the potential challenges of applying human pluripotent stem cell and organoid technologies for safety and efficiency evaluation of emerging genome editing tools.     

Current knowledge on the multiform reconstitution of intestinal stem cell niche

The development of “mini-guts” organoid originates from the identification of Lgr5⁺ intestinal stem cells (ISCs) and circumambient signalings within their specific niche at the crypt bottom. These in vitro self-renewing “mini-guts”, also named enteroids or colonoids, undergo perpetual proliferation and regulated differentiation, which results in a high-performance, self-assembling and physiological organoid platform in diverse areas of intestinal research and therapy. The triumphant reconstitution of ISC niche in vitro also relies on Matrigel, a heterogeneous sarcoma extract. Despite the promising prospect of organoids research, their expanding applications are hampered by the canonical culture pattern, which reveals limitations such as inaccessible lumen, confine scale, batch to batch variation and low reproducibility. The tumor-origin of Matrigel also raises biosafety concerns in clinical treatment. However, the convergence of breakthroughs in cellular biology and bioengineering contribute to multiform reconstitution of the ISC niche. Herein, we review the recent advances in the microfabrication of intestinal organoids on hydrogel systems.     

Focus: Personalized Medicine: Value of Organoids from Comparative Epithelia Models

Organoids have tremendous therapeutic potential. They were recently defined as a collection of organ-specific cell types, which self-organize through cell-sorting, develop from stem cells, and perform an organ specific function. The ability to study organoid development and growth in culture and manipulate their genetic makeup makes them particularly suitable for studying development, disease, and drug efficacy. Organoids show great promise in personalized medicine. From a single patient biopsy, investigators can make hundreds of organoids with the genetic landscape of the patient of origin. This genetic similarity makes organoids an ideal system in which to test drug efficacy. While many investigators assume human organoids are the ultimate model system, we believe that the generation of epithelial organoids of comparative model organisms has great potential. Many key transport discoveries were made using marine organisms. In this paper, we describe how deriving organoids from the spiny dogfish shark, zebrafish, and killifish can contribute to the fields of comparative biology and disease modeling with future prospects for personalized medicine.     

类器官的研究进展及应用

随着人类生物学研究的不断深入,需建立新的模型系统为研究提供了有力的工具。虽然传统的研究模型已被广泛应用,但难以准确反映组织、器官在机体中的生理现象。类器官 (Organoid) 是来源于干细胞或器官祖细胞的三维细胞聚集体,可分化和自组织形成具有人体相应器官的部分特定功能和结构。由于类器官具有人源性,可模拟器官发育和形成,在体外长期扩增中具有基因组稳定性,并能够形成活体生物库进行高通量筛选等优势,成为近年来备受关注的体外模型。目前,利用类器官模型结合新兴的基因编辑、器官芯片、单细胞RNA测序技术等,能够突破传统模型的瓶颈,在器官水平上为疾病模型的建立、药物研发、精准医疗以及再生医学等提供有价值的信息。文中就类器官分类及特性、研究应用、与其他技术结合应用及展望这4个方面进行综述。