RESUMEN
Coronary microvascular disease (CMD) and its progression towards major adverse coronary events pose a significant health challenge. Accurate in vitro investigation of CMD requires a robust cell model that faithfully represents the cells within the cardiac microvasculature. Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) offer great potential; however, they are traditionally derived via differentiation protocols that are not readily scalable and are not specified towards the microvasculature. Here, we report the development and comprehensive characterisation of a scalable 3D protocol enabling the generation of phenotypically stable cardiac hPSC-microvascular-like ECs (hPSC-CMVECs) and cardiac pericyte-like cells. These were derived by growing vascular organoids within 3D stirred tank bioreactors and subjecting the emerging 3D hPSC-ECs to high-concentration VEGF-A treatment (3DV). Not only did this promote phenotypic stability of the 3DV hPSC-ECs; single cell-RNA sequencing (scRNA-seq) revealed the pronounced expression of cardiac endothelial- and microvascular-associated genes. Further, the generated mural cells attained from the vascular organoid exhibited markers characteristic of cardiac pericytes. Thus, we present a suitable cell model for investigating the cardiac microvasculature as well as the endothelial-dependent and -independent mechanisms of CMD. Moreover, owing to their phenotypic stability, cardiac specificity, and high angiogenic potential, the cells described within would also be well suited for cardiac tissue engineering applications.
Asunto(s)
Diferenciación Celular , Células Endoteliales , Microvasos , Células Madre Pluripotentes , Humanos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/citología , Microvasos/citología , Microvasos/metabolismo , Pericitos/citología , Pericitos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/farmacología , Organoides/citología , Organoides/irrigación sanguínea , Organoides/metabolismoRESUMEN
Diabetes presents a pressing healthcare crisis, necessitating innovative solutions. Organoid technologies have rapidly advanced, leading to the emergence of bioengineering islet organoids as an unlimited source of insulin-producing cells for treating insulin-dependent diabetes. This advancement surpasses the need for cadaveric islet transplantation. However, clinical translation of this approach faces two major limitations: immature endocrine function and the absence of a perfusable vasculature compared to primary human islets. In this review, we summarize the latest developments in bioengineering functional islet organoids in vitro and promoting vascularization of organoid grafts before and after transplantation. We highlight the crucial roles of the vasculature in ensuring long-term survival, maturation, and functionality of islet organoids. Additionally, we discuss key considerations that must be addressed before clinical translation of islet organoid-based therapy, including functional immaturity, undesired heterogeneity, and potential tumorigenic risks.
Asunto(s)
Diabetes Mellitus Tipo 1 , Insulinas , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Humanos , Organoides/irrigación sanguínea , Diabetes Mellitus Tipo 1/terapia , BioingenieríaRESUMEN
BACKGROUND: Kidney organoids derived from human pluripotent stem cells (HiPSCs) hold huge applications for drug screening, disease modeling, and cell transplanting therapy. However, these applications are limited since kidney organoid cannot maintain complete morphology and function like human kidney. Kidney organoids are not well differentiated since the core of the organoid lacked oxygen, nutrition, and vasculature, which creates essential niches. Hypoxia-inducible factor-1 α (HIF-1α) serves as a critical regulator in vascularization and cell survival under hypoxia environment. Less is known about the role of HIF-1α in kidney organoids in this regard. This study tried to investigate the effect of HIF-1α in kidney organoid vascularization and related disease modeling. METHODS: For the vascularization study, kidney organoids were generated from human induced pluripotent stem cells. We overexpressed HIF-1α via plasmid transfection or treated DMOG (Dimethyloxallyl Glycine, an agent for HIF-1α stabilization and accumulation) in kidney progenitor cells to detect the endothelium. For the disease modeling study, we treated kidney organoid with cisplatin under hypoxia environment, with additional HIF-1α transfection. RESULT: HIF-1α overexpression elicited kidney organoid vascularization. The endothelial cells and angiotool analysis parameters were increased in HIF-1α plasmid-transfected and DMOG-treated organoids. These angiogenesis processes were partially blocked by VEGFR inhibitors, semaxanib or axitinib. Cisplatin-induced kidney injury (Cleaved caspase 3) was protected by HIF-1α through the upregulation of CD31 and SOD2. CONCLUSION: We demonstrated that HIF-1α elicited the process of kidney organoid vascularization and protected against cisplatin-induced kidney organoid injury in hypoxia environment.
Asunto(s)
Angiogénesis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Riñón , Modelos Biológicos , Organoides , Organoides/irrigación sanguínea , Organoides/metabolismo , Riñón/metabolismo , Células Madre Multipotentes , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Humanos , Plásmidos/genética , Expresión Génica , Células Madre/citología , Células Madre/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Inhibidores de la Angiogénesis/farmacología , Angiogénesis/efectos de los fármacos , Angiogénesis/fisiopatología , Axitinib/farmacología , Células Cultivadas , Cisplatino/farmacología , Hipoxia de la Célula , Enfermedades Renales/fisiopatologíaRESUMEN
Pluripotent stem cell-derived organoids have transformed our ability to recreate complex three-dimensional models of human tissue. However, the directed differentiation methods used to create them do not afford the ability to introduce cross-germ-layer cell types. Here, we present a bottom-up engineering approach to building vascularized human tissue by combining genetic reprogramming with chemically directed organoid differentiation. As a proof of concept, we created neuro-vascular and myo-vascular organoids via transcription factor overexpression in vascular organoids. We comprehensively characterized neuro-vascular organoids in terms of marker gene expression and composition, and demonstrated that the organoids maintain neural and vascular function for at least 45 days in culture. Finally, we demonstrated chronic electrical stimulation of myo-vascular organoid aggregates as a potential path toward engineering mature and large-scale vascularized skeletal muscle tissue from organoids. Our approach offers a roadmap to build diverse vascularized tissues of any type derived entirely from pluripotent stem cells.
Asunto(s)
Vasos Sanguíneos/citología , Organoides/irrigación sanguínea , Organoides/citología , Organoides/fisiología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Ingeniería de Tejidos/métodos , Vasos Sanguíneos/fisiología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Humanos , Neovascularización Fisiológica , Tejido Parenquimatoso/fisiología , Factores de Transcripción/metabolismoRESUMEN
Vascularization of tissues, organoids and organ-on-chip models has been attempted using endothelial cells. However, the cultured endothelial cells lack the capacity to interact with other somatic cell types, which is distinct from developing vascular cells in vivo. Recently, it was demonstrated that blood vessel organoids (BVOs) recreate the structure and functions of developing human blood vessels. However, the tissue-specific adaptability of BVOs had not been assessed in somatic tissues. Herein, we investigated whether BVOs infiltrate human cerebral organoids and form a blood-brain barrier. As a result, vascular cells arising from BVOs penetrated the cerebral organoids and developed a vessel-like architecture composed of CD31+ endothelial tubes coated with SMA+ or PDGFR+ mural cells. Molecular markers of the blood-brain barrier were detected in the vascularized cerebral organoids. We revealed that BVOs can form neural-specific blood-vessel networks that can be maintained for over 50 days.
Asunto(s)
Vasos Sanguíneos/fisiología , Encéfalo/irrigación sanguínea , Neovascularización Fisiológica/fisiología , Organoides/irrigación sanguínea , Vasos Sanguíneos/citología , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Técnicas de Cocultivo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio/citología , Endotelio/metabolismo , Humanos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Organoides/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
The vasculature of stem-cell-derived liver organoids can be engineered using methods that recapitulate embryonic liver development. Hepatic organoids with a vascular network offer great application prospects for drug screening, disease modeling, and therapeutics. However, the application of stem cell-derived organoids is hindered by insufficient vascularization and maturation. Here, we review different theories about the origin of hepatic cells and the morphogenesis of hepatic vessels to provide potential approaches for organoid generation. We also review the main protocols for generating vascularized liver organoids from stem cells and consider their potential and limitations in the generation of vascularized liver organoids.
Asunto(s)
Hígado/patología , Organoides/irrigación sanguínea , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Evaluación Preclínica de Medicamentos/métodos , Ingeniería Genética/métodos , Hepatocitos/patología , Humanos , Hígado/crecimiento & desarrollo , Organogénesis/fisiología , Organoides/crecimiento & desarrollo , Organoides/metabolismo , Células Madre/metabolismoRESUMEN
Prevascularized 3D microtissues have been shown to be an effective cell delivery vehicle for cardiac repair. To this end, our lab has explored the development of self-organizing, prevascularized human cardiac organoids by co-seeding human cardiomyocytes with cardiac fibroblasts, endothelial cells, and stromal cells into agarose microwells. We hypothesized that this prevascularization process is facilitated by the endogenous upregulation of hypoxia-inducible factor (HIF) pathway in the avascular 3D microtissues. In this study, we used Molidustat, a selective PHD (prolyl hydroxylase domain enzymes) inhibitor that stabilizes HIF-α, to treat human cardiac organoids, which resulted in 150 ± 61% improvement in endothelial expression (CD31) and 220 ± 20% improvement in the number of lumens per organoids. We hypothesized that the improved endothelial expression seen in Molidustat treated human cardiac organoids was dependent upon upregulation of VEGF, a well-known downstream target of HIF pathway. Through the use of immunofluorescent staining and ELISA assays, we determined that Molidustat treatment improved VEGF expression of non-endothelial cells and resulted in improved co-localization of supporting cell types and endothelial structures. We further demonstrated that Molidustat treated human cardiac organoids maintain cardiac functionality. Lastly, we showed that Molidustat treatment improves survival of cardiac organoids when exposed to both hypoxic and ischemic conditions in vitro. For the first time, we demonstrate that targeted HIF-α stabilization provides a robust strategy to improve endothelial expression and lumen formation in cardiac microtissues, which will provide a powerful framework for prevascularization of various microtissues in developing successful cell transplantation therapies.
Asunto(s)
Fibroblastos/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Miocitos Cardíacos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Organoides , Pirazoles/farmacología , Triazoles/farmacología , Técnicas de Cocultivo , Humanos , Organoides/irrigación sanguínea , Organoides/metabolismoRESUMEN
Along with emergence of the organoids, their application in biomedical research has been currently one of the most fascinating themes. For the past few years, scientists have made significant contributions to deriving organoids representing the whole brain and specific brain regions. Coupled with somatic cell reprogramming and CRISPR/Cas9 editing, the organoid technologies were applied for disease modeling and drug screening. The methods to develop organoids further improved for rapid and efficient generation of cerebral organoids. Additionally, refining the methods to develop the regionally specified brain organoids enabled the investigation of development and interaction of the specific brain regions. Recent studies started resolving the issue in the lack of non-neuroectodermal cells in brain organoids, including vascular endothelial cells and microglia, which play fundamental roles in neurodevelopment and are involved in the pathophysiology of acute and chronic neural disorders. In this review, we highlight recent advances of neuronal organoid technologies, focusing on the region-specific brain organoids and complementation with endothelial cells and microglia, and discuss their potential applications to neuronal diseases.
Asunto(s)
Encéfalo/citología , Células Endoteliales/citología , Microglía/citología , Organoides/citología , Animales , Encéfalo/anatomía & histología , Encéfalo/embriología , Técnicas de Reprogramación Celular/ética , Técnicas de Reprogramación Celular/métodos , Cuerpos Embrioides/citología , Feto/anatomía & histología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteínas Hedgehog/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Neuronas/citología , Especificidad de Órganos , Organogénesis/efectos de los fármacos , Organoides/irrigación sanguínea , Retina/citología , Retina/embriología , Médula Espinal/citología , Médula Espinal/embriología , Vía de Señalización WntRESUMEN
As the field of organoid development matures, the need to transplant organoids to evaluate and characterize their functionality grows. Decades of research developing islet organoid transplantation for the treatment of type 1 diabetes can contribute substantially to accelerating diverse tissue organoid transplantation. Biomaterials-based organoid delivery methods offer the potential to maximize organoid survival and engraftment. In this protocol, we describe a vasculogenic degradable hydrogel vehicle and a method to deliver organoids to intraperitoneal tissue. Further, we describe a method to fluorescently label and image functional vasculature within the graft as a tool to investigate organoid engraftment.
Asunto(s)
Islotes Pancreáticos/irrigación sanguínea , Microscopía Confocal , Neovascularización Fisiológica , Organoides/irrigación sanguínea , Organoides/trasplante , Ingeniería de Tejidos , Inductores de la Angiogénesis/farmacología , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Hidrogeles , Islotes Pancreáticos/metabolismo , Trasplante de Islotes Pancreáticos , Maleimidas/química , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Organoides/metabolismo , Polietilenglicoles/química , Ratas , Fijación del Tejido , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
The kidneys are essential organs that filter the blood, removing urinary waste while maintaining fluid and electrolyte homeostasis. Current conventional research models such as static cell cultures and animal models are insufficient to grasp the complex human in vivo situation or lack translational value. To accelerate kidney research, novel research tools are required. Recent developments have allowed the directed differentiation of induced pluripotent stem cells to generate kidney organoids. Kidney organoids resemble the human kidney in vitro and can be applied in regenerative medicine and as developmental, toxicity, and disease models. Although current studies have shown great promise, challenges remain including the immaturity, limited reproducibility, and lack of perfusable vascular and collecting duct systems. This review gives an overview of our current understanding of nephrogenesis that enabled the generation of kidney organoids. Next, the potential applications of kidney organoids are discussed followed by future perspectives. This review proposes that advancement in kidney organoid research will be facilitated through our increasing knowledge on nephrogenesis and combining promising techniques such as organ-on-a-chip models.
Asunto(s)
Riñón/citología , Organoides/citología , Investigación Biomédica Traslacional/tendencias , Animales , Diferenciación Celular , Linaje de la Célula , Técnicas de Reprogramación Celular , Predicción , Humanos , Células Madre Pluripotentes Inducidas/citología , Riñón/embriología , Túbulos Renales Colectores/embriología , Túbulos Renales Colectores/ultraestructura , Ratones , Modelos Animales , Neovascularización Fisiológica , Organogénesis , Organoides/irrigación sanguínea , Organoides/trasplante , Medicina Regenerativa/métodos , Medicina Regenerativa/tendencias , Investigación Biomédica Traslacional/métodos , Uréter/embriología , Uréter/ultraestructuraRESUMEN
Organoids are three-dimensional (3D) constructs generated in stem cell cultures and are thought to mimic tissue and organ development in situ. However, until recently, they often exclusively recapitulated the development of the organ`s parenchyma without the major components of the organ stroma. Here, we describe a protocol to incorporate stromal components, first of all blood vessels, by co-culturing with induced pluripotent stem cell-derived mesodermal progenitor cells. For complete details on the use and execution of this protocol, please refer to Wörsdörfer et al. (2019).
Asunto(s)
Técnicas de Cultivo Tridimensional de Células/métodos , Células Madre Pluripotentes Inducidas/citología , Mesodermo/citología , Tejido Nervioso , Organoides , Animales , Células Cultivadas , Técnicas de Cocultivo/métodos , Humanos , Ratones , Tejido Nervioso/irrigación sanguínea , Tejido Nervioso/citología , Organoides/irrigación sanguínea , Organoides/citologíaRESUMEN
The increased interest in organoid research in recent years has contributed to an improved understanding of diseases that are currently untreatable. Various organoids, including kidney, brain, retina, liver, and spinal cord, have been successfully developed and serve as potential sources for regenerative medicine studies. However, the application of organoids has been limited by their lack of tissue components such as nerve and blood vessels that are essential to organ physiology. In this study, we used three-dimensional co-culture methods to develop colonic organoids that contained enteric nerves and blood vessels. The development of enteric nerves and blood vessels was confirmed phenotypically and genetically by the use of immunofluorescent staining and Western blotting. Colonic organoids that contain essential tissue components could serve as a useful model for the study of colon diseases and help to overcome current bottlenecks in colon disease research.
Asunto(s)
Vasos Sanguíneos/metabolismo , Técnicas de Cultivo de Célula/métodos , Colon/citología , Sistema Nervioso Entérico/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Neovascularización Fisiológica , Neurogénesis/fisiología , Organoides/crecimiento & desarrollo , Vasos Sanguíneos/crecimiento & desarrollo , Células Cultivadas , Sistema Nervioso Entérico/crecimiento & desarrollo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Neurogénesis/efectos de los fármacos , Neurogénesis/genética , Organoides/irrigación sanguínea , Organoides/efectos de los fármacos , Organoides/metabolismoRESUMEN
Despite the complexity and structural sophistication that 3D organoid models provide, their lack of vascularization and perfusion limit the capability of these models to recapitulate organ physiology effectively. A microfluidic platform named IFlowPlate is engineered, which can be used to culture up to 128 independently perfused and vascularized colon organoids in vitro. Unlike traditional microfluidic devices, the vascularized organoid-on-chip device with an "open-well" design does not require any external pumping systems and allows tissue extraction for downstream analyses, such as histochemistry or even in vivo transplantation. By optimizing both the extracellular matrix (ECM) and the culture media formulation, patient-derived colon organoids are co-cultured successfully within a self-assembled vascular network, and it is found that the colon organoids grow significantly better in the platform under constant perfusion versus conventional static condition. Furthermore, a colon inflammation model with an innate immune function where circulating monocytes can be recruited from the vasculature, differentiate into macrophage, and infiltrate the colon organoids in response to tumor necrosis factor (TNF)- inflammatory cytokine stimulation is developed using the platform. With the ability to grow vascularized colon organoids under intravascular perfusion, the IFlowPlate platform could unlock new possibilities for screening potential therapeutic targets or modeling relevant diseases.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Colon/citología , Dispositivos Laboratorio en un Chip , Neovascularización Fisiológica , Organoides/irrigación sanguínea , Organoides/citología , Humanos , PerfusiónRESUMEN
Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2)3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) 'resets' these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens4,5. In three-dimensional matrices-which do not have the constraints of bioprinted scaffolds-the 'reset' vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting.
Asunto(s)
Vasos Sanguíneos/citología , Carcinogénesis , Células Endoteliales/citología , Hemodinámica , Neoplasias/irrigación sanguínea , Organogénesis , Organoides/irrigación sanguínea , Vasos Sanguíneos/crecimiento & desarrollo , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Cromatina/metabolismo , Epigénesis Genética , Epigenómica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas In Vitro , Islotes Pancreáticos/irrigación sanguínea , Modelos Biológicos , Especificidad de Órganos , RNA-Seq , Análisis de la Célula Individual , Factores de Transcripción , TranscriptomaRESUMEN
Modeling the processes of neuronal progenitor proliferation and differentiation to produce mature cortical neuron subtypes is essential for the study of human brain development and the search for potential cell therapies. We demonstrated a novel paradigm for the generation of vascularized organoids (vOrganoids) consisting of typical human cortical cell types and a vascular structure for over 200 days as a vascularized and functional brain organoid model. The observation of spontaneous excitatory postsynaptic currents (sEPSCs), spontaneous inhibitory postsynaptic currents (sIPSCs), and bidirectional electrical transmission indicated the presence of chemical and electrical synapses in vOrganoids. More importantly, single-cell RNA-sequencing analysis illustrated that vOrganoids exhibited robust neurogenesis and that cells of vOrganoids differentially expressed genes (DEGs) related to blood vessel morphogenesis. The transplantation of vOrganoids into the mouse S1 cortex resulted in the construction of functional human-mouse blood vessels in the grafts that promoted cell survival in the grafts. This vOrganoid culture method could not only serve as a model to study human cortical development and explore brain disease pathology but also provide potential prospects for new cell therapies for nervous system disorders and injury.
Asunto(s)
Técnicas de Cultivo de Célula , Neurogénesis , Organoides/irrigación sanguínea , Telencéfalo/embriología , Animales , Células Madre Embrionarias , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Pluripotentes Inducidas , Ratones Endogámicos NOD , Ratones SCID , Organoides/metabolismo , Organoides/trasplanteRESUMEN
BACKGROUND: The utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the vascularization and subsequent maturation of human pluripotent stem cell-derived kidney organoids after renal subcapsular transplantation. This raises the question of whether the glomeruli also become functional upon transplantation. METHODS: We transplanted kidney organoids under the renal capsule of the left kidney in immunodeficient mice followed by the implantation of a titanium imaging window on top of the kidney organoid. To assess glomerular function in the transplanted human pluripotent stem cell-derived kidney tissue 1, 2, and 3 weeks after transplantation, we applied high-resolution intravital multiphoton imaging through the imaging window during intravenous infusion of fluorescently labeled low and high molecular mass dextran molecules or albumin. RESULTS: After vascularization, glomerular structures in the organoid displayed dextran and albumin size selectivity across their glomerular filtration barrier. We also observed evidence of proximal tubular dextran reuptake. CONCLUSIONS: Our results demonstrate that human pluripotent stem cell-derived glomeruli can develop an appropriate barrier function and discriminate between molecules of varying size. These characteristics together with tubular presence of low molecular mass dextran provide clear evidence of functional filtration. This approach to visualizing glomerular filtration function will be instrumental for translation of organoid technology for clinical applications as well as for disease modeling.
Asunto(s)
Células Madre Pluripotentes Inducidas/trasplante , Glomérulos Renales/metabolismo , Organoides/trasplante , Albúminas/metabolismo , Animales , Dextranos/metabolismo , Genes Reporteros , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Microscopía Intravital/métodos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microscopía de Fluorescencia por Excitación Multifotónica , Organoides/irrigación sanguínea , Organoides/metabolismo , Tamaño de la Partícula , Técnica de Ventana Cutánea , Imagen de Lapso de Tiempo/métodosRESUMEN
Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.
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Riñón/irrigación sanguínea , Riñón/crecimiento & desarrollo , Organoides/irrigación sanguínea , Organoides/crecimiento & desarrollo , Animales , Riñón/citología , Microscopía , Modelos Biológicos , Organoides/citología , Células Madre Pluripotentes/citología , Técnicas de Cultivo de Tejidos , Trasplante HeterólogoRESUMEN
Organ failure manifests severe symptoms affecting the whole body that may cause death. However, the number of organ donors is not enough for patients requiring transplantation worldwide. Illegal transplantation is also sometimes conducted. To help address this concern, primary hepatocytes are clinically transplanted in the liver. However, donor shortage and host rejection via instant blood-mediated inflammatory reactions are worrisome. Induced pluripotent stem cell-derived hepatocyte-like cells have been developed as an alternative treatment. Recently, organoid technology has been developed to investigate the pathology and mechanism of organoids in cultures. Organoids can be transplanted with vascularization and connected to host blood vessels, and functionally mature better in vivo than in vitro. Hepatic organoids improve pathology in liver disease models. In this review, we introduce induced pluripotent stem cell- and organoid-based therapies against liver diseases considering present and future perspectives.
Asunto(s)
Hepatocitos/citología , Células Madre Pluripotentes Inducidas/trasplante , Hepatopatías/terapia , Fallo Hepático/terapia , Regeneración Hepática , Organoides/citología , Células Madre Adultas/trasplante , Animales , Diferenciación Celular , Hepatocitos/trasplante , Humanos , Organoides/irrigación sanguínea , Organoides/trasplante , Trasplante de Células Madre/métodosRESUMEN
Maintaining long-term euglycemia after intraportal islet transplantation is hampered by the considerable islet loss in the peri-transplant period attributed to inflammation, ischemia and poor angiogenesis. Here, we show that viable and functional islet organoids can be successfully generated from dissociated islet cells (ICs) and human amniotic epithelial cells (hAECs). Incorporation of hAECs into islet organoids markedly enhances engraftment, viability and graft function in a mouse type 1 diabetes model. Our results demonstrate that the integration of hAECs into islet cell organoids has great potential in the development of cell-based therapies for type 1 diabetes. Engineering of functional mini-organs using this strategy will allow the exploration of more favorable implantation sites, and can be expanded to unlimited (stem-cell-derived or xenogeneic) sources of insulin-producing cells.
Asunto(s)
Diabetes Mellitus Tipo 1/terapia , Células Epiteliales/metabolismo , Trasplante de Islotes Pancreáticos/métodos , Organoides/trasplante , Ingeniería de Tejidos/métodos , Amnios/citología , Animales , Supervivencia Celular , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inducido químicamente , Células Epiteliales/trasplante , Supervivencia de Injerto , Xenoinjertos/irrigación sanguínea , Xenoinjertos/metabolismo , Xenoinjertos/trasplante , Humanos , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Ratones , Ratones SCID , Organoides/irrigación sanguínea , Organoides/metabolismo , Ratas , Ratas Sprague-Dawley , Medicina Regenerativa/métodos , Esferoides Celulares , Estreptozocina , Técnicas de Cultivo de Tejidos/métodos , Trasplante Heterólogo/métodosRESUMEN
Human cortical organoids (hCOs), derived from human embryonic stem cells (hESCs), provide a platform to study human brain development and diseases in complex three-dimensional tissue. However, current hCOs lack microvasculature, resulting in limited oxygen and nutrient delivery to the inner-most parts of hCOs. We engineered hESCs to ectopically express human ETS variant 2 (ETV2). ETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. Importantly, the presence of vasculature-like structures resulted in enhanced functional maturation of organoids. We found that vascularized hCOs (vhCOs) acquired several blood-brain barrier characteristics, including an increase in the expression of tight junctions, nutrient transporters and trans-endothelial electrical resistance. Finally, ETV2-induced endothelium supported the formation of perfused blood vessels in vivo. These vhCOs form vasculature-like structures that resemble the vasculature in early prenatal brain, and they present a robust model to study brain disease in vitro.