Generation and characterisation of scalable and stable human pluripotent stem cell-derived microvascular-like endothelial cells for cardiac applications.

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.

Bioengineering and vascularization strategies for islet organoids: advancing toward diabetes therapy.

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.

HIF-1α promotes kidney organoid vascularization and applications in disease modeling.

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.

Human Blood Vessel Organoids Penetrate Human Cerebral Organoids and Form a Vessel-Like System.

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.

Targeting HIF-α for robust prevascularization of human cardiac organoids.

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.

A Method for Organoid Transplantation and Whole-Mount Visualization of Post-Engraftment Vascularization.

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.

IFlowPlate-A Customized 384-Well Plate for the Culture of Perfusable Vascularized Colon Organoids.

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.

Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis.

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.

Vascularized human cortical organoids (vOrganoids) model cortical development in vivo.

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.

In Vivo Assessment of Size-Selective Glomerular Sieving in Transplanted Human Induced Pluripotent Stem Cell-Derived Kidney Organoids.

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.

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging.

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.

Present and Future Perspectives of Using Human-Induced Pluripotent Stem Cells and Organoid Against Liver Failure.

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.

Insulin-producing organoids engineered from islet and amniotic epithelial cells to treat diabetes.

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.

Generation of Human PSC-Derived Kidney Organoids with Patterned Nephron Segments and a De Novo Vascular Network.

Human pluripotent stem cell-derived kidney organoids recapitulate developmental processes and tissue architecture, but intrinsic limitations, such as lack of vasculature and functionality, have greatly hampered their application. Here we establish a versatile protocol for generating vascularized three-dimensional (3D) kidney organoids. We employ dynamic modulation of WNT signaling to control the relative proportion of proximal versus distal nephron segments, producing a correlative level of vascular endothelial growth factor A (VEGFA) to define a resident vascular network. Single-cell RNA sequencing identifies a subset of nephron progenitor cells as a potential source of renal vasculature. These kidney organoids undergo further structural and functional maturation upon implantation. Using this kidney organoid platform, we establish an in vitro model of autosomal recessive polycystic kidney disease (ARPKD), the cystic phenotype of which can be effectively prevented by gene correction or drug treatment. Our studies provide new avenues for studying human kidney development, modeling disease pathogenesis, and performing patient-specific drug validation.

Methods for Generating Vascularized Islet-Like Organoids Via Self-Condensation.

Despite the promise of emerging organoid-based approaches, building additional complexity, such as the vascular network, remains a major challenge toward regenerative therapy. Recently, we developed a complex organoid engineering method by "self-condensation," wherein mesenchymal cell-dependent contraction enables large-scale condensation from heterotypic multiple progenitors. Here, we describe the adaptation of this protocol for generating three-dimensional (3D) pancreatic condensates from dissociated ß cell lines (MIN6) together with blood vessel-forming progenitors. This protocol achieves 3D pancreatic islet-like organoid self-organization with endothelialized networks through mesenchymal stem cell-dependent contraction. Transplantation of pancreatic islet-like organoids treats diabetes in mice effectively. Given the donor shortage associated with clinical islet transplantation, our approach offers a promising alternative toward therapeutic organoid transplantation. © 2018 by John Wiley & Sons, Inc.

Generation of human vascularized brain organoids.

The aim of this study was to vascularize brain organoids with a patient's own endothelial cells (ECs). Induced pluripotent stem cells (iPSCs) of one UC Davis patient were grown into whole-brain organoids. Simultaneously, iPSCs from the same patient were differentiated into ECs. On day 34, the organoid was re-embedded in Matrigel with 250 000 ECs. Vascularized organoids were grown in vitro for 3-5 weeks or transplanted into immunodeficient mice on day 54, and animals were perfused on day 68. Coating of brain organoids on day 34 with ECs led to robust vascularization of the organoid after 3-5 weeks in vitro and 2 weeks in vivo. Human CD31-positive blood vessels were found inside and in-between rosettes within the center of the organoid after transplantation. Vascularization of brain organoids with a patient's own iPSC-derived ECs is technically feasible.

Tissue engineered pre-vascularized buccal mucosa equivalents utilizing a primary triculture of epithelial cells, endothelial cells and fibroblasts.

Artificial generated buccal mucosa equivalents are a promising approach for the reconstruction of urethral defects. Limiting in this approach is a poor blood vessel supply after transplantation, resulting in increased morbidity and necrosis. We generated a pre-vascularized buccal mucosa equivalent in a tri-culture of primary buccal epithelial cells, fibroblasts and microvascular endothelial cells, using a native collagen membrane as a scaffold. A successful pre-vascularization and dense formation of capillary-like structures at superficial areas was demonstrated. The lumen size of pre-formed blood vessels corresponded to the capillary size in vivo (10-30 µm). Comparing native with a highly cross-linked collagen membrane we found a distinct higher formation of capillary-like structures on the native membrane, apparently caused by higher secretion of angiogenic factors such as PDGF, IL-8 and angiopoietin by the cells. These capillary-like structures became functional blood vessels through anastomosis with the host vasculature after implantation in nude mice. This in vitro method should result in an accelerated blood supply to the biomaterial with cells after transplantation and increase the succes rates of the implant material.

Effect of prevascularization on in vivo vascularization of poly(propylene fumarate)/fibrin scaffolds.

The importance of vascularization in the field of bone tissue engineering has been established by previous studies. The present work proposes a novel poly(propylene fumarate) (PPF)/fibrin composite scaffold for the development of vascularized neobone tissue. The effect of prevascularization (i.e., in vitro pre-culture prior to implantation) with human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) on in vivo vascularization of scaffolds was determined. Five conditions were studied: no pre-culture (NP), 1 week pre-culture (1P), 2 week pre-culture (2P), 3 week pre-culture (3P), and scaffolds without cells (control, C). Scaffolds were implanted subcutaneously in a severe combined immunodeficiency (SCID) mouse model for 9 days. During in vitro studies, CD31 staining showed a significant increase in vascular network area over 3 weeks of culture. Vascular density was significantly higher in vivo when comparing the NP and 3P groups. Immunohistochemical staining of human CD-31 expression indicated spreading of vascular networks with increasing pre-culture time. These vascular networks were perfused with mouse blood indicated by perfused lectin staining in human CD-31 positive vessels. Our results demonstrate that in vitro prevascularization supports in vivo vascularization in PPF/fibrin scaffolds.

Tissue-engineered urinary conduits.

The role of tissue engineering in the cystectomy population rests on the principle of sparing healthy intestinal tissue while replacing diseased bladder. Over the last 25 years advances in cell biology and material science have improved the quality and durability of bladder replacement in animals. The neo-urinary conduit ([NUC]-Tengion) employs autologous fat smooth muscle cells which are seeded onto synthetic, biodegradable scaffolds. This seeded construct is then implanted in the patient and purportedly regenerates native urinary tissue to serve as a passive channel connecting the ureters to the skin surface. Preclinical animal studies as well as the first phase I human trial implanting the NUC are reviewed. While the ultimate goal of creating a durable, effective, tissue-engineered conduit is still in its infancy, important technical and experimental strides have been made.

Basement membrane-rich organoids with functional human blood vessels are permissive niches for human breast cancer metastasis.

Metastatic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention.