A functional genetic toolbox for human tissue-derived organoids.
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ABSTRACT:
SUBMITTER: Sun D
PROVIDER: S-EPMC8553336 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:The generation of reporter lines for cell identity, lineage, and physiologic state has provided a powerful tool in advancing the dissection of mouse kidney morphogenesis at a molecular level. Although use of this approach is not an option for studying human development in vivo, its application in human induced pluripotent stem cells (iPSCs) is now feasible. METHODS:We used CRISPR/Cas9 gene editing to generate ten fluorescence reporter iPSC lines designed to identify nephron progenitors, podocytes, proximal and distal nephron, and ureteric epithelium. Directed differentiation to kidney organoids was performed according to published protocols. Using immunofluorescence and live confocal microscopy, flow cytometry, and cell sorting techniques, we investigated organoid patterning and reporter expression characteristics. RESULTS:Each iPSC reporter line formed well patterned kidney organoids. All reporter lines showed congruence of endogenous gene and protein expression, enabling isolation and characterization of kidney cell types of interest. We also demonstrated successful application of reporter lines for time-lapse imaging and mouse transplantation experiments. CONCLUSIONS:We generated, validated, and applied a suite of fluorescence iPSC reporter lines for the study of morphogenesis within human kidney organoids. This fluorescent iPSC reporter toolbox enables the visualization and isolation of key populations in forming kidney organoids, facilitating a range of applications, including cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing approaches. These tools offer promise for enhancing our understanding of this model system and its correspondence with human kidney morphogenesis.
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Project description:Total thyroidectomy as part of thyroid cancer treatment results in hypothyroidism requiring lifelong daily thyroid hormone replacement. Unbalanced hormone levels result in persistent complaints such as fatigue, constipation, and weight increase. Therefore, we aimed to investigate a patient-derived thyroid organoid model with the potential to regenerate the thyroid gland. Murine and human thyroid-derived cells were cultured as organoids capable of self-renewal and which expressed proliferation and putative stem cell and thyroid characteristics, without a change in the expression of thyroid tumor-related genes. These organoids formed thyroid-tissue-resembling structures in culture. (Xeno-)transplantation of 600,000 dispersed organoid cells underneath the kidney capsule of a hypothyroid mouse model resulted in the generation of hormone-producing thyroid-resembling follicles. This study provides evidence that thyroid-lineage-specific cells can form organoids that are able to self-renew and differentiate into functional thyroid tissue. Subsequent (xeno-)transplantation of these thyroid organoids demonstrates a proof of principle for functional miniature gland formation.
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Project description:In vitro human pluripotent stem cell (hPSC) derived tissues are excellent models to study certain aspects of normal human development. Current research in the field of hPSC derived tissues reveals these models to be inherently fetal-like on both a morphological and gene expression level. In this review we briefly discuss current methods for differentiating lung and intestinal tissue from hPSCs into individual 3-dimensional units called organoids. We discuss how these methods mirror what is known about in vivo signaling pathways of the developing embryo. Additionally, we will review how the inherent immaturity of these models lends them to be particularly valuable in the study of immature human tissues in the clinical setting of premature birth. Human lung organoids (HLOs) and human intestinal organoids (HIOs) not only model normal development, but can also be utilized to study several important diseases of prematurity such as respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), and necrotizing enterocolitis (NEC).
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Project description:Diffusion is a limiting factor in regenerating large tissues (100-200 μm) due to reduced nutrient supply and waste removal leading to low viability of the regenerating cells as neovascularization of the implant by the host is a slow process. Thus, generating prevascularized tissue engineered constructs, in which endothelial (ECs) and mural (MCs) cells, such as smooth muscle cells (SMCs), and pericytes (PCs), are preassembled into functional in vitro vessels capable of rapidly connecting to the host vasculature could overcome this obstacle. Toward this purpose, using feeder-free and low serum conditions, we developed a simple, efficient and rapid in vitro approach to induce the differentiation of human pluripotent stem cells-hPSCs (human embryonic stem cells and human induced pluripotent stem cells) to defined SMC populations (contractile and synthetic hPSC-SMCs) by extensively characterizing the cellular phenotype (expression of CD44, CD73, CD105, NG2, PDGFRβ, and contractile proteins) and function of hPSC-SMCs. The latter were phenotypically and functionally stable for at least 8 passages, and could stabilize vessel formation and inhibit vessel network regression, when co-cultured with ECs in vitro. Subsequently, using a methylcellulose-based hydrogel system, we generated spheroids consisting of EC/hPSC-SMC (vascular organoids), which were extensively phenotypically characterized. Moreover, the vascular organoids served as focal starting points for the sprouting of capillary-like structures in vitro, whereas their delivery in vivo led to rapid generation of a complex functional vascular network. Finally, we investigated the vascularization potential of these vascular organoids, when embedded in hydrogels composed of defined extracellular components (collagen/fibrinogen/fibronectin) that can be used as scaffolds in tissue engineering applications. In summary, we developed a robust method for the generation of defined SMC phenotypes from hPSCs. Fabrication of vascularized tissue constructs using hPSC-SMC/EC vascular organoids embedded in chemically defined matrices is a significant step forward in tissue engineering and regenerative medicine.
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Project description:PurposeHuman pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histologic analysis in which multiple specimens fixed at different times are used to reconstruct developmental processes, repeated analysis of the same living organoids provides a more direct means to characterize changes. New live imaging modalities can provide insights into retinal organoid structure and metabolic function during in vitro growth. This study employed live tissue imaging to characterize retinal organoid development, including metabolic changes accompanying photoreceptor differentiation.MethodsLive hPSC-derived retinal organoids at different developmental stages were examined for microanatomic organization and metabolic function by phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). Features were compared to those revealed by histologic staining, immunostaining, and microcomputed tomography (micro-CT) of fixed organoid tissue.ResultsWe used FLIM and HSpec to detect changes in metabolic activity as organoids differentiated into organized lamellae. FLIM detected increased glycolytic activity and HSpec detected retinol and retinoic acid accumulation in the organoid outer layer, coinciding with photoreceptor genesis. OCT enabled imaging of lamellae formed during organoid maturation. Micro-CT revealed three-dimensional structure, but failed to detect lamellae.ConclusionsLive imaging modalities facilitate real-time and nondestructive imaging of retinal organoids as they organize into lamellar structures. FLIM and HSpec enable rapid detection of lamellar structure and photoreceptor metabolism. Live imaging techniques may aid in the continuous evaluation of retinal organoid development in diverse experimental and cell therapy settings.
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Project description:BackgroundHuman induced pluripotent stem (iPS) cell-derived enterocyte-like cells (ELCs) are expected to be useful for evaluating the intestinal absorption and metabolism of orally administered drugs. However, it is difficult to generate large amounts of ELCs with high quality because they cannot proliferate and be passaged.MethodsTo solve the issue above, we have established intestinal organoids from ELCs generated using our protocol. Furthermore, monolayers were produced from the organoids. We evaluated the usefulness of the monolayers by comparing their functions with those of the original ELCs and the organoids.ResultsWe established organoids from ELCs (ELC-org) that could be passaged and maintained for more than a year. When ELC-org were dissociated into single cells and seeded on cell culture inserts (ELC-org-mono), they formed a tight monolayer in 3 days. Both ELC-org and ELC-org-mono were composed exclusively of epithelial cells. Gene expressions of many drug-metabolizing enzymes and drug transporters in ELC-org-mono were enhanced, as compared with those in ELC-org, to a level comparable to those in adult human small intestine. The CYP3A4 activity level in ELC-org-mono was comparable or higher than that in primary cryopreserved human small intestinal cells. ELC-org-mono had the efflux activities of P-gp and BCRP. Importantly, ELC-org-mono maintained high intestinal functions without any negative effects even after long-term culture (for more than a year) or cryopreservation. RNA-seq analysis showed that ELC-org-mono were more mature as intestinal epithelial cells than ELCs or ELC-org.ConclusionsWe have successfully improved the function and convenience of ELCs by utilizing organoid technology.
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Project description:Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.
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Project description:Kidney organoids generated from induced pluripotent stem cells (iPSC) have proven valuable for studies of kidney development, disease, and therapeutic screening. However, specific applications have been hampered by limited expansion capacity, immaturity, off-target cells, and inability to access the apical side. Here, we apply recently developed tubuloid protocols to purify and propagate kidney epithelium from d7+18 (post nephrogenesis) iPSC-derived organoids. The resulting 'iPSC organoid-derived (iPSCod)' tubuloids can be exponentially expanded for at least 2.5 mo, while retaining expression of important tubular transporters and segment-specific markers. This approach allows for selective propagation of the mature tubular epithelium, as immature cells, stroma, and undesirable off-target cells rapidly disappeared. iPSCod tubuloids provide easy apical access, which enabled functional evaluation and demonstration of essential secretion and electrolyte reabsorption processes. In conclusion, iPSCod tubuloids provide a different, complementary human kidney model that unlocks opportunities for functional characterization, disease modeling, and regenerative nephrology.
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Project description:Kidney organoids derived from human or rodent pluripotent stem cells have glomerular structures and differentiated/polarized nephron segments. Although there is an increasing understanding of the patterns of expression of transcripts and proteins within kidney organoids, there is a paucity of data regarding functional protein expression, in particular on transporters that mediate the vectorial transport of solutes. Using cells derived from kidney organoids, we examined the functional expression of key ion channels that are expressed in distal nephron segments: the large-conductance Ca2+-activated K+ (BKCa) channel, the renal outer medullary K+ (ROMK, Kir1.1) channel, and the epithelial Na+ channel (ENaC). RNA-sequencing analyses showed that genes encoding the pore-forming subunits of these transporters, and for BKCa channels, key accessory subunits, are expressed in kidney organoids. Expression and localization of selected ion channels was confirmed by immunofluorescence microscopy and immunoblot analysis. Electrophysiological analysis showed that BKCa and ROMK channels are expressed in different cell populations. These two cell populations also expressed other unidentified Ba2+-sensitive K+ channels. BKCa expression was confirmed at a single channel level, based on its high conductance and voltage dependence of activation. We also found a population of cells expressing amiloride-sensitive ENaC currents. In summary, our results show that human kidney organoids functionally produce key distal nephron K+ and Na+ channels.NEW & NOTEWORTHY Our results show that human kidney organoids express key K+ and Na+ channels that are expressed on the apical membranes of cells in the aldosterone-sensitive distal nephron, including the large-conductance Ca2+-activated K+ channel, renal outer medullary K+ channel, and epithelial Na+ channel.
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Project description:Genetic cardiomyopathies are characterized by changes in the function and structure of the myocardium. The development of a novel in vitro model could help to better emulate healthy and diseased human heart conditions and may improve the understanding of disease mechanisms. In this study, for the first time, we demonstrated the generation of cardiac organoids using a triculture approach of human induced pluripotent stem-cell-derived cardiomyocytes (hiPS-CMs)-from healthy subjects and cardiomyopathy patients-human cardiac microvascular endothelial cells (HCMECs) and human cardiac fibroblasts (HCFs). We assessed the organoids' suitability as a 3D cellular model for the representation of phenotypical features of healthy and cardiomyopathic hearts. We observed clear differences in structure and beating behavior between the organoid groups, depending on the type of hiPS-CMs (healthy versus cardiomyopathic) used. Organoids may thus prove a promising tool for the design and testing of patient-specific treatments as well as provide a platform for safer and more efficacious drug development.
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