Generation of human pluripotent stem cell-derived fused organoids with oligodendroglia and myelin.
Ontology highlight
ABSTRACT:
SUBMITTER: Kim H
PROVIDER: S-EPMC8038946 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease.
| S-EPMC4370217 | biostudies-literature
Project description:BackgroundThe use of a personalized liver organoid derived from human-induced pluripotent stem cells (HuiPSCs) is advancing the use of in vitro disease models for the design of specific, effective therapies for individuals. Collecting patient peripheral blood cells for HuiPSC generation is preferable because it is less invasive; however, the capability of blood cell-derived HuiPSCs for hepatic differentiation and liver organoid formation remains uncertain. Moreover, the currently available methods for liver organoid formation require a multistep process of cell differentiation or a combination of hepatic endodermal, endothelial and mesenchymal cells, which is a major hurdle for the application of personalized liver organoids in high-throughput testing of drug toxicity and safety. To demonstrate the capability of blood cell-derived HuiPSCs for liver organoid formation without support from endothelial and mesenchymal cells.MethodsThe peripheral blood-derived HuiPSCs first differentiated into hepatic endoderm (HE) in two-dimensional (2D) culture on Matrigel-coated plates under hypoxia for 10 days. The HE was then collected and cultured in 3D culture using 50% Matrigel under ambient oxygen. The maturation of hepatocytes was further induced by adding hepatocyte growth medium containing HGF and oncostatin M on top of the 3D culture and incubating the culture for an additional 12-17 days. The function of the liver organoids was assessed using expression analysis of hepatocyte-specific gene and proteins. Albumin (ALB) synthesis, glycogen and lipid storage, and metabolism of indocyanine were evaluated. The spatial distribution of albumin was examined using immunofluorescence and confocal microscopy.ResultsCD34+ hematopoietic cell-derived HuiPSCs were capable of differentiating into definitive endoderm expressing SOX17 and FOXA2, hepatic endoderm expressing FOXA2, hepatoblasts expressing AFP and hepatocytes expressing ALB. On day 25 of the 2D culture, cells expressed SOX17, FOXA2, AFP and ALB, indicating the presence of cellular heterogeneity. In contrast, the hepatic endoderm spontaneously formed a spherical, hollow structure in a 3D culture of 50% Matrigel, whereas hepatoblasts and hepatocytes could not form. Microscopic observation showed a single layer of polygonal-shaped cells arranged in a 3D structure. The hepatic endoderm-derived organoid synthesis ALB at a higher level than the 2D culture but did not express definitive endoderm-specific SOX17, indicating the greater maturity of the hepatocytes in the liver organoids. Confocal microscopic images and quantitative ELISA confirmed albumin synthesis in the cytoplasm of the liver organoid and its secretion. Overall, 3D culture of the hepatic endoderm is a relatively fast, simple, and less laborious way to generate liver organoids from HuiPSCs that is more physiologically relevant than 2D culture.
| S-EPMC7580584 | biostudies-literature
Project description:Human induced pluripotent stem cell (hiPSC)-derived cerebral organoids (COs) can serve as an in vitro model for studying normal and pathologic human brain development. Here, we optimized existing protocols to streamline the generation of forebrain COs from hiPSCs. We employ these COs to define the impact of disease-causing mutations on cell fate, differentiation, maturation, and morphology relevant to neurodevelopmental disorders. Although limited to forebrain CO identity, this schema requires minimal external interference and is amenable to low-throughput biochemical assays. For complete details on the use and execution of this profile, please refer to Anastasaki et al. (2020) and Wegscheid et al. (2021).
| S-EPMC8844852 | biostudies-literature
Project description:Genetic liver disease modeling is difficult because it is challenging to access patient tissue samples and to develop practical and relevant model systems. Previously, we developed novel proliferative and functional liver organoids from pluripotent stem cells; however, the protocol requires improvement for standardization and reproducible mass production. Here, we improved the method such that it is suitable for scalable expansion and relatively homogenous production, resulting in an efficient and reproducible process. Moreover, three medium components critical for long-term expansion were defined. Detailed transcriptome analysis revealed that fibroblast growth factor signaling, the essential pathway for hepatocyte proliferation during liver regeneration, was mainly enriched in proliferative liver organoids. Short hairpin RNA-mediated knockdown of FGFR4 impaired the generation and proliferation of organoids. Finally, glycogen storage disease type Ia (GSD1a) patient-specific liver organoids were efficiently and reproducibly generated using the new protocol. They well maintained disease-specific phenotypes such as higher lipid and glycogen accumulation in the liver organoids and lactate secretion into the medium consistent with the main pathologic characteristics of patients with GSD1a. Therefore, our newly established liver organoid platform can provide scalable and practical personalized disease models and help to find new therapies for incurable liver diseases including genetic liver diseases.
| S-EPMC10739970 | biostudies-literature
Project description:The hypothalamus is a region of the brain that plays an important role in regulating body functions and behaviors. There is a growing interest in human pluripotent stem cells (hPSCs) for modeling diseases that affect the hypothalamus. Here, we established an hPSC-derived hypothalamus organoid differentiation protocol to model the cellular diversity of this brain region. Using an hPSC line with a tyrosine hydroxylase (TH)-TdTomato reporter for dopaminergic neurons (DNs) and other TH-expressing cells, we interrogated DN-specific pathways and functions in electrophysiologically active hypothalamus organoids. Single-cell RNA sequencing (scRNA-seq) revealed diverse neuronal and non-neuronal cell types in mature hypothalamus organoids. We identified several molecularly distinct hypothalamic DN subtypes that demonstrated different developmental maturities. Our in vitro 3D hypothalamus differentiation protocol can be used to study the development of this critical brain structure and can be applied to disease modeling to generate novel therapeutic approaches for disorders centered around the hypothalamus.
| S-EPMC10460991 | biostudies-literature
Project description:Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world's population and are largely due to chronic Helicobacter pylori infection. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells. We show that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signalling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signalling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signalling and induction of epithelial proliferation. Together, these studies describe a new and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease.
| S-EPMC4270898 | biostudies-literature
Project description:A human cell-based liver model capable of long-term expansion and mature hepatic function is a fundamental requirement for pre-clinical drug development. We previously established self-renewing and functionally mature human pluripotent stem cell-derived liver organoids as an alternate to primary human hepatocytes. In this study, we tested long-term prolonged culture of organoids to increase their maturity. Organoid growing at the edge of Matrigel started to deteriorate two weeks after culturing, and the expression levels of the functional mature hepatocyte marker ALB were decreased at four weeks of culture. Replating the organoids weekly at a 1：2 ratio in fresh Matrigel, resulted in healthier morphology with a thicker layer compared to organoids maintained on the same Matrigel and significantly increased ALB expression until three weeks, although, it decreased sharply at four weeks. The levels of the fetal hepatocyte marker AFP were considerably increased in long-term cultures of organoids. Therefore, we performed serial passaging of organoids, whereby they were mechanically split weekly at a 1：3∼1：5 ratio in fresh Matrigel. The organoids expanded so far over passage 55, or 1 year, without growth retardation and maintained a normal karyotype after long-term cryopreservation. Differentiation potentials were maintained or increased after long-term passaging, while AFP expression considerably decreased after passaging. Therefore, these data demonstrate that organoids can be exponentially expanded by serial passaging, while maintaining long-term functional maturation potential. Thus, hepatic organoids can be a practical and renewable cell source for human cell-based and personalized 3D liver models.
| S-EPMC7378903 | biostudies-literature
Project description:Recent advances in stem cell research have resulted in methods to generate kidney organoids from human pluripotent stem cells (hPSCs), which contain cells of multiple lineages including nephron epithelial cells. Methods to purify specific types of cells from differentiated hPSCs, however, have not been established well. For bioengineering, cell transplantation, and disease modeling, it would be useful to establish those methods to obtain pure populations of specific types of kidney cells. Here, we report a simple two-step differentiation protocol to generate kidney tubular organoids from hPSCs with direct purification of KSP (kidney specific protein)-positive cells using anti-KSP antibody. We first differentiated hPSCs into mesoderm cells using a glycogen synthase kinase-3β inhibitor for 3 days, then cultured cells in renal epithelial growth medium to induce KSP+ cells. We purified KSP+ cells using flow cytometry with anti-KSP antibody, which exhibited characteristics of all segments of kidney tubular cells and cultured KSP+ cells in 3D Matrigel, which formed tubular organoids in vitro. The formation of tubular organoids by KSP+ cells induced the acquisition of functional kidney tubules. KSP+ cells also allowed for the generation of chimeric kidney cultures in which human cells self-assembled into 3D tubular structures in combination with mouse embryonic kidney cells.
| S-EPMC5159864 | biostudies-literature
Project description:Otic neurons, also known as spiral ganglion neurons (SGNs) in mammalian cochlea, transmit electrical signals from sensory hair cells to cochlear nuclei of the auditory system. SGNs are sensitive to toxic insults, vulnerable to get irreversible damaged and hardly regenerate after damage, causing persistent sensorineural hearing loss. Yet, to get authentic SGNs for research or therapeutic purpose remains challenging. Here we developed a protocol to generate human otic neuronal organoids (hONOs) from human pluripotent stem cells (hESCs), in which hESCs were step-wisely induced to SGNs of the corresponding stages according to their developmental trajectory. The hONOs were enriched for SGN-like cells at early stage, and for both neurons and astrocytes, Schwann cells or supporting cells thereafter. In these hONOs, we also determined the existence of typical Type I and Type II SGNs. Mature hONOs (at differentiation Day 60) formed neural network, featured by giant depolarizing potential (GDP)-like events and rosette-organized regions-elicited calcium traces. Electrophysiological analysis confirmed the existence of glutamate-responsive neurons in these hONOs. The otic neuronal organoids generated in this study provide an ideal model to study SGNs and related disorders, facilitating therapeutic development for sensorineural hearing loss.
| S-EPMC10212712 | biostudies-literature
Project description:The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system but involutes with age, resulting in reduced naive T cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro, but functional thymic epithelial cells (TECs) only form months after transplantation of TEPs in vivo. We show the generation of TECs in vitro in isogenic stem cell-derived thymic organoids (sTOs) consisting of TEPs, hematopoietic progenitor cells, and mesenchymal cells, differentiated from the same hPSC line. sTOs support T cell development, express key markers of negative selection, including the autoimmune regulator (AIRE) protein, and facilitate regulatory T cell development. sTOs provide the basis for functional patient-specific thymic organoid models, allowing for the study of human thymus function, T cell development, and transplant immunity.
| S-EPMC10147832 | biostudies-literature