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Nature Materials Feb 2021In recent years considerable progress has been made in the development of faithful procedures for the differentiation of human pluripotent stem cells (hPSCs). An... (Review)
Review
In recent years considerable progress has been made in the development of faithful procedures for the differentiation of human pluripotent stem cells (hPSCs). An important step in this direction has also been the derivation of organoids. This technology generally relies on traditional three-dimensional culture techniques that exploit cell-autonomous self-organization responses of hPSCs with minimal control over the external inputs supplied to the system. The convergence of stem cell biology and bioengineering offers the possibility to provide these stimuli in a controlled fashion, resulting in the development of naturally inspired approaches to overcome major limitations of this nascent technology. Based on the current developments, we emphasize the achievements and ongoing challenges of bringing together hPSC organoid differentiation, bioengineering and ethics. This Review underlines the need for providing engineering solutions to gain control of self-organization and functionality of hPSC-derived organoids. We expect that this knowledge will guide the community to generate higher-grade hPSC-derived organoids for further applications in developmental biology, drug screening, disease modelling and personalized medicine.
Topics: Bioengineering; Humans; Organoids; Pluripotent Stem Cells
PubMed: 33199860
DOI: 10.1038/s41563-020-00804-4 -
The Journal of Clinical Investigation Nov 2023Over the last decade, several organoid models have evolved to acquire increasing cellular, structural, and functional complexity. Advanced lung organoid platforms... (Review)
Review
Over the last decade, several organoid models have evolved to acquire increasing cellular, structural, and functional complexity. Advanced lung organoid platforms derived from various sources, including adult, fetal, and induced pluripotent stem cells, have now been generated, which more closely mimic the cellular architecture found within the airways and alveoli. In this regard, the establishment of novel protocols with optimized stem cell isolation and culture conditions has given rise to an array of models able to study key cellular and molecular players involved in lung injury and repair. In addition, introduction of other nonepithelial cellular components, such as immune, mesenchymal, and endothelial cells, and employment of novel precision gene editing tools have further broadened the range of applications for these systems by providing a microenvironment and/or phenotype closer to the desired in vivo scenario. Thus, these developments in organoid technology have enhanced our ability to model various aspects of lung biology, including pathogenesis of diseases such as chronic obstructive pulmonary disease, pulmonary fibrosis, cystic fibrosis, and infectious disease and host-microbe interactions, in ways that are often difficult to undertake using only in vivo models. In this Review, we summarize the latest developments in lung organoid technology and their applicability for disease modeling and outline their strengths, drawbacks, and potential avenues for future development.
Topics: Endothelial Cells; Induced Pluripotent Stem Cells; Lung; Pluripotent Stem Cells; Organoids
PubMed: 37966116
DOI: 10.1172/JCI170500 -
Trends in Molecular Medicine Dec 2018Brain organoids are 3D self-assembled structures composed of hundreds of thousands to millions of cells that resemble the cellular organization and transcriptional and... (Review)
Review
Brain organoids are 3D self-assembled structures composed of hundreds of thousands to millions of cells that resemble the cellular organization and transcriptional and epigenetic signature of a developing human brain. Advancements using brain organoids have been made to elucidate the genetic basis of certain neurodevelopmental disorders, such as microcephaly and autism; and to investigate the impact of environmental factors to the brain, such as during Zika virus infection. It remains to be explored how far brain organoids can functionally mature and process external information. An improved brain organoid model might reproduce important aspects of the human brain in a more reproducible and high-throughput fashion. This novel and complementary approach in the neuroscience toolbox opens perspectives to understand the fundamental features of the human neurodevelopment, with implications to personalize therapeutic opportunities for neurological disorders.
Topics: Animals; Brain; Humans; Neural Stem Cells; Organoids; Pluripotent Stem Cells; Zika Virus Infection
PubMed: 30377071
DOI: 10.1016/j.molmed.2018.09.005 -
Nature Jun 2020The skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily...
The skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily fluids, guarding against external stresses and mediating the sensation of touch and pain. Reconstructing appendage-bearing skin in cultures and in bioengineered grafts is a biomedical challenge that has yet to be met. Here we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use stepwise modulation of the transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During an incubation period of 4-5 months, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis and pigmented hair follicles that are equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking the neural circuitry associated with human touch. Single-cell RNA sequencing and direct comparison to fetal specimens suggest that the skin organoids are equivalent to the facial skin of human fetuses in the second trimester of development. Moreover, we show that skin organoids form planar hair-bearing skin when grafted onto nude mice. Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo. We anticipate that our skin organoids will provide a foundation for future studies of human skin development, disease modelling and reconstructive surgery.
Topics: Animals; Ectoderm; Female; Hair; Hair Color; Hair Follicle; Head; Heterografts; Humans; Mice; Mice, Nude; Organoids; Pluripotent Stem Cells; RNA-Seq; Single-Cell Analysis; Skin; Skin Transplantation
PubMed: 32494013
DOI: 10.1038/s41586-020-2352-3 -
Gastroenterology Feb 2021Preclinical identification of compounds at risk of causing drug induced liver injury (DILI) remains a significant challenge in drug development, highlighting a need for...
BACKGROUND & AIMS
Preclinical identification of compounds at risk of causing drug induced liver injury (DILI) remains a significant challenge in drug development, highlighting a need for a predictive human system to study complicated DILI mechanism and susceptibility to individual drug. Here, we established a human liver organoid (HLO)-based screening model for analyzing DILI pathology at organoid resolution.
METHODS
We first developed a reproducible method to generate HLO from storable foregut progenitors from pluripotent stem cell (PSC) lines with reproducible bile transport function. The qRT-PCR and single cell RNA-seq determined hepatocyte transcriptomic state in cells of HLO relative to primary hepatocytes. Histological and ultrastructural analyses were performed to evaluate micro-anatomical architecture. HLO based drug-induced liver injury assays were transformed into a 384 well based high-speed live imaging platform.
RESULTS
HLO, generated from 10 different pluripotent stem cell lines, contain polarized immature hepatocytes with bile canaliculi-like architecture, establishing the unidirectional bile acid transport pathway. Single cell RNA-seq profiling identified diverse and zonal hepatocytic populations that in part emulate primary adult hepatocytes. The accumulation of fluorescent bile acid into organoid was impaired by CRISPR-Cas9-based gene editing and transporter inhibitor treatment with BSEP. Furthermore, we successfully developed an organoid based assay with multiplexed readouts measuring viability, cholestatic and/or mitochondrial toxicity with high predictive values for 238 marketed drugs at 4 different concentrations (Sensitivity: 88.7%, Specificity: 88.9%). LoT positively predicts genomic predisposition (CYP2C9∗2) for Bosentan-induced cholestasis.
CONCLUSIONS
Liver organoid-based Toxicity screen (LoT) is a potential assay system for liver toxicology studies, facilitating compound optimization, mechanistic study, and precision medicine as well as drug screening applications.
Topics: Cell Line; Chemical and Drug Induced Liver Injury; Drug Evaluation, Preclinical; Hepatocytes; High-Throughput Screening Assays; Humans; Liver; Organoids; Pluripotent Stem Cells; Toxicity Tests, Acute
PubMed: 33039464
DOI: 10.1053/j.gastro.2020.10.002 -
Cell Stem Cell Dec 2018Kidney organoids derived from human pluripotent stem cells have great utility for investigating organogenesis and disease mechanisms and, potentially, as a replacement... (Comparative Study)
Comparative Study
Kidney organoids derived from human pluripotent stem cells have great utility for investigating organogenesis and disease mechanisms and, potentially, as a replacement tissue source, but how closely organoids derived from current protocols replicate adult human kidney is undefined. We compared two directed differentiation protocols by single-cell transcriptomics of 83,130 cells from 65 organoids with single-cell transcriptomes of fetal and adult kidney cells. Both protocols generate a diverse range of kidney cells with differing ratios, but organoid-derived cell types are immature, and 10%-20% of cells are non-renal. Reconstructing lineage relationships by pseudotemporal ordering identified ligands, receptors, and transcription factor networks associated with fate decisions. Brain-derived neurotrophic factor (BDNF) and its cognate receptor NTRK2 were expressed in the neuronal lineage during organoid differentiation. Inhibiting this pathway improved organoid formation by reducing neurons by 90% without affecting kidney differentiation, highlighting the power of single-cell technologies to characterize and improve organoid differentiation.
Topics: Cell Differentiation; Cells, Cultured; Humans; Kidney; Organogenesis; Organoids; Pluripotent Stem Cells; Single-Cell Analysis; Transcriptome
PubMed: 30449713
DOI: 10.1016/j.stem.2018.10.010 -
Journal of Hematology & Oncology Jan 2020Patient-derived tumor xenografts (PDXs), in which tumor fragments surgically dissected from cancer patients are directly transplanted into immunodeficient mice, have... (Review)
Review
Patient-derived tumor xenografts (PDXs), in which tumor fragments surgically dissected from cancer patients are directly transplanted into immunodeficient mice, have emerged as a useful model for translational research aimed at facilitating precision medicine. PDX susceptibility to anti-cancer drugs is closely correlated with clinical data in patients, from whom PDX models have been derived. Accumulating evidence suggests that PDX models are highly effective in predicting the efficacy of both conventional and novel anti-cancer therapeutics. This also allows "co-clinical trials," in which pre-clinical investigations in vivo and clinical trials could be performed in parallel or sequentially to assess drug efficacy in patients and PDXs. However, tumor heterogeneity present in PDX models and in the original tumor samples constitutes an obstacle for application of PDX models. Moreover, human stromal cells originally present in tumors dissected from patients are gradually replaced by host stromal cells as the xenograft grows. This replacement by murine stroma could preclude analysis of human tumor-stroma interactions, as some mouse stromal cytokines might not affect human carcinoma cells in PDX models. The present review highlights the biological and clinical significance of PDX models and three-dimensional patient-derived tumor organoid cultures of several kinds of solid tumors, such as those of the colon, pancreas, brain, breast, lung, skin, and ovary.
Topics: Animals; Antineoplastic Agents; Disease Models, Animal; Humans; Neoplasms; Organ Culture Techniques; Organoids; Tumor Microenvironment; Xenograft Model Antitumor Assays
PubMed: 31910904
DOI: 10.1186/s13045-019-0829-z -
Nature Materials Feb 2021Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for...
Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here, we apply extrusion-based three-dimensional cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate the relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, three-dimensional bioprinting enables precise manipulation of biophysical properties, including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.
Topics: Bioprinting; Humans; Kidney Tubules, Proximal; Organoids; Pluripotent Stem Cells
PubMed: 33230326
DOI: 10.1038/s41563-020-00853-9 -
Nature Jun 2019Experimental models of the human brain are needed for basic understanding of its development and disease. Human brain organoids hold unprecedented promise for this...
Experimental models of the human brain are needed for basic understanding of its development and disease. Human brain organoids hold unprecedented promise for this purpose; however, they are plagued by high organoid-to-organoid variability. This has raised doubts as to whether developmental processes of the human brain can occur outside the context of embryogenesis with a degree of reproducibility that is comparable to the endogenous tissue. Here we show that an organoid model of the dorsal forebrain can reliably generate a rich diversity of cell types appropriate for the human cerebral cortex. We performed single-cell RNA-sequencing analysis of 166,242 cells isolated from 21 individual organoids, finding that 95% of the organoids generate a virtually indistinguishable compendium of cell types, following similar developmental trajectories and with a degree of organoid-to-organoid variability comparable to that of individual endogenous brains. Furthermore, organoids derived from different stem cell lines show consistent reproducibility in the cell types produced. The data demonstrate that reproducible development of the complex cellular diversity of the central nervous system does not require the context of the embryo, and that establishment of terminal cell identity is a highly constrained process that can emerge from diverse stem cell origins and growth environments.
Topics: Cell Line; Cerebral Cortex; Female; Fetus; Humans; Induced Pluripotent Stem Cells; Male; Organoids; Prosencephalon; RNA-Seq; Reproducibility of Results; Single-Cell Analysis; Time Factors; Tissue Culture Techniques; Transcriptome
PubMed: 31168097
DOI: 10.1038/s41586-019-1289-x -
Nature Oct 2022Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease. However, organoids lack the connectivity...
Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease. However, organoids lack the connectivity that exists in vivo, which limits maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered.
Topics: Animals; Animals, Newborn; Autistic Disorder; Humans; Long QT Syndrome; Motivation; Neural Pathways; Neurons; Optogenetics; Organoids; Rats; Reward; Somatosensory Cortex; Stem Cells; Syndactyly
PubMed: 36224417
DOI: 10.1038/s41586-022-05277-w