A fully automated high throughput-workflow for human neural organoids
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE119060 | GEO | 2020/09/28
REPOSITORIES: GEO
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Project description:Recent advances in three dimensional (3D) culture systems have led to the generation of brain organoids that share resemblance to different parts of the human brains; however, a 3D organoid model of the midbrain that contains functional midbrain dopaminergic (mDA) neurons has not been reported. In this study, we develop a method to differentiate human PSCs into a large multicellular organoid-like structure that contains distinct layers of neuronal cells with a transcriptomic profile that resembles human prenatal midbrain. Importantly, we detected electrically active and functionally mature mDA neurons, and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using non-3D methods or in the MLOs generated from mouse embryonic stem cells, our human MLOs uniquely produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a novel tractable in vitro system to study the human midbrain and its related diseases.
2016-08-24 | E-MTAB-4868 | biostudies-arrayexpress
Project description:Underdeveloped lungs are the primary cause of death in premature infants, however, little is known about stem and progenitor cell maintenance during human lung development. In this study, we have identified that FGF7, Retinoic Acid and CHIR-99021, a small molecule that inhibits GSK3 to activate Wnt signaling, support in vitro maintenance of primary human fetal lung bud tip progenitor cells in a progenitor state. Furthermore, these factors are sufficient to derive a population of human bud tip-like progenitor cells in 3D organoid structures from human pluripotent stem cells (hPSC). Functional studies showed that hPSC-derived bud tip progenitor organoids do not contain any mesenchymal cell types, maintain multilineage potential in vitro and are able to engraft into the airways of injured mice and respond to systemic factors. We performed RNA-sequencing to assess the degree of similarity in global gene expression profiles between the full human fetal lung (59-127 days gestation), isolated human fetal bud tip progenitors, organoids grown from primary fetal bud tip progenitors, and hPSC-derived bud tip organoids. Results showed that hPSC-derived organoids have molecular profiles similar to organoids generated from primary human fetal lung tissue. Gene expression differences between hPSC-derived bud tip organoids and fetal progenitor organoids may be related to the presence of contaminating mesenchymal cells in primary cultures. hPSC-derived bud tip organoids are generated from a well-defined human cell sources, offering a distinct advantage over rare primary tissue as a means to study human specific lung development, homeostasis and disease.<br>Sample Nomenclature - Description<br> -------------------------------------------------------------------------<br> Peripheral fetal lung the distal/peripheral portion of the fetal lung (i.e., distal 0.5 cm) was excised from the rest of the lung using a scalpel. This includes all components of the lung (e.g., epithelial, mesenchymal, vascular). <br>Isolated fetal bud tip the bud peripheral portion of the fetal lung was excised with a scalpel and subjected to enzymatic digestion and microdissection. The epithelium was dissected and separated from the mesenchyme, but a small amount of associated mesenchyme likely remained. <br>Fetal progenitor organoid 3D organoid structures that arose from culturing isolated fetal epithelial bud tips. <br>Foregut spheroid 3D foregut endoderm structure as described in Dye et al. (2015). Gives rise to patterned lung organoid (PLO) when grown in 3F medium. <br> Patterned lung organoid (PLO) lung organoids that were generated by differentiating hPSCs, as described throughout the manuscript. <br> Bud tip organoid organoids derived from PLOs, enriched for SOX2/SOX9 co-expressing cells, and grown/passaged in 3F medium.
2018-11-17 | E-MTAB-6023 | biostudies-arrayexpress
Project description:Murine small intestinal crypts of six independent mice were isolated and cultured as 3D-Organoids. Expression of NICD and loss of p53 mutagenesis was induced by 24h treatment with 4OH-tamoxifen in three of the cultures, resulting in three mutant organoid lines. The other three organoid lines were not exposed to tamoxifen and served as controls. Of each organoid line a duplicate was taken resulting in six control and six mutant samples that were compared in the analysis.
2021-06-04 | E-MTAB-6588 | biostudies-arrayexpress
Project description:In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal (CRC) patients. For most, organoids were also generated from adjacent normal tissue. The organoids closely resemble the original tumor. The spectrum of genetic changes observed within the 'living biobank' agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to robotized, high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43 (rather than in APC). Organoid technology may fill the gap between cancer genetics and patient trials, complement cell line- and xenograft-based drug studies and allow personalized therapy design. We generated organoids from healthy tissue and coloncarcinoma tissue. The organoids were trypsinized, plated in matrigel and overlaid with medium. After three days, RNA was isolated using Qiagen RNAeasy. Medium conditions are the same for all organoids, irrespective of their origin.
2015-05-07 | E-GEOD-64392 | biostudies-arrayexpress
Project description:We previously established long-term 3D organoid culture systems for several murine tissues (intestine, stomach, pancreas and liver) as well as human intestine and pancreas. Here, we describe culture conditions to generate long-term 3D culture from human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori. Human gastric cultures can expand indefinitely in 3D Matrigel. Cultures can be generated from normal tissue, from single sorted stem cells, or from tumor tissue. Organoids maintain many characteristics of the respective tissue in terms of histology, marker expression and euploidy. Organoids from normal tissue express markers of four lineages of the stomach and self-organize in gland and pit-domains. They can be directed to specifically express either lineages of the gastric gland, or the gastric pit by addition of Nicotinamide and withdrawal of Wnt. While gastric pit lineages react marginally to bacterial infection, gastric gland lineages mount a strong inflammatory response. The gastric culture system provides a unique tool to study gastric pathologies. We generated 2 sets of experiments. The first set contains organoids in 4 conditions: (1) organoids in expansion condition ENRWFGNiTi ("gland-type organoids") from 3 donors, (2) organoids as in 1 but differentiated for 4 days in differentiation condition ENR_FGNiTi ("pit'type organoids"), (3) organoids as in 1 but infected with Helicobacter pylori strain P12 MOI 50 for 2 h, (4) organoids as in 2 but infected as in 3. All 4 conditions were tested on the same organoid line in parallel. This experiment was conducted independently with cultures from 3 different donors. The second set of experiments compares freshly isolated glands with organoids. Samples from 2 patients were analyzed. Each patient received a total gastrectomy. From each patient, glands from corpus region or pyloric antrum were isolated. From each isolation, one aliquod was stored for microarray analysis and one aliquod used to generate organoids. Organoids and glands were subsequently lysed and analyzed in parallel.
2014-10-13 | E-GEOD-60557 | biostudies-arrayexpress
Project description:Generation of long-term, genetically-stable organoid cultures of extra-embryonic fetal trophoblast cells from human early placentas. Methylation profiling of the human trophoblast organoids and early placenta tissue.
2018-11-28 | E-MTAB-7204 | biostudies-arrayexpress
Project description:Acetylcholine (ACh) has been considered a neurotransmitter residing in central, parasympathetic and neuromuscular synapses of mammals. Here, experiments using crypt-villus organoids that lack nerve and immune cells in culture led us to suggest that endogenous ACh is synthesized in the intestinal epithelium to evoke growth and differentiation of the organoids through activation of muscarinic ACh receptors (mAChRs). The extracts of the cultured organoids exhibit a noticeable capacity for ACh synthesis that is sensitive to a potent inhibitor of choline acetyltransferase (ChAT). Imaging mass spectrometry reveals distribution of endogenous ACh that is localized in intestinal epithelial layer in the cultured organoids as well as in mouse small intestinal epithelium in vivo, suggesting non-neural resources of ACh. Treatment of organoids with carbachol down-regulates growth of organoids and expression of marker gene for each epithelial cell. On the other hand, antagonists for mAChRs enhances growth and differentiation of organoids, indicating involvement of mAChRs in regulating proliferation and differentiation of Lgr5-positive stem cells. Collectively, our data provide evidence that endogenous ACh released from intestinal epithelium maintains homeostasis of intestinal epithelial cell growth and differentiation via mAChRs in mice. Gene expression patters of gut, crypt, y-organoid and o-organoid, respectively
2013-08-15 | E-GEOD-49864 | biostudies-arrayexpress
Project description:Human astrocytes have reported to reprogram into neurons, but they have yet to be induced to three-dimensional (3D) neural tissue for neural organogenesis. Here, we demonstrate a remarkable strategy for 3D organoid generation by direct reprogramming human astrocytes. By combining overexpression OCT4, suppression p53 and small molecules CHIR99021, SB431542, RepSox and Y27632, termed as Op53-CSBRY, we successfully reprogramed human astrocytes into neural ectodermal cells and further induced human 3D-brain organoid. Those organoids can be finally induced into spinal cord organoids by activating FGF, SHH and BMP signaling. The grafts of Human astrocyte derived spinal cord organoids (hADSC-Organs) can survived, differentiated into spinal cord neurons, migrated long distance, formed synaptic connectivity with host neurons, bridged complete injury spinal cord tissue in mice. This method indicates that human astrocytes could be directly triggered neural organogenesis, and may hold a great promising to support local astrocytes in situ organogenesis after brain damages, such as stroke and spinal cord injury.
2024-02-01 | GSE168635 | GEO
Project description:Two-dimensional (2D) monolayer cell cultures are inevitable for drug development to identify drug candidate molecules because they are extremely potent for screening procedures. They can be used to predict in vivo drug responses for a variety of targets and pathways but they also face disadvantages like a lack of cell-to-cell and cell-to-matrix interaction as well as the loss of tissue-specific architecture. To overcome the drawbacks of 2D systems, three-dimensional (3D) cell culture was designed to provide cells a more physiological environment. In light of the multiple benefits of 3D cell culture it is important to note that this cultivation system is not automated and thus not ideal for high throughput screening (HTS) so far. An automated technique is critical for reproducible and standardized in vitro cultivation and it can greatly speed up preclinical research and total drug development. This procedure also requires a quality control system to monitor results and ensure that it is suitable for customers´ applications. For this purpose, the physiological condition of 2 D and 3D cell cultures will be assessed analyzing gene expression profiles of human mesenchymal stem cells (hMSCs). This will be accomplished by extracting total ribonucleic acid (RNA) from 2D and 3D cells followed by Affymetrix microarrays to get transcription profiles. The resulting data from both cell systems can be compared, and changes in cell behaviour caused by the 3D system can be observed.
2022-06-30 | GSE207210 | GEO
Project description:Background & Aims Patient-derived gastrointestinal organoids are powerful tools for studying human physiology and disease. However, generating organoids requires prompt isolation and culture of fresh tissue, which is labor and time intensive, placing restraints on basic and clinical research. For example organoid biobanking efforts, or the ability to obtain specimens from distant locations that lack the expertise to culture organoids, is limited by current approaches. We sought to develop a method by which tissue biopsies from patients could be cryo-preserved, stored, or shipped, and later thawed in order to establish live organoid cultures. Methods A method for freezing and recovery, along with straightforward isolation methods using readily available materials were developed. Epithelial isolation and culture conditions were optimized to promote cell survival and the establishment of long-term culture post-thawing. Results Patient biopsies from stomach, duodenum, ileum, colon and from adenomateous colonic polyps were frozen, subsequently thawed and used to successfully establish patient-specific epithelium-only organoid cultures with 100% efficiency (n=31 independent patient biopsies from different regions of the GI tract). Frozen tissue could be shipped internationally and across the United States and used to establish successful organoid cultures. Organoid cultures could be expanded, passaged and frozen down for long-term storage. RNA-sequencing demonstrates that organoids derived from fresh tissue or from frozen biopsies are >99% transcriptionally identical. Conclusions Cryo-preservation of human tissue biopsies followed by the establishment of long-term, expandable cultures has negligible effect on transcription when compared to freshly prepared organoids, and will be of immense benefit to research and for clinical applications. This technique will allow for the establishment of biobanks of human tissues that can be revived and cultured in the future as well as transported across the globe for research, therapeutic or diagnostic use in remote labs and/or clinics.
2018-06-30 | E-MTAB-6516 | biostudies-arrayexpress