Control Organoids Research Articles

Label-free three-photon imaging of intact human cerebral organoids for tracking early events in brain development and deficits in Rett syndrome.

Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (~2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging.

Establishment and characterization of human pluripotent stem cells-derived brain organoids to model cerebellar diseases

The establishment of robust human brain organoids to model cerebellar diseases is essential to study new therapeutic strategies for cerebellum-associated disorders. Machado-Joseph disease (MJD) is a cerebellar hereditary neurodegenerative disease, without therapeutic options able to prevent the disease progression. In the present work, control and MJD induced-pluripotent stem cells were used to establish human brain organoids. These organoids were characterized regarding brain development, cell type composition, and MJD-associated neuropathology markers, to evaluate their value for cerebellar diseases modeling. Our data indicate that the organoids recapitulated, to some extent, aspects of brain development, such as astroglia emerging after neurons and the presence of ventricular-like zones surrounded by glia and neurons that are found only in primate brains. Moreover, the brain organoids presented markers of neural progenitors proliferation, neuronal differentiation, inhibitory and excitatory synapses, and firing neurons. The established brain organoids also exhibited markers of cerebellar neurons progenitors and mature cerebellar neurons. Finally, MJD brain organoids showed higher ventricular-like zone numbers, an indication of lower maturation, and an increased number of ataxin-3-positive aggregates, compared with control organoids. Altogether, our data indicate that the established organoids recapitulate important characteristics of human brain development and exhibit cerebellar features, constituting a resourceful tool for testing therapeutic approaches for cerebellar diseases.

Abstract 2673: Combined inhibition of PI3K and HDAC1/2 as a novel treatment strategy for RAS/RAF wildtype colorectal cancer in a patient derived organoid model

Abstract Background: Treating colorectal cancer (CRC) using targeted therapies based on each patient’s individual mutational profile continues to gain interest. Oncogenic mutations of the PIK3CA gene leads to tumor initiation and progression. Copanlisib inhibits the PI3K pathway and has proven to be an effective treatment strategy for various cancers characterized by overactivation of the PI3K signaling cascade. However, resistance mechanisms to targeted therapies such as copanlisib are frequently encountered, and can be overcome through combination with another molecule such as romidepsin, a histone deacetylase (HDAC) inhibitor. Here we aim to identify a novel therapeutic regimen for improved treatment of the molecular subtypes of CRC that can be targeted with PI3K inhibition. Methods: Organoids were derived from colorectal cancer tumor tissue from two different biopsy sites from one patient with PIK3CA mutant rectal cancer (RC46A and RC46B), as well as from a patient with APC and TP53 mutant CRC (MTB74) under approved IRB protocols. All organoids were plated and allowed to mature for 24 to 48 hours before treatment and baseline images were obtained. The organoids were imaged again 48 hours later, and response was determined by measuring the change in organoid diameter using ImageJ. Additionally, organoids were collected for immunoblotting to examine alterations in the PI3K pathway as well as histone acetylation, and proteins involved in apoptosis. Results: Control organoids demonstrated increased median relative changes in diameter of +53.38%, +43.04% and +46.2% for RC46A, RC46B and MTB74, respectively. In comparison, the combination treatment group exhibited median relative changes for RC46A, RC46B and MTB74 as -29.19%, -46.46% and -100% (p<0.001). These results were supported with immunoblot analysis that revealed a synergistic increase in histone acetylation with the combination treatment. Single agent copanlisib treatment elicited alterations in PI3K signaling including downregulation of pS6, pAKT, p4EBP1 as expected. Lastly, an increase in cleaved PARP was observed in combination treated organoids which is indicative of increased apoptosis. Conclusions: As molecular diagnostic technology continues to advance, treating each individual cancer with a targeted therapy based on molecular profiling is becoming more feasible. Dual inhibition of the PI3K pathway and HDAC is effective in reducing cell growth and proliferation of CRC organoids characterized by PIK3CA mutations, as well as other molecular subtypes such as cancers with APC and TP53 mutations. This combination strategy shows promise for improving treatment response and reducing resistance in patients across these molecular subtypes. Citation Format: Alyssa K. DeZeeuw, Rebecca A. DeStefanis, Susan N. Payne, Autumn M. Olson, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. Combined inhibition of PI3K and HDAC1/2 as a novel treatment strategy for RAS/RAF wildtype colorectal cancer in a patient derived organoid model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2673.

Screening of Organophosphate Flame Retardants with Placentation-Disrupting Effects in Human Trophoblast Organoid Model and Characterization of Adverse Pregnancy Outcomes in Mice.

Background:Abnormal placental development may result in adverse pregnancy outcomes and metabolic diseases in adulthood; however, it remains unknown whether and how xenobiotics affect human placentation.Objectives:This study aimed to screen and identify placentation-disrupting chemicals in commonly used organophosphate flame retardants (OPFRs) and, if identified, to investigate potential adverse effects on placentation in relation to adverse pregnancy outcomes and metabolic disorder in offspring in mice.Methods:We devised a high-throughput immunofluorescence screening assay based on human trophoblast organoids and used it to screen OPFRs that inhibit the proliferation of organoids. One identified chemical was assessed for its effects on placentation by evaluating villous cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts using immunofluorescence and a mitochondrial stress test after 2 d of exposure. A 10-d exposure study was further performed to observe the dynamic effect of the OPFR on the structure of the organoids. RNA-sequencing and western blotting experiments were performed to explore the associated pathways, and a potential binding protein was identified by immunoprecipitation and in vitro kinase activity assays. Animal studies were performed to determine whether the findings in organoids could be replicated in mice and to observe adverse pregnancy outcomes.Results:The proliferation of organoids exposed to three aryl-OPFRs was significantly lower than the proliferation of control organoids. Further analysis demonstrated that one such chemical, 2-ethylhexyl-diphenyl phosphate (EHDPP), disrupted placentation in organoids. Mechanistically, EHDPP interfered with insulin-like growth factor 1 receptor (IGF1R) to inhibit aerobic respiration. Mice exposed to EHDPP at a physiological human concentrations exhibited immature and mature placental disorders, which correlated with fetal growth restriction, implantation failure, stillbirth, and impaired glucose tolerance.Conclusions:The human trophoblast organoid model showed that the commonly used OPFRs disrupted placentation via IGF1R, indicating that its use may contribute to adverse pregnancy outcomes and metabolic disorders in offspring. https://doi.org/10.1289/EHP10273

Endometrial organoids derived from Mayer-Rokitansky-Küster-Hauser syndrome patients provide insights into disease-causing pathways.

ABSTRACTThe uterus is responsible for the nourishment and mechanical protection of the developing embryo and fetus and is an essential part in mammalian reproduction. Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome is characterized by agenesis of the uterus and upper part of the vagina in females with normal ovarian function. Although heavily studied, the cause of the disease is still enigmatic. Current research in the field of MRKH mainly focuses on DNA-sequencing efforts and, so far, has been unable to decipher the nature and heterogeneity of the disease, thereby holding back scientific and clinical progress. Here, we developed long-term expandable organoid cultures from endometrium found in uterine rudiment horns of MRKH patients. Phenotypically, they share great similarity with healthy control organoids and are surprisingly fully hormone responsive. Transcriptome analyses, however, identified an array of dysregulated genes that point to potentially disease-causing pathways altered during the development of the female reproductive tract. We consider the endometrial organoid cultures to be a powerful research tool that promise to enable an array of studies into the pathogenic origins of MRKH syndrome and possible treatment opportunities to improve patient quality of life.

A17 TRP53 REGULATES ESLF-RENEWAL AND CELL DIFFERENTIATION IN KRT15+ INTESTINAL STEM CELLS

Abstract Background Different pools of stem cells assure the intestinal epithelium homeostasis and regeneration. We recently reported that Krt15+ intestinal cells harbor self-renewal, multipotent and regenerative capacities, characteristics consistent with a stem cell population. p53 has been associated with the development of various types of cancer, and recent studies suggested it also regulates adult stem cell behavior in some tissues. However, its role in the maintenance of intestinal stem cells has not been explicitly covered. Therefore, we hypothesize that Trp53 loss specifically in Krt15+ intestinal stem cells will perturb the epithelial homeostasis and differentiation. Aims Identify the role of Trp53 in the regulation of intestinal stem cell and intestinal homeostasis Methods To induce Trp53 loss specifically in Krt15+ cells, we generated Krt15 CrePR1;Trp53fl/fl ( Krt15△Trp53) mice and induced Cre recombination by injecting RU486 (PR agonist). Mice were euthanized after Cre recombination at different time points. Organoid cultures were established from control and experimental mice. Results Two-month post Cre recombination, we observed no major morphological changes in the intestinal epithelium. However, we observed that crypts isolated from Krt15△Trp53 mice died rapidly and had difficulties forming organoids. When hyperactivating the Wnt/beta-catenin pathway through CHIR treatment, we observed that budding capacity was maintained in experimental organoids while control organoids formed mainly cysts as expected. We also observed an increased in Paneth cell-specific genes, suggesting an enrichment in Paneth cells. Interestingly, the expression of EpHB2 receptor and its target genes was decreased in organoids derived from Krt15△Trp53 mice. Ephrin signaling plays a crucial role in intestinal homeostasis by regulating cell position and migration, and previous studies suggest that ephrin receptors are a target of p53. Indeed, twelve-month post Cre recombination, altered architecture of crypt and villi was noted in the small intestinal epithelium of Krt15△Trp53 mice. Differentiation towards secretory cell types, specifically Paneth, goblet, and tuft cells, are significantly increased in Krt15△Trp53 mice. Interestingly, we also observed crypt cells expressing both goblet and Paneth cell markers suggesting dysfunction in the cell fate decision. Finally, decreased Notch pathway activation was also observed supporting that Trp53 loss, specifically in Krt15+ cells, leads to a dysregulation of secretory cell fate. Conclusions Loss of Trp53 specifically in Krt15+ cells affects the small intestinal homeostasis and secretory cell differentiation. Decreased Notch and ephrin signaling support the possible role of Trp53 in cell fate decision and self-renewal. Funding Agencies None

Establishment of Adenomyosis Organoids as a Preclinical Model to Study Infertility.

Adenomyosis is related to infertility and miscarriages, but so far there are no robust in vitro models that reproduce its pathological features to study the molecular mechanisms involved in this disease. Endometrial organoids are in vitro 3D models that recapitulate the native microenvironment and reproduce tissue characteristics that would allow the study of adenomyosis pathogenesis and related infertility disorders. In our study, human endometrial biopsies from adenomyosis (n = 6) and healthy women (n = 6) were recruited. Organoids were established and hormonally differentiated to recapitulate midsecretory and gestational endometrial phases. Physiological and pathological characteristics were evaluated by immunohistochemistry, immunofluorescence, qRT-PCR, and ELISA. Secretory and gestational organoids recapitulated in vivo glandular epithelial phenotype (pan-cytokeratin, Muc-1, PAS, Laminin, and Ki67) and secretory and gestational features (α-tubulin, SOX9, SPP1, PAEP, LIF, and 17βHSD2 expression and SPP1 secretion). Adenomyosis organoids showed higher expression of TGF-β2 and SMAD3 and increased gene expression of SPP1, PAEP, LIF, and 17βHSD2 compared with control organoids. Our results demonstrate that organoids derived from endometria of adenomyosis patients and differentiated to secretory and gestational phases recapitulate native endometrial-tissue-specific features and disease-specific traits. Adenomyosis-derived organoids are a promising in vitro preclinical model to study impaired implantation and pregnancy disorders in adenomyosis and enable personalized drug screening.

A Potential Treatment of Congenital Sodium Diarrhea in Patients With Activating GUCY2C Mutations.

INTRODUCTION:Gain-of-function mutations in guanylyl cyclase C (GCC) result in persistent diarrhea with perinatal onset. We investigated a specific GCC inhibitor, SSP2518, for its potential to treat this disorder.METHODS:We investigated the effect of SSP2518 on GCC-mediated intracellular cyclic guanosine monophosphate (cGMP) levels and on GCC-mediated chloride secretion in intestinal organoids from 3 patients with distinct activating GCC mutations and from controls, with and without stimulation of GCC with heat-stable enterotoxin.RESULTS:Patient-derived organoids had significantly higher basal cGMP levels than control organoids, which were lowered by SSP2518 to levels found in control organoids. In addition, SSP2518 significantly reduced cGMP levels and chloride secretion in patient-derived and control organoids (P < 0.05 for all comparisons) after heat-stable enterotoxin stimulation.DISCUSSION:We reported in this study that the GCC inhibitor SSP2518 normalizes cGMP levels in intestinal organoids derived from patients with GCC gain-of-function mutations and markedly reduces cystic fibrosis transmembrane conductance regulator–dependent chloride secretion, the driver of persistent diarrhea.

Epithelial Regeneration Ability of Crohn's Disease Assessed Using Patient-Derived Intestinal Organoids.

Little is known about the ability for epithelial regeneration and wound healing in patients with inflammatory bowel diseases. We evaluated the epithelial proliferation and wound healing ability of patients with Crohn’s disease (CD) using patient-derived intestinal organoids. Human intestinal organoids were constructed in a three-dimensional intestinal crypt culture of enteroscopic biopsy samples from controls and CD patients. The organoid-forming efficiency of ileal crypts derived from CD patients was reduced compared with those from control subjects (p < 0.001). Long-term cultured organoids (≥6 passages) derived from controls and CD patients showed an indistinguishable microscopic appearance and culturing behavior. Under TNFα-enriched conditions (30 ng/mL), the organoid reconstitution rate and cell viability of CD patient-derived organoids were significantly lower than those of the control organoids (p < 0.05 for each). The number of EdU+ cells was significantly lower in TNFα-treated organoids derived from CD patients than in TNFα-treated control organoids (p < 0.05). In a wound healing assay, the unhealed area in TNFα-treated CD patient-derived organoids was significantly larger than that of TNFα-treated control organoids (p < 0.001). The wound healing ability of CD patient-derived organoids is reduced in TNFα-enriched conditions, due to reduced cell proliferation. Epithelial regeneration ability may be impaired in patients with CD.

Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids.

SummaryMutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX-LCA and other CRX retinopathies.

S100P Expression via DNA Hypomethylation Promotes Cell Growth in the Sessile Serrated Adenoma/Polyp-Cancer Sequence

Background/Aims: Sessile serrated adenomas/polyps (SSA/Ps) are a putative precursor lesion of colon cancer. Although the relevance of DNA hypermethylation in the SSA/P-cancer sequence is well documented, the role of DNA hypomethylation is unknown. We investigated the biological relevance of DNA hypomethylation in the SSA/P-cancer sequence by using 3-dimensional organoids of SSA/P. Methods: We first analyzed hypomethylated genes using datasets from our previous DNA methylation array analysis on 7 SSA/P and 2 cancer in SSA/P specimens. Expression levels of hypomethylated genes in SSA/P specimens were determined by RT-PCR and immunohistochemistry. We established 3-dimensional SSA/P organoids and performed knockdown experiments using a lentiviral shRNA vector. DNA hypomethylation at CpG sites of the gene was quantitated by MassARRAY analysis. Results: The mean number of hypomethylated genes in SSA/P and cancer in SSA/P was 41.6 ± 27.5 and 214 ± 19.8, respectively, showing a stepwise increment in hypomethylation during the SSA/P-cancer sequence. S100P, S100α2, PKP3, and MUC2 were most commonly hypomethylated in SSA/P specimens. The mRNA and protein expression levels of S100P, S100α2, and MUC2 were significantly elevated in SSA/P compared with normal colon tissues, as revealed by RT-PCR and immunohistochemistry, respectively. Among these, mRNA and protein levels were highest for S100P. Knockdown of the S100P gene using a lentiviral shRNA vector in 3-dimensional SSA/P organoids inhibited cell growth by >50% (p < 0.01). The mean diameter of SSA/P organoids with S100P gene knockdown was significantly smaller compared with control organoids. MassARRAY analysis of DNA hypomethylation in the S100P gene revealed significant hypomethylation at specific CpG sites in intron 1, exon 1, and the 5′-flanking promoter region. Conclusion: These results suggest that DNA hypomethylation, including S100P hypomethylation, is supposedly associated with the SSA/P-cancer sequence. S100P overexpression via DNA hypomethylation plays an important role in promoting cell growth in the SSA/P-cancer sequence.

Enhanced Neuronal Activity and Asynchronous Calcium Transients Revealed in a 3D Organoid Model of Alzheimer’s Disease

Advances in the development of three-dimensional (3D) brain organoids maintained in vitro have provided excellent opportunities to study brain development and neurodegenerative disorders, including Alzheimer's disease (AD). However, there remains a need to generate AD organoids bearing patient-specific genomic backgrounds that can functionally recapitulate the key features observed in the AD patient's brain. To address this need, we described a strategy to generate self-organizing 3D cerebral organoids which develop a functional neuronal network connectivity. This was achieved by neuroectoderm induction of human pluripotent stem cell (hPSCs) aggregates and subsequent differentiation into desired neuroepithelia and mature neurons in a 3D Matrigel matrix. Using this approach, we successfully generated AD cerebral organoids from human pluripotent stem cells (hPSCs) derived from a familial AD patient with a common mutation in presenilin 2 (PSEN2N141I). An isogenic control with an identical genetic background but wild-type PSEN2 was generated using CRISPR/Cas9 technology. Both control and AD organoids were characterized by analyzing their morphology, the Aβ42/Aβ40 ratio, functional neuronal network activity, drug sensitivity, and the extent of neural apoptosis. The spontaneous activity of the network and its synchronization was measured in the organoids via calcium imaging. We found that compared with the mutation-corrected control organoids, AD organoids had a higher Aβ42/Aβ40 ratio, asynchronous calcium transients, and enhanced neuronal hyperactivity, successfully recapitulating an AD-like pathology at the molecular, cellular, and network level in a human genetic context. Moreover, two drugs which increase neuronal activity, 4-aminopyridine (4-AP) and bicuculline methochloride, induced high-frequency synchronized network bursting to a similar extent in both organoids. Therefore, our study presents a promising organoid-based biosystem for the study of the pathophysiology of AD and a platform for AD drug development.

Manipulate intestinal organoids with niobium carbide nanosheets.

Multifunctional two-dimensional nanosheet materials have attracted attention in biomedical fields due to their unique physiochemical and biological properties. Interactions between intestinal stem cells and Engineered Nanomaterials (ENMs) are an essential area in research with the growing diagnosis of gastrointestinal (GI) diseases. One unique type of two-dimensional metal carbide nanomaterial, niobium carbide (Nb2 C), has shown promising properties for potential applications in this field, such as biocompatibility, stability, and high photothermal conversion efficiency. In this study, Nb2 C nanosheets were prepared by spark plasma sintering and HF etching. Various concentrations of Nb2 C nanosheets were placed inside intestinal organoids, which mimic the real functions of an intestinal system. These organoids were formed from intestinal crypts that were isolated from mice and grew into self-maintained systems. Through growth analysis, surface area calculations, and cell viability tests, it was concluded that an optimal concentration of nanosheets exists that may offer stimulation to intestinal cells while having no toxic effects. A high concentration of nanosheets in the organoids inhibited growth, whereas the control and low concentration of nanosheets showed no reduced growth rate. When placed under infrared exposure, the organoids with nanosheets offered stimulation and showed more viability after time as compared to the control organoids with no nanosheets. These results show overall potential benefits of placing low concentration Nb2 C nanosheets in intestinal systems to protect and stimulate cell survivability when undergoing various treatments.

Beta-amyloid deposition around hepatic bile ducts is a novel pathobiological and diagnostic feature of biliary atresia

Biliary atresia (BA) is a poorly understood and devastating obstructive bile duct disease of newborns. It is often diagnosed late, is incurable and frequently requires liver transplantation. In this study, we aimed to investigate the underlying pathogenesis and molecular signatures associated with BA. We combined organoid and transcriptomic analysis to gain new insights into BA pathobiology using patient samples and a mouse model of BA. Liver organoids derived from patients with BA and a rhesus rotavirus A-infected mouse model of BA, exhibited aberrant morphology and disturbed apical-basal organization. Transcriptomic analysis of BA organoids revealed a shift from cholangiocyte to hepatocyte transcriptional signatures and altered beta-amyloid-related gene expression. Beta-amyloid accumulation was observed around the bile ducts in BA livers and exposure to beta-amyloid induced the aberrant morphology in control organoids. The novel observation that beta-amyloid accumulates around bile ducts in the livers of patients with BA has important pathobiological implications, as well as diagnostic potential. Biliary atresia is a poorly understood and devastating obstructive bile duct disease of newborns. It is often diagnosed late, is incurable and frequently requires liver transplantation. Using human and mouse 'liver mini-organs in the dish', weunexpectedly identified beta-amyloid deposition - the main pathological feature of Alzheimer's disease and cerebral amyloid angiopathy - around bile ducts in livers from patients with biliary atresia. This finding reveals a novel pathogenic mechanism that could have important diagnostic and therapeutic implications.

Characterization of Human Colon Organoids From Inflammatory Bowel Disease Patients.

Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders, where epithelial defects drive, at least in part, some of the pathology. We reconstituted human intestinal epithelial organ, by using three-dimension culture of human colon organoids. Our aim was to characterize morphological and functional phenotypes of control (non-IBD) organoids, compared to inflamed organoids from IBD patients. The results generated describe the epithelial defects associated with IBD in primary organoid cultures, and evaluate the use of this model for pharmacological testing of anti-inflammatory approaches. Human colonic tissues were obtained from either surgical resections or biopsies, all harvested in non-inflammatory zones. Crypts were isolated from controls (non-IBD) and IBD patients and were cultured up to 12-days. Morphological (size, budding formation, polarization, luminal content), cell composition (proliferation, differentiation, immaturity markers expression), and functional (chemokine and tight junction protein expression) parameters were measured by immunohistochemistry, RT-qPCR or western-blot. The effects of inflammatory cocktail or anti-inflammatory treatments were studied in controls and IBD organoid cultures respectively. Organoid cultures from controls or IBD patients had the same cell composition after 10 to 12-days of culture, but IBD organoid cultures showed an inflammatory phenotype with decreased size and budding capacity, increased cell death, luminal debris, and inverted polarization. Tight junction proteins were also significantly decreased in IBD organoid cultures. Inflammatory cytokine cocktail reproduced this inflammatory phenotype in non-IBD organoids. Clinically used treatments (5-ASA, glucocorticoids, anti-TNF) reduced some, but not all parameters. Inflammatory phenotype is associated with IBD epithelium, and can be studied in organoid cultures. This model constitutes a reliable human pre-clinical model to investigate new strategies targeting epithelial repair.

Rapid Prototyping of Multilayer Microphysiological Systems.

Microfluidic organs-on-chips aim to realize more biorelevant in vitro experiments compared to traditional two-dimensional (2D) static cell culture. Often such devices are fabricated via poly(dimethylsiloxane) (PDMS) soft lithography, which offers benefits (e.g., high feature resolution) along with drawbacks (e.g., prototyping time/costs). Here, we report benchtop fabrication of multilayer, PDMS-free, thermoplastic organs-on-chips via laser cut and assembly with double-sided adhesives that overcome some limitations of traditional PDMS lithography. Cut and assembled chips are economical to prototype ($2 per chip), can be fabricated in parallel within hours, and are Luer compatible. Biocompatibility was demonstrated with epithelial line Caco-2 cells and primary human small intestinal organoids. Comparable to control static Transwell cultures, Caco-2 and organoids cultured on chips formed confluent monolayers expressing tight junctions with low permeability. Caco-2 cells-on-chip differentiated ∼4 times faster, including increased mucus, compared to controls. To demonstrate the robustness of cut and assemble, we fabricated a dual membrane, trilayer chip integrating 2D and 3D compartments with accessible apical and basolateral flow chambers. As proof of concept, we cocultured a human, differentiated monolayer and intact 3D organoids within multilayered contacting compartments. The epithelium exhibited 3D tissue structure and organoids expanded close to the adjacent monolayer, retaining proliferative stem cells over 10 days. Taken together, cut and assemble offers the capability to rapidly and economically manufacture microfluidic devices, thereby presenting a compelling fabrication technique for developing organs-on-chips of various geometries to study multicellular tissues.

Midbrain Organoids: A New Tool to Investigate Parkinson's Disease.

The study of human 3D cell culture models not only bridges the gap between traditional 2D in vitro experiments and in vivo animal models, it also addresses processes that cannot be recapitulated by either of these traditional models. Therefore, it offers an opportunity to better understand complex biology including brain development. The brain organoid technology provides a physiologically relevant context, which holds great potential for its application in modeling neurological diseases. Here, we compare different methods to obtain highly specialized structures that resemble specific features of the human midbrain. Regionally patterned neural stem cells (NSCs) were utilized to derive such human midbrain-specific organoids (hMO). The resulting neural tissue exhibited abundant neurons with midbrain dopaminergic neuron identity, as well as astroglia and oligodendrocyte differentiation. Within the midbrain organoids, neurite myelination, and the formation of synaptic connections were observed. Regular neuronal fire patterning and neural network synchronicity were determined by multielectrode array recordings. In addition to electrophysiologically functional neurons producing and secreting dopamine, responsive neuronal subtypes, such as GABAergic and glutamatergic neurons were also detected. In order to model disorders like Parkinson’s disease (PD) in vitro, midbrain organoids carrying a disease specific mutation were derived and compared to healthy control organoids to investigate relevant neurodegenerative pathophysiology. In this way midbrain-specific organoids constitute a powerful tool for human-specific in vitro modeling of neurological disorders with a great potential to be utilized in advanced therapy development.

Extracellular Vesicles Derived from Neural Progenitor Cells\u2013\u2013a Preclinical Evaluation for Stroke Treatment in Mice

Stem cells such as mesenchymal stem cells (MSCs) enhance neurological recovery in preclinical stroke models by secreting extracellular vesicles (EVs). Since previous reports have focused on the application of MSC-EVs only, the role of the most suitable host cell for EV enrichment and preclinical stroke treatment remains elusive. The present study aimed to evaluate the therapeutic potential of EVs derived from neural progenitor cells (NPCs) following experimental stroke. Using the PEG technique, EVs were enriched and characterized by electron microscopy, proteomics, rt-PCR, nanosight tracking analysis, and Western blotting. Different dosages of NPC-EVs displaying a characteristic profile in size, shape, cargo protein, and non-coding RNA contents were incubated in the presence of cerebral organoids exposed to oxygen-glucose deprivation (OGD), significantly reducing cell injury when compared with control organoids. Systemic administration of NPC-EVs in male C57BL6 mice following experimental ischemia enhanced neurological recovery and neuroregeneration for as long as 3 months. Interestingly, the therapeutic impact of such NPC-EVs was found to be not inferior to MSC-EVs. Flow cytometric analyses of blood and brain samples 7 days post-stroke demonstrated increased blood concentrations of B and T lymphocytes after NPC-EV delivery, without affecting cerebral cell counts. Likewise, a biodistribution analysis after systemic delivery of NPC-EVs revealed the majority of NPC-EVs to be found in extracranial organs such as the liver and the lung. This proof-of-concept study supports the idea of EVs being a general concept of stem cell–induced neuroprotection under stroke conditions, where EVs contribute to reverting the peripheral post-stroke immunosuppression.

Harnessing The Biology of Intestinal Organoids To Accelerate Drug Discovery in Inflammatory Bowel Disease: A One Health Approach

Rationale and SignificanceCanine inflammatory bowel disease (IBD) refers to a group of chronic gastrointestinal (GI) disorders of unknown cause and pathogenesis which mimic the spectrum of chronic enteropathies in human patients with IBD. There is currently no cure for IBD and many therapeutic interventions developed over the past 3 decades have consistently failed to demonstrate evidence of efficacy in clinical trials. There is, therefore, a critical need to develop robust drug screening tools to accelerate the availability of effective and safe therapeutic strategies for management of IBD. With the aim of modeling spontaneous GI disease in dogs and humans, intestinal stem cell (ISC)‐derived organoids are emerging as a promising ex vivo tool in the study of IBD pathogenesis. Our working testable hypothesis is that canine organoids will faithfully reproduce structural and functional changes of the intestinal epithelium in dogs with IBD, which will enable more accurate prediction of therapeutic drug efficacy and safety. Long‐term, this model will provide critical information for the design of canine clinical trials, and ultimately generate preclinical data for similar studies in humans with IBD. Altogether, we therefore expect to contribute to decreased morbidity/mortality, and improved quality of life in dogs and patients with IBD.MethodsISCs isolated from endoscopic biopsies of two healthy dogs and two dogs with active IBD were differentiated into intestinal organoids. Ileal organoids and matching tissues were probed by RNA in situ hybridization and immunohistochemistry for phenotypic changes in IBD. A panel of six phenotypic markers identified different epithelial cell lineages (LGR5+: intestinal stem cell, ALP: enterocyte, PAS: goblet cell, NeuroG3: enteroendocrine cell), epithelial barrier integrity (ZO‐1) and cell proliferation (Ki‐67). Functional features of IBD organoids were investigated by cystic fibrosis transmembrane conductance regulator (CFTR) organoid swelling assay to measure Cl− channel‐water conductance.ResultsOur preliminary findings suggest that IBD organoids exhibit increased cell proliferation, intestinal stem cells, enteroendocrine cells, and expression of tight junction protein as compared to healthy organoids. Similar trends were observed when comparing phenotypic marker expression in original intestinal tissues. Forskolin significantly (P &lt; 0.05) increased swelling of IBD organoids after 1 and 4 hr vs. controls indicating functional CFTR in organoids.ConclusionOverall, our data show that ileal organoids derived from dogs with IBD recapitulate both the phenotypic and physiological features of diseased tissue compared to healthy tissue, demonstrating its utility as an ex vivo model for investigating mechanism and therapeutic strategies in IBD. These preliminary results will be further validated by additional intestinal biopsies from 14 dogs with IBD in an ongoing clinical trials.Support or Funding Information Research Support: IG BiosciencesPreliminary phenotypic characterization of IBD organoids.(Left) Representative RNA ISH of ALP and NeuroG3 staining in ileal organoids at x40 magnification (ratio of total area). Scale bar: 50 μm. (Right) Summary box plots of ALP and PAS expression in IBD vs. Control (CTR) organoids. Data from 10 distinct microscopic fields were aggregated per dog.Figure 1

MP09-07 USE OF 3D VIDEO MICROSCOPY OF HUMAN-DERIVED PROSTATE ORGANOIDS TO ASSESS FEATURES OF EARLY INVASIVE PROSTATE CANCER

INTRODUCTION AND OBJECTIVE: Patient-derived organoids provide a method for creating human prostate cancer cell lines in culture in three dimensions. We established living organoids using normal and prostate cancer tissue from hormone-naive patients obtained at the time of surgery. We assessed with an image analysis system, dynamic, 3D morphologic transformations of the organoids. Our long-term goal is to assess prognostic features of early invasive disease. METHODS: Needle cores from fresh radical prostatectomy specimens were harvested at the time of surgery. Gross tumor and normal prostate as controls were sampled. The de-identified samples are processed as described by Drost et al. Briefly, cells from the digested tissue are suspended within an extracellular matrix (ECM) gel, which give rise to differentiated prostate “mini-glands” or “mini-tumors” over ∼1-2 wks. Organoids are also biobanked. To assay dynamic spreading of cells from the compact mini-gland structure, mature organoids are isolated from the gel and plated on dishes coated with ECM. Time-lapse images are acquired and processed into binary masks for analysis. Spreading of prostate cells is estimated by the following formula of circularity: C = 4*pi*A/P^2, where C is circularity, A is area and P is perimeter. For a perfect circle, C = 1. RESULTS: We have biobanked prostate-derived organoids using primary tumor and matched normal controls from 47 patients. The samples represent a range of pathologic grades stratified by Gleason score (GS): GS6 n=8 (17%), GS7 n=33 (70%), GS8 n=1 (2%), and GS9 n=5 (11%). We tested 3D dynamic movement onto two ECM proteins: Laminin-332, normally secreted by epithelial cells, and Laminin-511, which coats smooth muscle cells and nerve axons within the gland. For control organoids, average time to dynamic change threshold was 12hr on Laminin-332 and 20hr on Laminin-511 using a circularity of 0.6 as a cut-off. CONCLUSIONS: This study demonstrates feasibility in assaying cellular behaviors of living human organoid cultures for prostate tumors and matched normal tissue. It will be interesting to correlate invasive behavior to the clinical grading and staging of the primary neoplasm. We predict further studies will reveal that organoids derived from higher grade tumors will spread on Laminin-511, reflective of their ability to invade through the smooth muscle stroma along nerves to escape the prostate gland. Source of Funding: NCI P30 CA 23074, Arizona Biomedical Research Centre ADHS15-090301, and DoD PCRP W81XWH-19-1-0455. K.D.M. is supported by the ARCS Foundation.