Multi-lineage heart-chip models drug cardiotoxicity and enhances maturation of human stem cell-derived cardiovascular cells.

Cardiovascular toxicity causes adverse drug reactions and may lead to drug removal from the pharmaceutical market. Cancer therapies can induce life-threatening cardiovascular side effects such as arrhythmias, muscle cell death, or vascular dysfunction. New technologies have enabled cardiotoxic compounds to be identified earlier in drug development. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) and vascular endothelial cells (ECs) can screen for drug-induced alterations in cardiovascular cell function and survival. However, most existing hiPSC models for cardiovascular drug toxicity utilize two-dimensional, immature cells grown in static culture. Improved in vitro models to mechanistically interrogate cardiotoxicity would utilize more adult-like, mature hiPSC-derived cells in an integrated system whereby toxic drugs and protective agents can flow between hiPSC-ECs that represent systemic vasculature and hiPSC-CMs that represent heart muscle (myocardium). Such models would be useful for testing the multi-lineage cardiotoxicities of chemotherapeutic drugs such as VEGFR2/PDGFR-inhibiting tyrosine kinase inhibitors (VPTKIs). Here, we develop a multi-lineage, fully-integrated, cardiovascular organ-chip that can enhance hiPSC-EC and hiPSC-CM functional and genetic maturity, model endothelial barrier permeability, and demonstrate long-term functional stability. This microfluidic organ-chip harbors hiPSC-CMs and hiPSC-ECs on separate channels that can be subjected to active fluid flow and rhythmic biomechanical stretch. We demonstrate the utility of this cardiovascular organ-chip as a predictive platform for evaluating multi-lineage VPTKI toxicity. This study may lead to the development of new modalities for the evaluation and prevention of cancer therapy-induced cardiotoxicity.

Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects.

Using induced pluripotent stem cells (iPSCs) to understand the mechanisms of neurological disease holds great promise; however, there is a lack of well-curated lines from a large array of participants. Answer ALS has generated over 1,000 iPSC lines from control and amyotrophic lateral sclerosis (ALS) patients along with clinical and whole-genome sequencing data. The current report summarizes cell marker and gene expression in motor neuron cultures derived from 92 healthy control and 341 ALS participants using a 32-day differentiation protocol. This is the largest set of iPSCs to be differentiated into motor neurons, and characterization suggests that cell composition and sex are significant sources of variability that need to be carefully controlled for in future studies. These data are reported as a resource for the scientific community that will utilize Answer ALS data for disease modeling using a wider array of omics being made available for these samples.

Tissue clearing of human iPSC-derived organ-chips enables high resolution imaging and analysis.

Engineered microfluidic organ-chips enable increased cellular diversity and function of human stem cell-derived tissues grown in vitro. These three dimensional (3D) cultures, however, are met with unique challenges in visualization and quantification of cellular proteins. Due to the dense 3D nature of cultured nervous tissue, classical methods of immunocytochemistry are complicated by sub-optimal light and antibody penetrance as well as image acquisition parameters. In addition, complex polydimethylsiloxane scaffolding surrounding the tissue of interest can prohibit high resolution microscopy and spatial analysis. Hyperhydration tissue clearing methods have been developed to mitigate similar challenges of in vivo tissue imaging. Here, we describe an adaptation of this approach to efficiently clear human pluripotent stem cell-derived neural tissues grown on organ-chips. We also describe critical imaging considerations when designing signal intensity-based approaches to complex 3D architectures inherent in organ-chips. To determine morphological and anatomical features of cells grown in organ-chips, we have developed a reliable protocol for chip sectioning and high-resolution microscopic acquisition and analysis.

Human iPSC-derived fallopian tube organoids with BRCA1 mutation recapitulate early-stage carcinogenesis.

Germline pathogenic mutations in BReast CAncer (BRCA1) genes are thought to drive normal fallopian tube epithelial (FTE) cell transformation to high-grade serous ovarian cancer. No human models capture the sequence of events for disease initiation and progression. Here, we generate induced pluripotent stem cells (iPSCs) from healthy individuals and young ovarian cancer patients with germline pathogenic BRCA1 mutations (BRCA1mut). Following differentiation into FTE organoids, BRCA1mut lines exhibit cellular abnormalities consistent with neoplastic transformation compared to controls. BRCA1mut organoids show an increased production of cancer-specific proteins and survival following transplantation into mice. Organoids from women with the most aggressive ovarian cancer show the greatest pathology, indicating the potential value to predict clinical severity prior to disease onset. These human FTE organoids from BRCA1mut carriers provide a faithful physiological in vitro model of FTE lesion generation and early carcinogenesis. This platform can be used for personalized mechanistic and drug screening studies.

Microglial transcription profiles in mouse and human are driven by APOE4 and sex.

Apolipoprotein E4 (APOE4) is the strongest genetic risk factor for sporadic Alzheimer's disease (AD). APOE4 is known to affect the function of microglia, but to what extent this gene drives microglial gene expression has thus far not been examined. Using a transgenic mouse model of AD that expresses human APOE, we identify a unique transcriptional profile associated with APOE4 expression. We also show a sex and APOE interaction, such that both female sex and APOE4 drive expression of this gene profile. We confirm these findings in human cells, using microglia derived from induced pluripotent stem cells (iMGL). Moreover, we find that these interactions are driven in part by genes related to metal processing, and we show that zinc treatment has APOE genotype-dependent effects on iMGL. These data identify a sex- and APOE4-associated microglial transcription profile and highlight the importance of considering interactive risk factors such as sex and environmental exposures.

High larvicidal efficacy of yeast-encapsulated orange oil against Aedes aegypti strains from Brazil.

BACKGROUND: Botanical substances such as essential oils (EOs) have demonstrated insecticidal properties and are a valid option for vector control. However, free EOs are unreliable as mosquito larvicides due their easy degradation by environmental exposure to ultraviolet light and higher temperatures. Here, we assessed the efficacy of a mosquito larvicide based on orange oil in a yeast-based delivery system against Aedes aegypti strains with different resistance status towards chemical neurotoxic insecticides. This larvicide preparation was physicochemically characterized in a previous report. METHODS: Larvae of four Ae. aegypti strains from different regions of Brazil and different resistance profiles for deltamethrin (pyrethroid) and temephos (organophosphate) were tested against yeast-encapsulated orange oil (YEOO) in laboratory conditions for measurement of LC50 and LC90 values. The same assays were performed with the Belo Horizonte strain under environmental conditions (natural light and temperature). The resistance profiles of these strains were compared to the Rockefeller reference strain in all conditions. RESULTS: YEOO was found to be a highly active larvicide (LC50 < 50 mg/L) against all Ae. aegypti strains tested in both laboratory conditions (LC50 = 8.1-24.7 mg/L) and environmental conditions with natural light and temperature fluctuation (LC50 = 20.0-49.9 mg/L). Moreover, all strains were considered susceptible (RR < 5) to YEOO, considering resistance ratios calculated based on the Rockefeller strain. The resistance ratios were only higher than 2.5 for LC90-95 of Belo Horizonte in the laboratory, probably due the higher heterogeneity associated with older egg papers (> 5 months). CONCLUSION: YEOO demonstrates high larvicidal activity against Ae. aegypti strains with resistant phenotypes for deltamethrin (PY) and temephos (OP). This larvicidal activity suggests the potential for the development of YEOO as an alternative intervention to synthetic insecticides in integrated vector management programs, for populations with resistance to commonly used insecticides.

Cross-Comparison of Human iPSC Motor Neuron Models of Familial and Sporadic ALS Reveals Early and Convergent Transcriptomic Disease Signatures.

Induced pluripotent stem cell (iPSC)-derived neural cultures from amyotrophic lateral sclerosis (ALS) patients can model disease phenotypes. However, heterogeneity arising from genetic and experimental variability limits their utility, impacting reproducibility and the ability to track cellular origins of pathogenesis. Here, we present methodologies using single-cell RNA sequencing (scRNA-seq) analysis to address these limitations. By repeatedly differentiating and applying scRNA-seq to motor neurons (MNs) from healthy, familial ALS, sporadic ALS, and genome-edited iPSC lines across multiple patients, batches, and platforms, we account for genetic and experimental variability toward identifying unified and reproducible ALS signatures. Combining HOX and developmental gene expression with global clustering, we anatomically classified cells into rostrocaudal, progenitor, and postmitotic identities. By relaxing statistical thresholds, we discovered genes in iPSC-MNs that were concordantly dysregulated in postmortem MNs and yielded predictive ALS markers in other human and mouse models. Our approach thus revealed early, convergent, and MN-resolved signatures of ALS.

Recent advances in human iPSC-derived models of the blood-brain barrier.

The blood-brain barrier (BBB) is a critical component of the central nervous system that protects neurons and other cells of the brain parenchyma from potentially harmful substances found in peripheral circulation. Gaining a thorough understanding of the development and function of the human BBB has been hindered by a lack of relevant models given significant species differences and limited access to in vivo tissue. However, advances in induced pluripotent stem cell (iPSC) and organ-chip technologies now allow us to improve our knowledge of the human BBB in both health and disease. This review focuses on the recent progress in modeling the BBB in vitro using human iPSCs.

Multi-lineage Human iPSC-Derived Platforms for Disease Modeling and Drug Discovery.

Human induced pluripotent stem cells (hiPSCs) provide a powerful platform for disease modeling and have unlocked new possibilities for understanding the mechanisms governing human biology, physiology, and genetics. However, hiPSC-derivatives have traditionally been utilized in two-dimensional monocultures, in contrast to the multi-systemic interactions that influence cells in the body. We will discuss recent advances in generating more complex hiPSC-based systems using three-dimensional organoids, tissue-engineering, microfluidic organ-chips, and humanized animal systems. While hiPSC differentiation still requires optimization, these next-generation multi-lineage technologies can augment the biomedical researcher's toolkit and enable more realistic models of human tissue function.

Generation of a Human iPSC-Based Blood-Brain Barrier Chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB's role in health and disease. We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

Yeast-encapsulated essential oils: a new perspective as an environmentally friendly larvicide.

BACKGROUND: Effective mosquito control approaches incorporate both adult and larval stages. For the latter, physical, biological, and chemical control have been used with varying results. Successful control of larvae has been demonstrated using larvicides including insect growth regulators, e.g. the organophosphate temephos, as well as various entomopathogenic microbial species. However, a variety of health and environmental issues are associated with some of these. Laboratory trials of essential oils (EO) have established the larvicidal activity of these substances, but there are currently no commercially available EO-based larvicides. Here we report on the development of a new approach to mosquito larval control using a novel, yeast-based delivery system for EO. METHODS: Food-grade orange oil (OO) was encapsulated into yeast cells following an established protocol. To prevent environmental contamination, a proprietary washing strategy was developed to remove excess EO that is adsorbed to the cell exterior during the encapsulation process. The OO-loaded yeast particles were then characterized for OO loading, and tested for efficacy against Aedes aegypti larvae. RESULTS: The composition of encapsulated OO extracted from the yeast microparticles was demonstrated not to differ from that of un-encapsulated EO when analyzed by high performance liquid chromatography. After lyophilization, the oil in the larvicide comprised 26-30 percentage weight (wt%), and is consistent with the 60-65% reduction in weight observed after the drying process. Quantitative bioassays carried with Liverpool and Rockefeller Ae. aegypti strains in three different laboratories presented LD50 of 5.1 (95% CI: 4.6-5.6) to 27.6 (95% CI: 26.4-28.8) mg/l, for L1 and L3/L4 mosquito larvae, respectively. LD90 ranged between 18.9 (95% CI: 16.4-21.7) mg/l (L1 larvae) to 76.7 (95% CI: 69.7-84.3) mg/l (L3/L4 larvae). CONCLUSIONS: The larvicide based on OO encapsulated in yeast was shown to be highly active (LD50 < 50 mg/l) against all larval stages of Ae. aegypti. These results demonstrate its potential for incorporation in an integrated approach to larval source management of Ae. aegypti. This novel approach can enable development of affordable control strategies that may have significant impact on global health.

Modeling Intestinal Epithelial Response to Interferon-γ in Induced Pluripotent Stem Cell-Derived Human Intestinal Organoids.

Human intestinal organoids (HIOs) are increasingly being used to model intestinal responses to various stimuli, yet few studies have confirmed the fidelity of this modeling system. Given that the interferon-gamma (IFN-γ) response has been well characterized in various other cell types, our goal was to characterize the response to IFN-γ in HIOs derived from induced pluripotent stem cells (iPSCs). To achieve this, iPSCs were directed to form HIOs and subsequently treated with IFN-γ. Our results demonstrate that IFN-γ phosphorylates STAT1 but has little effect on the expression or localization of tight and adherens junction proteins in HIOs. However, transcriptomic profiling by microarray revealed numerous upregulated genes such as IDO1, GBP1, CXCL9, CXCL10 and CXCL11, which have previously been shown to be upregulated in other cell types in response to IFN-γ. Notably, "Response to Interferon Gamma" was determined to be one of the most significantly upregulated gene sets in IFN-γ-treated HIOs using gene set enrichment analysis. Interestingly, similar genes and pathways were upregulated in publicly available datasets contrasting the gene expression of in vivo biopsy tissue from patients with IBD against healthy controls. These data confirm that the iPSC-derived HIO modeling system represents an appropriate platform to evaluate the effects of various stimuli and specific environmental factors responsible for the alterations in the intestinal epithelium seen in various gastrointestinal conditions such as inflammatory bowel disease.

Human iPSC-Derived Blood-Brain Barrier Chips Enable Disease Modeling and Personalized Medicine Applications.

The blood-brain barrier (BBB) tightly regulates the entry of solutes from blood into the brain and is disrupted in several neurological diseases. Using Organ-Chip technology, we created an entirely human BBB-Chip with induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (iBMECs), astrocytes, and neurons. The iBMECs formed a tight monolayer that expressed markers specific to brain vasculature. The BBB-Chip exhibited physiologically relevant transendothelial electrical resistance and accurately predicted blood-to-brain permeability of pharmacologics. Upon perfusing the vascular lumen with whole blood, the microengineered capillary wall protected neural cells from plasma-induced toxicity. Patient-derived iPSCs from individuals with neurological diseases predicted disease-specific lack of transporters and disruption of barrier integrity. By combining Organ-Chip technology and human iPSC-derived tissue, we have created a neurovascular unit that recapitulates complex BBB functions, provides a platform for modeling inheritable neurological disorders, and advances drug screening, as well as personalized medicine.

Enhanced Utilization of Induced Pluripotent Stem Cell-Derived Human Intestinal Organoids Using Microengineered Chips.

BACKGROUND AND AIMS: Human intestinal organoids derived from induced pluripotent stem cells have tremendous potential to elucidate the intestinal epithelium's role in health and disease, but it is difficult to directly assay these complex structures. This study sought to make this technology more amenable for study by obtaining epithelial cells from induced pluripotent stem cell-derived human intestinal organoids and incorporating them into small microengineered Chips. We then investigated if these cells within the Chip were polarized, had the 4 major intestinal epithelial subtypes, and were biologically responsive to exogenous stimuli. METHODS: Epithelial cells were positively selected from human intestinal organoids and were incorporated into the Chip. The effect of continuous media flow was examined. Immunocytochemistry and in situ hybridization were used to demonstrate that the epithelial cells were polarized and possessed the major intestinal epithelial subtypes. To assess if the incorporated cells were biologically responsive, Western blot analysis and quantitative polymerase chain reaction were used to assess the effects of interferon (IFN)-γ, and fluorescein isothiocyanate-dextran 4 kDa permeation was used to assess the effects of IFN-γ and tumor necrosis factor-α on barrier function. RESULTS: The optimal cell seeding density and flow rate were established. The continuous administration of flow resulted in the formation of polarized intestinal folds that contained Paneth cells, goblet cells, enterocytes, and enteroendocrine cells along with transit-amplifying and LGR5+ stem cells. Administration of IFN-γ for 1 hour resulted in the phosphorylation of STAT1, whereas exposure for 3 days resulted in a significant upregulation of IFN-γ related genes. Administration of IFN-γ and tumor necrosis factor-α for 3 days resulted in an increase in intestinal permeability. CONCLUSIONS: We demonstrate that the Intestine-Chip is polarized, contains all the intestinal epithelial subtypes, and is biologically responsive to exogenous stimuli. This represents a more amenable platform to use organoid technology and will be highly applicable to personalized medicine and a wide range of gastrointestinal conditions.

Integration of Platinum Group Metal-Free Catalysts and Bilirubin Oxidase into a Hybrid Material for Oxygen Reduction: Interplay of Chemistry and Morphology.

Catalytic activity toward the oxygen reduction reaction (ORR) of platinum group metal-free (PGM-free) electrocatalysts integrated with an enzyme (bilirubin oxidase, BOx) in neutral media was studied. The effects of chemical and morphological characteristics of PGM-free materials on the enzyme enhancement of the overall ORR kinetics was investigated. The surface chemistry of the PGM-free catalyst was studied using X-ray Photoelectron Spectroscopy. Catalyst surface morphology was characterized using two independent methods: length-scale specific image analysis and nitrogen adsorption. Good agreement of macroscopic and microscopic morphological properties was found. Enhancement of ORR activity by the enzyme is influenced by chemistry and surface morphology of the catalyst itself. Catalysts with a higher nitrogen content, specifically pyridinic moieties, showed the greatest enhancement. Furthermore, catalysts with a higher fraction of surface roughness in the range of 3-5 nm exhibited greater performance enhancement than catalysts lacking features of this size.

Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

Subependymal giant cell astrocytoma in a genetically negative tuberous sclerosis complex adult: Case report.

INTRODUCTION: The well-described entity of Subependymal Giant Cell Astrocytoma (SEGA) in the setting of Tuberous Sclerosis Complex (TSC) is profound in current literature. It has been described in children as well as adults with or without identifiable clinical presentations of tuberous sclerosis. To our knowledge there has not been any report of a negative genetic workup of Tuberous Sclerosis Complex in an adult patient presenting with an isolated SEGA. CASE REPORT: We present a case of a 25-year-old female with no medical history who presented to the emergency room for headaches. Further workup included gadolinium enhanced MRI of the brain which revealed a homogenously enhancing mass in the left lateral ventricle with eccentric calcification and resultant obstructive hydrocephalus. A left frontal craniotomy with an interhemispheric transcallosal approach was taken for complete removal of the mass. DISCUSSION: Final pathological diagnosis was SEGA with suggestive cell population, positive GFAP and positive synaptophysin. Genetic testing included TSC1 (MLPA, DNA Sequencing) and TSC2 (MLPA, DNA Sequencing), which were all negative. The panel did not identify mutations associated with Tuberous Sclerosis. CONCLUSION: Rare cases of isolated SEGA have been reported in patients who do not have typical features of tuberous sclerosis, and may represent minimal penetrance of the disease with an attenuated phenotype. Negative genetic testing, as demonstrated, can be seen in adults with isolated SEGA. With a negative genetic workup of TSC, regular follow up may still be necessary; however this may prove to be low yield for identifying any TSC features in the future.

Application of the Discrete Wavelet Transform to SEM and AFM Micrographs for Quantitative Analysis of Complex Surfaces.

The discrete wavelet transform (DWT) has found significant utility in process monitoring, filtering, and feature isolation of SEM, AFM, and optical images. Current use of the DWT for surface analysis assumes initial knowledge of the sizes of the features of interest in order to effectively isolate and analyze surface components. Current methods do not adequately address complex, heterogeneous surfaces in which features across multiple size ranges are of interest. Further, in situations where structure-to-property relationships are desired, the identification of features relevant for the function of the material is necessary. In this work, the DWT is examined as a tool for quantitative, length-scale specific surface metrology without prior knowledge of relevant features or length-scales. A new method is explored for determination of the best wavelet basis to minimize variation in roughness and skewness measurements with respect to change in position and orientation of surface features. It is observed that the size of the wavelet does not directly correlate with the size of features on the surface, and a method to measure the true length-scale specific roughness of the surface is presented. This method is applied to SEM and AFM images of non-precious metal catalysts, yielding new length-scale specific structure-to-property relationships for chemical speciation and fuel cell performance. The relationship between SEM and AFM length-scale specific roughness is also explored. Evidence is presented that roughness distributions of SEM images, as measured by the DWT, is representative of the true surface roughness distribution obtained from AFM.

Surgical treatment of a lenticulostriate artery aneurysm. Case report.

Aneurysms arising from a lenticulostriate artery (LSA) are uncommon. Their causes include hypertension, moyamoya disease, infection, systemic lupus erythematosis, and flow-related saccular aneurysms. Options for treating these aneurysms are limited. The authors present a case in which an LSA aneurysm was identified in a 69-year-old woman with no significant medical history, who experienced a sudden onset of right hemiparesis and aphasia due to a basal ganglia hemorrhage. The different causes and treatment options available for these rare and difficult-to-treat aneurysms are discussed.

Adjunctive endovascular techniques in the management of postoperative carotid artery pseudoaneurysms--useful armamentarium for vascular surgeons--three case reports.

Postoperative carotid artery pseudoaneurysms are rare. The traditional treatment of choice has been operative repair, which can present a significant technical challenge owing to the reoperative neck inflammation and potential cranial nerve injuries. The authors report 3 cases of postoperative carotid pseudoaneurysms that were successfully managed by use of various adjunctive endovascular techniques. The adjunctive endovascular maneuvers included the following: 1) endoluminal balloon placement for preoperative test occlusion and intraoperative proximal control to facilitate operative dissection in the first patient with a carotid pseudoaneurysm; 2) endoluminal stent-graft placement to exclude a large expanding carotid pseudoaneurysm in the second patient; and 3) endoluminal coil placement along with balloon occlusion to achieve complete hemostasis in the third patient, who presented with a hemorrhaging carotid pseudoaneurysm. Successful outcomes were achieved in all 3 patients by use of adjunctive endovascular techniques. These cases underscore the role of adjunctive endovascular treatment as an armamentarium for vascular surgeons in the treatment of complex carotid pseudoaneurysms.