Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues.

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and skeletal muscle diseases, contractile force and kinetics constitute key metrics for assessing muscle function. New and improved methods for generating engineered muscle tissues (EMTs) from induced pluripotent stem cells have made in vitro disease modeling more reliable for contractile tissues; however, reproducibly fabricating tissues from suspended cell cultures and measuring their contractility is challenging. Such techniques are often plagued with high failure rates and require complex instrumentation and customized data analysis routines. A new platform and device that utilizes 3D EMTs in conjunction with a label-free, highly-parallel, and automation-friendly contractility assay circumvent many of these obstacles. The platform enables facile and reproducible fabrication of 3D EMTs using virtually any cell source. Tissue contractility is then measured via an instrument that simultaneously measures 24 tissues without the need for complex software analysis routines. The instrument can reliably measure micronewton changes in force, allowing for dose-dependent compound screening to measure the effect of a drug or therapeutic on contractile output. Engineered tissues made with this device are fully functional, generating twitch and tetanic contractions upon electrical stimulation, and can be analyzed longitudinally in culture over weeks or months. Here, we show data from cardiac muscle EMTs under acute and chronic dosing with known toxicants, including a drug (BMS-986094) that was pulled from clinical trials after patient fatalities due to unanticipated cardiotoxicity. Altered skeletal muscle function in engineered tissues in response to treatment with a myosin inhibitor is also presented. This platform enables the researcher to integrate complex, information-rich bioengineered model systems into their drug discovery workflow with minimal additional training or skills required.

High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing.

Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic.

Knock-Down of HDAC2 in Human Induced Pluripotent Stem Cell Derived Neurons Improves Neuronal Mitochondrial Dynamics, Neuronal Maturation and Reduces Amyloid Beta Peptides.

Histone deacetylase 2 (HDAC2) is a major HDAC protein in the adult brain and has been shown to regulate many neuronal genes. The aberrant expression of HDAC2 and subsequent dysregulation of neuronal gene expression is implicated in neurodegeneration and brain aging. Human induced pluripotent stem cell-derived neurons (hiPSC-Ns) are widely used models for studying neurodegenerative disease mechanisms, but the role of HDAC2 in hiPSC-N differentiation and maturation has not been explored. In this study, we show that levels of HDAC2 progressively decrease as hiPSCs are differentiated towards neurons. This suppression of HDAC2 inversely corresponds to an increase in neuron-specific isoforms of Endophilin-B1, a multifunctional protein involved in mitochondrial dynamics. Expression of neuron-specific isoforms of Endophilin-B1 is accompanied by concomitant expression of a neuron-specific alternative splicing factor, SRRM4. Manipulation of HDAC2 and Endophilin-B1 using lentiviral approaches shows that the knock-down of HDAC2 or the overexpression of a neuron-specific Endophilin-B1 isoform promotes mitochondrial elongation and protects against cytotoxic stress in hiPSC-Ns, while HDAC2 knock-down specifically influences genes regulating mitochondrial dynamics and synaptogenesis. Furthermore, HDAC2 knock-down promotes enhanced mitochondrial respiration and reduces levels of neurotoxic amyloid beta peptides. Collectively, our study demonstrates a role for HDAC2 in hiPSC-neuronal differentiation, highlights neuron-specific isoforms of Endophilin-B1 as a marker of differentiating hiPSC-Ns and demonstrates that HDAC2 regulates key neuronal and mitochondrial pathways in hiPSC-Ns.

Advances and Current Challenges Associated with the Use of Human Induced Pluripotent Stem Cells in Modeling Neurodegenerative Disease.

One of the most profound advances in the last decade of biomedical research has been the development of human induced pluripotent stem cell (hiPSC) models for identification of disease mechanisms and drug discovery. Human iPSC technology has the capacity to revolutionize healthcare and the realization of personalized medicine, but differentiated tissues derived from stem cells come with major criticisms compared to native tissue, including variability in genetic backgrounds, a lack of functional maturity, and differences in epigenetic profiles. It is widely believed that increasing complexity will lead to improved clinical relevance, so methods are being developed that go from a single cell type to various levels of 2-D coculturing and 3-D organoids. As this inevitable trend continues, it will be essential to thoroughly understand the strengths and weaknesses of more complex models and to develop criteria for assessing biological relevance. We believe the payoff of robust, high-throughput, clinically meaningful human stem cell models could be the elimination of often inadequate animal models. To facilitate this transition, we will look at the challenges and strategies of complex model development through the lens of neurodegeneration to encapsulate where the disease-in-a-dish field currently is and where it needs to go to improve.

Physiological Aß Concentrations Produce a More Biomimetic Representation of the Alzheimer's Disease Phenotype in iPSC Derived Human Neurons.

Alzheimer's disease (AD) is characterized by slow, progressive neurodegeneration leading to severe neurological impairment, but current drug development efforts are limited by the lack of robust, human-based disease models. Amyloid-ß (Aß) is known to play an integral role in AD progression as it has been shown to interfere with neurological function. However, studies into AD pathology commonly apply Aß to neurons for short durations at nonphysiological concentrations to induce an exaggerated dysfunctional phenotype. Such methods are unlikely to elucidate early stage disease dysfunction, when treatment is still possible, since damage to neurons by these high concentrations is extensive. In this study, we investigated chronic, pathologically relevant Aß oligomer concentrations to induce an electrophysiological phenotype that is more representative of early AD progression compared to an acute high-dose application in human cortical neurons. The high, acute oligomer dose resulted in severe neuronal toxicity as well as upregulation of tau and phosphorylated tau. Chronic, low-dose treatment produced significant functional impairment without increased cell death or accumulation of tau protein. This in vitro phenotype more closely mirrors the status of early stage neural decline in AD pathology and could provide a valuable tool to further understanding of early stage AD pathophysiology and for screening potential therapeutic compounds.

Leptomeninges-Derived Induced Pluripotent Stem Cells and Directly Converted Neurons From Autopsy Cases With Varying Neuropathologic Backgrounds.

Patient-specific stem cell technology from skin and other biopsy sources has transformed in vitro models of neurodegenerative disease, permitting interrogation of the effects of complex human genetics on neurotoxicity. However, the neuropathologic changes that underlie cognitive and behavioral phenotypes can only be determined at autopsy. To better correlate the biology of derived neurons with age-related and neurodegenerative changes, we generated leptomeningeal cell lines from well-characterized research subjects that have undergone comprehensive postmortem neuropathologic examinations. In a series of proof of principle experiments, we reprogrammed autopsy leptomeningeal cell lines to human-induced pluripotent stem cells (hiPSCs) and differentiated these into neurons. We show that leptomeningeal-derived hiPSC lines can be generated from fresh and frozen leptomeninges, are pluripotent, and retain the karyotype of the starting cell population. Additionally, neurons differentiated from these hiPSCs are functional and produce measurable Alzheimer disease-relevant analytes (Aß and Tau). Finally, we used direct conversion protocols to transdifferentiate leptomeningeal cells to neurons. These resources allow the generation of in vitro models to test mechanistic hypotheses as well as diagnostic and therapeutic strategies in association with neuropathology, clinical and cognitive data, and biomarker studies, aiding in the study of late-onset Alzheimer disease and other age-related neurodegenerative diseases.

Morphological and functional characterization of human induced pluripotent stem cell-derived neurons (iCell Neurons) in defined culture systems.

Pre-clinical testing of drug candidates in animal models is expensive, time-consuming, and often fails to predict drug effects in humans. Industry and academia alike are working to build human-based in vitro test beds and advanced high throughput screening systems to improve the translation of preclinical results to human drug trials. Human neurons derived from induced pluripotent stems cells (hiPSCs) are readily available for use within these test-beds and high throughput screens, but there remains a need to robustly evaluate cellular behavior prior to their incorporation in such systems. This study reports on the characterization of one source of commercially available hiPSC-derived neurons, iCell(®) Neurons, for their long-term viability and functional performance to assess their suitability for integration within advanced in vitro platforms. The purity, morphology, survival, identity, and functional maturation of the cells utilizing different culture substrates and medium combinations were evaluated over 28 days in vitro (DIV). Patch-clamp electrophysiological data demonstrated increased capacity for repetitive firing of action potentials across all culture conditions. Significant differences in cellular maturity, morphology, and functional performance were observed in the different conditions, highlighting the importance of evaluating different surface types and growth medium compositions for application in specific in vitro protocols.

Growth of Serratia liquefaciens under 7 mbar, 0°C, and CO2-enriched anoxic atmospheres.

Twenty-six strains of 22 bacterial species were tested for growth on trypticase soy agar (TSA) or sea-salt agar (SSA) under hypobaric, psychrophilic, and anoxic conditions applied singly or in combination. As each factor was added to multi-parameter assays, the interactive stresses decreased the numbers of strains capable of growth and, in general, reduced the vigor of the strains observed to grow. Only Serratia liquefaciens strain ATCC 27592 exhibited growth at 7 mbar, 0°C, and CO2-enriched anoxic atmospheres. To discriminate between the effects of desiccation and hypobaria, vegetative cells of Bacillus subtilis strain 168 and Escherichia coli strain K12 were grown on TSA surfaces and simultaneously in liquid Luria-Bertani (LB) broth media. Inhibition of growth under hypobaria for 168 and K12 decreased in similar ways for both TSA and LB assays as pressures were reduced from 100 to 25 mbar. Results for 168 and K12 on TSA and LB are interpreted to indicate a direct low-pressure effect on microbial growth with both species and do not support the hypothesis that desiccation alone on TSA was the cause of reduced growth at low pressures. The growth of S. liquefaciens at 7 mbar, 0°C, and CO2-enriched anoxic atmospheres was surprising since S. liquefaciens is ecologically a generalist that occurs in terrestrial plant, fish, animal, and food niches. In contrast, two extremophiles tested in the assays, Deinococcus radiodurans strain R1 and Psychrobacter cryohalolentis strain K5, failed to grow under hypobaric (25 mbar; R1 only), psychrophilic (0°C; R1 only), or anoxic (< 0.1% ppO2; both species) conditions.

Microphysiological systems and low-cost microfluidic platform with analytics.

A multiorgan, functional, human in vitro assay system or 'Body-on-a-Chip' would be of tremendous benefit to the drug discovery and toxicology industries, as well as providing a more biologically accurate model for the study of disease as well as applied and basic biological research. Here, we describe the advances our team has made towards this goal, as well as the most pertinent issues facing further development of these systems. Description is given of individual organ models with appropriate cellular functionality, and our efforts to produce human iterations of each using primary and stem cell sources for eventual incorporation into this system. Advancement of the 'Body-on-a-Chip' field is predicated on the availability of abundant sources of human cells, capable of full differentiation and maturation to adult phenotypes, for which researchers are largely dependent on stem cells. Although this level of maturation is not yet achievable in all cell types, the work of our group highlights the high level of functionality that can be achieved using current technology, for a wide variety of cell types. As availability of functional human cell types for in vitro culture increases, the potential to produce a multiorgan in vitro system capable of accurately reproducing acute and chronic human responses to chemical and pathological challenge in real time will also increase.

Effects of simulated Mars conditions on the survival and growth of Escherichia coli and Serratia liquefaciens.

Escherichia coli and Serratia liquefaciens, two bacterial spacecraft contaminants known to replicate under low atmospheric pressures of 2.5 kPa, were tested for growth and survival under simulated Mars conditions. Environmental stresses of high salinity, low temperature, and low pressure were screened alone and in combination for effects on bacterial survival and replication, and then cells were tested in Mars analog soils under simulated Mars conditions. Survival and replication of E. coli and S. liquefaciens cells in liquid medium were evaluated for 7 days under low temperatures (5, 10, 20, or 30 degrees C) with increasing concentrations (0, 5, 10, or 20%) of three salts (MgCl(2), MgSO(4), NaCl) reported to be present on the surface of Mars. Moderate to high growth rates were observed for E. coli and S. liquefaciens at 30 or 20 degrees C and in solutions with 0 or 5% salts. In contrast, cell densities of both species generally did not increase above initial inoculum levels under the highest salt concentrations (10 and 20%) and the four temperatures tested, with the exception that moderately higher cell densities were observed for both species at 10% MgSO(4) maintained at 20 or 30 degrees C. Growth rates of E. coli and S. liquefaciens in low salt concentrations were robust under all pressures (2.5, 10, or 101.3 kPa), exhibiting a general increase of up to 2.5 orders of magnitude above the initial inoculum levels of the assays. Vegetative E. coli cells were maintained in a Mars analog soil for 7 days under simulated Mars conditions that included temperatures between 20 and -50 degrees C for a day/night diurnal period, UVC irradiation (200 to 280 nm) at 3.6 W m(-2) for daytime operations (8 h), pressures held at a constant 0.71 kPa, and a gas composition that included the top five gases found in the martian atmosphere. Cell densities of E. coli failed to increase under simulated Mars conditions, and survival was reduced 1 to 2 orders of magnitude by the interactive effects of desiccation, UV irradiation, high salinity, and low pressure (in decreasing order of importance). Results suggest that E. coli may be able to survive, but not grow, in surficial soils on Mars.