Chronic autoimmune demyelinating neuropathies are a group of rare neuromuscular disorders with complex, poorly characterized etiology. Here the authors describe a phenotypic, human-on-a-chip (HoaC) electrical conduction model of two rare autoimmune demyelinating neuropathies, chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy (MMN), and explore the efficacy of TNT005, a monoclonal antibody inhibitor of the classical complement pathway. Patient sera is shown to contain anti-GM1 IgM and IgG antibodies capable of binding to human primary Schwann cells and induced pluripotent stem cell-derived motoneurons (MNs). Patient autoantibody binding is sufficient to activate the classical complement pathway, resulting in detection of C3b and C5b-9. A HoaC model, using a microelectrode array with directed axonal outgrowth over the electrodes treated with patient sera, exhibits reductions in MN action potential frequency and conduction velocity. TNT005 rescued the serum-induced complement deposition and functional deficits while treatment with an isotype control antibody has no rescue effect. These data indicate that complement activation by CIDP and MMN patient serum is sufficient to mimic neurophysiological features of each disease and that complement inhibition with TNT005 is sufficient to rescue these pathological effects and provide efficacy data included in an investigational new drug application, demonstrating the model's translational potential.

Retinal in bacteriorhodopsin was replaced by several analogs. Changes in absorption and Raman spectra and in the capacity to function as a proton pump after reconstitution into liposomes, were recorded. We conclude from these studies that considerable alterations in chemical structure, particularly in the ring of retinal, can be tolerated with retention of some functional activity.

There are many neurological rare diseases where animal models have proven inadequate or do not currently exist. NGLY1 Deficiency, a congenital disorder of deglycosylation, is a rare disease that predominantly affects motor control, especially control of neuromuscular action. In this study, NGLY1-deficient, patient-derived induced pluripotent stem cells (iPSCs) were differentiated into motoneurons (MNs) to identify disease phenotypes analogous to clinical disease pathology with significant deficits apparent in the NGLY1-deficient lines compared to the control. A neuromuscular junction (NMJ) model was developed using patient and wild type (WT) MNs to study functional differences between healthy and diseased NMJs. Reduced axon length, increased and shortened axon branches, MN action potential (AP) bursting and decreased AP firing rate and amplitude were observed in the NGLY1-deficient MNs in monoculture. When transitioned to the NMJ-coculture system, deficits in NMJ number, stability, failure rate, and synchronicity with indirect skeletal muscle (SkM) stimulation were observed. This project establishes a phenotypic NGLY1 model for investigation of possible therapeutics and investigations into mechanistic deficits in the system.

The maturation and functional characteristics of human induced pluripotent stem cell (hiPSC)-cortical neurons has not been fully documented. This study developed a phenotypic model of hiPSC-derived cortical neurons, characterized their maturation process, and investigated its application for disease modeling with the integration of multi-electrode array (MEA) technology. Immunocytochemistry analysis indicated early-stage neurons (day 21) were simultaneously positive for both excitatory (vesicular glutamate transporter 1 [VGlut1]) and inhibitory (GABA) markers, while late-stage cultures (day 40) expressed solely VGlut1, indicating a purely excitatory phenotype without containing glial cells. This maturation process was further validated utilizing patch clamp and MEA analysis. Particularly, induced long-term potentiation (LTP) successfully persisted for 1 h in day 40 cultures, but only achieved LTP in the presence of the GABAA receptor antagonist picrotoxin in day 21 cultures. This system was also applied to epilepsy modeling utilizing bicuculline and its correction utilizing the anti-epileptic drug valproic acid.

Myasthenia gravis (MG) is a chronic and progressive neuromuscular disease where autoantibodies target essential proteins such as the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction (NMJ) causing muscle fatigue and weakness. Autoantibodies directed against nAChRs are proposed to work by three main pathological mechanisms of receptor disruption: blocking, receptor internalization, and downregulation. Current in vivo models using experimental autoimmune animal models fail to recapitulate the disease pathology and are limited in clinical translatability due to disproportionate disease severity and high animal death rates. The development of a highly sensitive antibody assay that mimics human disease pathology is desirable for clinical advancement and therapeutic development. To address this lack of relevant models, an NMJ platform derived from human iPSC differentiated motoneurons and primary skeletal muscle was used to investigate the ability of an anti-nAChR antibody to induce clinically relevant MG pathology in the serum-free, spatially organized, functionally mature NMJ platform. Treatment of the NMJ model with the anti-nAChR antibody revealed decreasing NMJ stability as measured by the number of NMJs before and after the synchrony stimulation protocol. This decrease in NMJ stability was dose-dependent over a concentration range of 0.01–20 μg/mL. Immunocytochemical (ICC) analysis was used to distinguish between pathological mechanisms of antibody-mediated receptor disruption including blocking, receptor internalization and downregulation. Antibody treatment also activated the complement cascade as indicated by complement protein 3 deposition near the nAChRs. Additionally, complement cascade activation significantly altered other readouts of NMJ function including the NMJ fidelity parameter as measured by the number of muscle contractions missed in response to increasing motoneuron stimulation frequencies. This synchrony readout mimics the clinical phenotype of neurological blocking that results in failure of muscle contractions despite motoneuron stimulations. Taken together, these data indicate the establishment of a relevant disease model of MG that mimics reduction of functional nAChRs at the NMJ, decreased NMJ stability, complement activation and blocking of neuromuscular transmission. This system is the first functional human in vitro model of MG to be used to simulate three potential disease mechanisms as well as to establish a preclinical platform for evaluation of disease modifying treatments (etiology).

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor's contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.

Recent findings suggest a pathologic role of skeletal muscle in amyotrophic lateral sclerosis (ALS) onset and progression. However, the exact mechanism by which this occurs remains elusive due to limited human-based studies. To this end, phenotypic ALS skeletal muscle models were developed from induced pluripotent stem cells (iPSCs) derived from healthy individuals (WT) and ALS patients harboring mutations in the superoxide dismutase 1 (SOD1) gene. Although proliferative, SOD1 myoblasts demonstrated delayed and reduced fusion efficiency compared to WT. Additionally, SOD1 myotubes exhibited significantly reduced length and cross-section. Also, SOD1 myotubes had loosely arranged myosin heavy chain and reduced acetylcholine receptor expression per immunocytochemical analysis. Functional analysis indicated considerably reduced contractile force and synchrony in SOD1 myotubes. Mitochondrial assessment indicated reduced inner mitochondrial membrane potential (ΔΨm) and metabolic plasticity in the SOD1-iPSC derived myotubes. This work presents the first well-characterized in vitro iPSC-derived muscle model that demonstrates SOD1 toxicity effects on human muscle regeneration, contractility and metabolic function in ALS. Current findings align with previous ALS patient biopsy studies and suggest an active contribution of skeletal muscle in NMJ dysfunction. Further, the results validate this model as a human-relevant platform for ALS research and drug discovery studies.

Abstract Introduction The quest to identify an effective therapeutic strategy for neurodegenerative diseases, such as mild congitive impairment (MCI) and Alzheimer's disease (AD), suffers from the lack of good human‐based models. Animals represent the most common models used in basic research and drug discovery studies. However, safe and effective compounds identified in animal studies often translate poorly to humans, yielding unsuccessful clinical trials. Methods A functional in vitro assay based on long‐term potentiation (LTP) was used to demonstrate that exposure to amyloid beta (Aβ42) and tau oligomers, or brain extracts from AD transgenic mice led to prominent changes in human induced pluripotent stem cells (hiPSC)‐derived cortical neurons, notably, without cell death. Results Impaired information processing was demonstrated by treatment of neuron‐MEA (microelectrode array) systems with the oligomers and brain extracts by reducing the effects of LTP induction. These data confirm the neurotoxicity of molecules linked to AD pathology and indicate the utility of this human‐based system to model aspects of AD in vitro and study LTP deficits without loss of viability; a phenotype that more closely models the preclinical or early stage of AD. Discussion In this study, by combining multiple relevant and important molecular and technical aspects of neuroscience research, we generated a new, fully human in vitro system to model and study AD at the preclinical stage. This system can serve as a novel drug discovery platform to identify compounds that rescue or alleviate the initial neuronal deficits caused by Aβ42 and/or tau oligomers, a main focus of clinical trials.

Myelination and node of Ranvier formation play an important role in the rapid conduction of nerve impulses, referred to as saltatory conduction, along axons in the peripheral nervous system. We report a human-human myelination model using human primary Schwann cells (SCs) and human-induced pluripotent stem-cell-derived motoneurons utilizing a serum-free medium supplemented with ascorbate to induce myelination, where 41.6% of SCs expressed the master transcription factor for myelination, early growth response protein 2. After 30 days in coculture, myelin segments were visualized using immunocytochemistry for myelin basic protein surrounding neurofilament-stained motor neuron axons, which was confirmed via 3D confocal Raman microscopy, a viable alternative for transmission electron microscopy analysis. The myelination efficiency was 65%, and clusters of voltage-gated sodium channels and the paranodal protein contactin-associated protein 1 indicated node of Ranvier formation. This model has applications to study remyelination and demyelinating diseases, including Charcot-Marie Tooth disorder, Guillian-Barre syndrome, and anti-myelin-associated glycoprotein peripheral neuropathy.

A functional, human, multiorgan, pumpless, immune system-on-a-chip featuring recirculating THP-1 immune cells with cardiomyocytes, skeletal muscle, and liver in separate compartments in a serum-free medium is developed. This in vitro platform can emulate both a targeted immune response to tissue-specific damage, and holistic proinflammatory immune response to proinflammatory compound exposure. The targeted response features fluorescently labeled THP-1 monocytes selectively infiltrating into an amiodarone-damaged cardiac module and changes in contractile force measurements without immune-activated damage to the other organ modules. In contrast to the targeted immune response, general proinflammatory treatment of immune human-on-a-chip systems with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) causes nonselective damage to cells in all three-organ compartments. Biomarker analysis indicates upregulation of the proinflammation cytokines TNF-α, IL-6, IL-10, MIP-1, MCP-1, and RANTES in response to LPS + IFN-γ treatment indicative of the M1 macrophage phenotype, whereas amiodarone treatment only leads to an increase in the restorative cytokine IL-6 which is a marker for the M2 phenotype. This system can be used as an alternative to humanized animal models to determine direct immunological effects of biological therapeutics including monoclonal antibodies, vaccines, and gene therapies, and the indirect effects caused by cytokine release from target tissues in response to a drug's pharmacokinetics (PK)/pharmacodynamics (PD) profile.

A pumpless, reconfigurable, multi-organ-on-a-chip system containing recirculating serum-free medium can be used to predict preclinical on-target efficacy, metabolic conversion, and measurement of off-target toxicity of drugs using functional biological microelectromechanical systems. In the first configuration of the system, primary human hepatocytes were cultured with two cancer-derived human bone marrow cell lines for antileukemia drug analysis in which diclofenac and imatinib demonstrated a cytostatic effect on bone marrow cancer proliferation. Liver viability was not affected by imatinib; however, diclofenac reduced liver viability by 30%. The second configuration housed a multidrug-resistant vulva cancer line, a non-multidrug-resistant breast cancer line, primary hepatocytes, and induced pluripotent stem cell-derived cardiomyocytes. Tamoxifen reduced viability of the breast cancer cells only after metabolite generation but did not affect the vulva cancer cells except when coadministered with verapamil, a permeability glycoprotein inhibitor. Both tamoxifen alone and coadministration with verapamil produced off-target cardiac effects as indicated by a reduction of contractile force, beat frequency, and conduction velocity but did not affect viability. These systems demonstrate the utility of a human cell-based in vitro culture system to evaluate both on-target efficacy and off-target toxicity for parent drugs and their metabolites; these systems can augment and reduce the use of animals and increase the efficiency of drug evaluations in preclinical studies.

Alzheimer's disease (AD) is the prevailing cause of age-associated cognitive decline, affecting more than 50 million people worldwide. Currently, there is no cure and few approved palliative treatment options for AD. These shortcomings can be partly attributed to a greater than 90% failure rate for drugs in clinical trial due to a lack of effective translational models. Therefore, it is imperative to develop more efficient and effective model systems to accelerate the evaluation process of novel therapeutic agents. Here, we combined hiPSCs-derived cortical neurons together with microelectrode arrays (MEAs) to analyze the deleterious effects of amyloid beta (Aβ) and tau oligomers on long-term potentiation (LTP) in vitro, thus establishing an alternative AD model. Using immunocytochemisty, patch-clamp electrophysiology and MEAs recording methods, our study demonstrated that dosing with Aβ1-42 oligomers, tau aggregates, or brain extracts from AD transgenic mice caused substential damage to the cortical neurons. Specifically, we observed a reduction in axon length and a significant deficit in cell physiological functions (p≤.005). Further, treatment with Aβ1-42 oligomers, tau aggregates, or brain extracts impaired increases in spontaneous action potential generation after an LTP induction protocol. These data support the conclusions observed in the literature regarding the deleterious effects of Aβ and tau on neuronal physiology without the invasive aspect of patch-clamp recording. In summary, the results presented here provide strong support for the application of hiPSCs combined with human-on-a-chip system as a relevant model to study AD pathology and neurodegenerative disease (ND) in general, and highlight its potential as a human-based model for drug screening and therapeutic intervention development.

Drug development for rare diseases, classified as diseases with a prevalence of < 200 000 patients, is limited by the high cost of research and low target population. Owing to a lack of representative disease models, research has been challenging for orphan drugs. Human-on-a-chip (HoaC) technology, which models human tissues in interconnected in vitro microfluidic devices, has the potential to lower the cost of preclinical studies and increase the rate of drug approval by introducing human phenotypic models early in the drug discovery process. Advances in HoaC technology can drive a new approach to rare disease research and orphan drug development.

Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and -1β serve as master transcriptional regulators of muscle mitochondrial functional capacity and are capable of enhancing muscle endurance when overexpressed in mice. We sought to determine whether muscle-specific transgenic overexpression of PGC-1β affects the detraining response following endurance training. First, we established and validated a mouse exercise-training-detraining protocol. Second, using multiple physiological and gene expression end points, we found that PGC-1β overexpression in skeletal muscle of sedentary mice fully recapitulated the training response. Lastly, PGC-1β overexpression during the detraining period resulted in partial prevention of the detraining response. Specifically, an increase in the plateau at which O 2 uptake (V̇o 2 ) did not change from baseline with increasing treadmill speed [peak V̇o 2 (ΔV̇o 2max )] was maintained in trained mice with PGC-1β overexpression in muscle 6 wk after cessation of training. However, other detraining responses, including changes in running performance and in situ half relaxation time (a measure of contractility), were not affected by PGC-1β overexpression. We conclude that while activation of muscle PGC-1β is sufficient to drive the complete endurance phenotype in sedentary mice, it only partially prevents the detraining response following exercise training, suggesting that the process of endurance detraining involves mechanisms beyond the reversal of muscle autonomous mechanisms involved in endurance fitness. In addition, the protocol described here should be useful for assessing early-stage proof-of-concept interventions in preclinical models of muscle disuse atrophy.