Modelling the innate immune system in microphysiological systems.

This critical review aims to highlight how modeling of the immune response has adapted over time to utilize microphysiological systems. Topics covered here will discuss the integral components of the immune system in various human body systems, and how these interactions are modeled using these systems. Through the use of microphysiological systems, we have not only expanded on foundations of basic immune cell information, but have also gleaned insight on how immune cells work both independently and collaboratively within an entire human body system.

Validation of a functional human AD model with four AD therapeutics utilizing patterned iPSC-derived cortical neurons integrated with microelectrode arrays.

Preclinical methods are needed for screening potential Alzheimer's disease (AD) therapeutics that recapitulate phenotypes found in the Mild Cognitive Impairment (MCI) stage or even before this stage of the disease. This would require a phenotypic system that reproduces cognitive deficits without significant neuronal cell death to mimic the clinical manifestations of AD during these stages. A potential functional parameter to be monitored is long-term potentiation (LTP), which is a correlate of learning and memory, that would be one of the first functions effected by AD onset. Mature human iPSC-derived cortical neurons and primary astrocytes were co-cultured on microelectrode arrays (MEA) where surface chemistry was utilized to create circuit patterns connecting two adjacent electrodes to model LTP function. LTP maintenance was significantly reduced in the presence of Amyloid-Beta 42 (Aß42) oligomers compared to the controls, however, co-treatment with AD therapeutics (Donepezil, Memantine, Rolipram and Saracatinib) corrected Aß42 induced LTP impairment. The results presented here illustrate the significance of the system as a validated platform that can be utilized to model and study MCI AD pathology, and potentially for the pre-MCI phase before the occurrence of significant cell death. It also has the potential to become an ideal platform for high content therapeutic screening for other neurodegenerative diseases.

Human IPSC-Derived PreBötC-Like Neurons and Development of an Opiate Overdose and Recovery Model.

Opioid overdose is the leading cause of drug overdose lethality, posing an urgent need for investigation. The key brain region for inspiratory rhythm regulation and opioid-induced respiratory depression (OIRD) is the preBötzinger Complex (preBötC) and current knowledge has mainly been obtained from animal systems. This study aims to establish a protocol to generate human preBötC neurons from induced pluripotent cells (iPSCs) and develop an opioid overdose and recovery model utilizing these iPSC-preBötC neurons. A de novo protocol to differentiate preBötC-like neurons from human iPSCs is established. These neurons express essential preBötC markers analyzed by immunocytochemistry and demonstrate expected electrophysiological responses to preBötC modulators analyzed by patch clamp electrophysiology. The correlation of the specific biomarkers and function analysis strongly suggests a preBötC-like phenotype. Moreover, the dose-dependent inhibition of these neurons' activity is demonstrated for four different opioids with identified IC50's comparable to the literature. Inhibition is rescued by naloxone in a concentration-dependent manner. This iPSC-preBötC mimic is crucial for investigating OIRD and combating the overdose crisis and a first step for the integration of a functional overdose model into microphysiological systems.

The Effect of Skeletal Muscle-Specific Creatine Treatment on ALS NMJ Integrity and Function.

Although skeletal muscle (hSKM) has been proven to be actively involved in Amyotrophic Lateral Sclerosis (ALS) neuromuscular junction (NMJ) dysfunction, it is rarely considered as a pharmacological target in preclinical drug discovery. This project investigated how improving ALS hSKM viability and function effects NMJ integrity. Phenotypic ALS NMJ human-on-a-chip models developed from patient-derived induced pluripotent stem cells (iPSCs) were used to study the effect of hSKM-specific creatine treatment on clinically relevant functional ALS NMJ parameters, such as NMJ numbers, fidelity, stability, and fatigue index. Results indicated comparatively enhanced NMJ numbers, fidelity, and stability, as well as reduced fatigue index, across all hSKM-specific creatine-treated systems. Immunocytochemical analysis of the NMJs also revealed improved post-synaptic nicotinic Acetylcholine receptor (AChR) clustering and cluster size in systems supplemented with creatine relative to the un-dosed control. This work strongly suggests hSKM as a therapeutic target in ALS drug discovery. It also demonstrates the need to consider all tissues involved in multi-systemic diseases, such as ALS, in drug discovery efforts. Finally, this work further establishes the BioMEMs NMJ platform as an effective means of performing mutation-specific drug screening, which is a step towards personalized medicine for rare diseases.

Mimicking the Tendon Microenvironment to Enhance Skeletal Muscle Adhesion and Longevity in a Functional Microcantilever Platform.

Microcantilever platforms are functional models for studying skeletal muscle force dynamics in vitro. However, the contractile force generated by the myotubes can cause them to detach from the cantilevers, especially during long-term experiments, thus impeding the chronic investigations of skeletal muscles for drug efficacy and toxicity. To improve the integration of myotubes with microcantilevers, we drew inspiration from the elastomeric proteins, elastin and resilin, that are present in the animal and insect worlds, respectively. The spring action of these proteins plays a critical role in force dampening in vivo. In animals, elastin is present in the collagenous matrix of the tendon which is the attachment point of muscles to bones. The tendon microenvironment consists of elastin, collagen, and an aqueous jelly-like mass of proteoglycans. In an attempt to mimic this tendon microenvironment, elastin, collagen, heparan sulfate proteoglycan, and hyaluronic acid were deposited on a positively charged silane substrate. This enabled the long-term survival of mechanically active myotubes on glass and silicon microcantilevers for over 28 days. The skeletal muscle cultures were derived from both primary and induced pluripotent stem cell (iPSC)-derived human skeletal muscles. Both types of myoblasts formed myotubes which survived for five weeks. Primary skeletal muscles and iPSC-derived skeletal muscles also showed a similar trend in fatigue index values. Upon integration with the microcantilever system, the primary muscle and iPSC-derived myotubes were tested successively over a one month period, thus paving the way for long-term chronic experiments on these systems for both drug efficacy and toxicity studies.

Development of a human malaria-on-a-chip disease model for drug efficacy and off-target toxicity evaluation.

A functional, multi-organ, serum-free system was developed for the culture of P. falciparum in an attempt to establish innovative platforms for therapeutic drug development. It contains 4 human organ constructs including hepatocytes, splenocytes, endothelial cells, as well as recirculating red blood cells which allow for infection with the parasite. Two strains of P. falciparum were used: the 3D7 strain, which is sensitive to chloroquine; and the W2 strain, which is resistant to chloroquine. The maintenance of functional cells was successfully demonstrated both in healthy and diseased conditions for 7 days in the recirculating microfluidic model. To demonstrate an effective platform for therapeutic development, systems infected with the 3D7 strain were treated with chloroquine which significantly decreased parasitemia, with recrudescence observed after 5 days. Conversely, when the W2 systems were dosed with chloroquine, parasitemia levels were moderately decreased when compared to the 3D7 model. The system also allows for the concurrent evaluation of off-target toxicity for the anti-malarial treatment in a dose dependent manner which indicates this model could be utilized for therapeutic index determination. The work described here establishes a new approach to the evaluation of anti-malarial therapeutics in a realistic human model with recirculating blood cells for 7 days.

Development of a functional human induced pluripotent stem cell-derived nociceptor MEA system as a pain model for analgesic drug testing.

The control of severe or chronic pain has relied heavily on opioids and opioid abuse and addiction have recently become a major global health crisis. Therefore, it is imperative to develop new pain therapeutics which have comparable efficacy for pain suppression but lack of the harmful effects of opioids. Due to the nature of pain, any in vivo experiment is undesired even in animals. Recent developments in stem cell technology has enabled the differentiation of nociceptors from human induced pluripotent stem cells. This study sought to establish an in vitro functional induced pluripotent stem cells-derived nociceptor culture system integrated with microelectrode arrays for nociceptive drug testing. Nociceptors were differentiated from induced pluripotent stem cells utilizing a modified protocol and a medium was designed to ensure prolonged and stable nociceptor culture. These neurons expressed nociceptor markers as characterized by immunocytochemistry and responded to the exogenous toxin capsaicin and the endogenous neural modulator ATP, as demonstrated with patch clamp electrophysiology. These cells were also integrated with microelectrode arrays for analgesic drug testing to demonstrate their utilization in the preclinical drug screening process. The neural activity was induced by ATP to mimic clinically relevant pathological pain and then the analgesics Lidocaine and the opioid DAMGO were tested individually and both induced immediate silencing of the nociceptive activity. This human-based functional nociceptive system provides a valuable platform for investigating pathological pain and for evaluating effective analgesics in the search of opioid substitutes.

Hyperglycemia Negatively Affects IPSC-Derived Myoblast Proliferation and Skeletal Muscle Regeneration and Function.

Diabetic myopathy is a co-morbidity diagnosed in most diabetes mellitus patients, yet its pathogenesis is still understudied, which hinders the development of effective therapies. This project aimed to investigate the effect of hyperglycemia on human myoblast physiology, devoid of other complicating factors, by utilizing human myoblasts derived from induced pluripotent stem cells (iPSCs), in a defined in vitro system. IPSC-derived myoblasts were expanded under three glucose conditions: low (5 mM), medium (17.5 mM) or high (25 mM). While hyperglycemic myoblasts demonstrated upregulation of Glut4 relative to the euglycemic control, myoblast proliferation demonstrated a glucose dose-dependent impedance. Further cellular analysis revealed a retarded cell cycle progression trapped at the S phase and G2/M phase and an impaired mitochondrial function in hyperglycemic myoblasts. Terminal differentiation of these hyperglycemic myoblasts resulted in significantly hypertrophic and highly branched myotubes with disturbed myosin heavy chain arrangement. Lastly, functional assessment of these myofibers derived from hyperglycemic myoblasts demonstrated comparatively increased fatigability. Collectively, the hyperglycemic myoblasts demonstrated deficient muscle regeneration capability and functionality, which falls in line with the sarcopenia symptoms observed in diabetic myopathy patients. This human-based iPSC-derived skeletal muscle hyperglycemic model provides a valuable platform for mechanistic investigation of diabetic myopathy and therapeutic development.

Classical Complement Pathway Inhibition in a "Human-On-A-Chip" Model of Autoimmune Demyelinating Neuropathies.

Chronic autoimmune demyelinating neuropathies are a group of rare neuromuscular disorders with complex, poorly characterized etiology. Here we 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 was shown to contain anti-GM1 IgM and IgG antibodies capable of binding to human primary Schwann cells and induced pluripotent stem cell derived motoneurons. Patient autoantibody binding was sufficient to activate the classical complement pathway resulting in detection of C3b and C5b-9 deposits. A HoaC model, using a microelectrode array with directed axonal outgrowth over the electrodes treated with patient sera, exhibited reductions in motoneuron action potential frequency and conduction velocity. TNT005 rescued the serum-induced complement deposition and functional deficits while treatment with an isotype control antibody had 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 was sufficient to rescue these pathological effects and provide efficacy data included in an investigational new drug application, demonstrating the model's translational potential.

ALS mutations in both human skeletal muscle and motoneurons differentially affects neuromuscular junction integrity and function.

There is evidence for the involvement of human skeletal muscle (hSKM) in ALS neuromuscular junction (NMJ) dysfunction. However, the specific avenue by which the hSKM contributes to NMJ disruption is not well understood due to limited human-based studies performed to investigate the subject. Thus, hSKM and human motoneurons (hMN) generated from induced pluripotent stem cells of healthy individuals (WT) and ALS patients with two different SOD1 mutations were integrated into functional NMJ systems to investigate and compare the pathological contribution of the hSKM and hMN to ALS NMJ disruption. Morphological assessment of ALS NMJs demonstrated reduced acetylcholine receptor clustering in the post-synaptic membrane of co-cultures with ALS hSKM (hSKMSOD1-hMNWT and hSKMSOD1-hMNSOD1). Significantly reduced functional NMJ numbers, NMJ stability, contraction fidelity and increased fatigue index were observed in all ALS co-cultures compared to WT. However, these disease phenotypes were comparatively more severe in microphysiologic systems with hSKMSOD1-hMNWT or hSKMSOD1-hMNSOD1 than those with hSKMWT-hMNSOD1 co-cultures. Results from this study affirm that the inherent pathological defects in ALS hSKM, independent of motoneurons, significantly contributes to NMJ dysfunction. As such, therapeutically targeting the ALS hSKM may be just as, if not more critical than, the hMN in alleviating disease phenotypes and attenuating disease progression.

A functional hiPSC-cortical neuron differentiation and maturation model and its application to neurological disorders.

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.

An induced pluripotent stem cell-derived NMJ platform for study of the NGLY1-Congenital Disorder of Deglycosylation.

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.

A Functional Human-on-a-Chip Autoimmune Disease Model of Myasthenia Gravis for Development of Therapeutics.

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).

A functional long-term 2D serum-free human hepatic in vitro system for drug evaluation.

Human in vitro hepatic models generate faster drug toxicity data with higher human predictability compared to animal models. However, for long-term studies, current models require the use of serum and 3D architecture, limiting their utility. Maintaining a functional long-term human in vitro hepatic culture that avoids complex structures and serum would improve the value of such systems for preclinical studies. This would also enable a more straightforward integration with current multi-organ devices to study human systemic toxicity to generate an alternative model to chronic animal evaluations. A human primary hepatocyte culture system was characterized for 28 days in 2D and serum-free defined conditions. Under the studied conditions, human primary hepatocytes maintained their characteristic morphology, hepatic markers and functions for 28 days. The acute and chronic administration of known drugs validated the sensitivity of the system for drug testing. This human 2D model represents a realistic system to evaluate hepatic function for long-term drug studies, without the need of animal serum, confounding variable in most models, and with less complexity and resultant cost compared to most 3D models. The defined culture conditions can easily be integrated into complex multi-organ in vitro models for studying systemic effects driven by the liver function for long-term evaluations.

Characterization of Functional Human Skeletal Myotubes and Neuromuscular Junction Derived-From the Same Induced Pluripotent Stem Cell Source.

In vitro generation of functional neuromuscular junctions (NMJs) utilizing the same induced pluripotent stem cell (iPSC) source for muscle and motoneurons would be of great value for disease modeling and tissue engineering. Although, differentiation and characterization of iPSC-derived motoneurons are well established, and iPSC-derived skeletal muscle (iPSC-SKM) has been reported, there is a general lack of systemic and functional characterization of the iPSC-SKM. This study performed a systematic characterization of iPSC-SKM differentiated using a serum-free, small molecule-directed protocol. Morphologically, the iPSC-SKM demonstrated the expression and appropriate distribution of acetylcholine, ryanodine and dihydropyridine receptors. Fiber type analysis revealed a mixture of human fast (Type IIX, IIA) and slow (Type I) muscle types and the absence of animal Type IIB fibers. Functionally, the iPSC-SKMs contracted synchronously upon electrical stimulation, with the contraction force comparable to myofibers derived from primary myoblasts. Most importantly, when co-cultured with human iPSC-derived motoneurons from the same iPSC source, the myofibers contracted in response to motoneuron stimulation indicating the formation of functional NMJs. By demonstrating comparable structural and functional capacity to primary myoblast-derived myofibers, this defined, iPSC-SKM system, as well as the personal NMJ system, has applications for patient-specific drug testing and investigation of muscle physiology and disease.

Functional skeletal muscle model derived from SOD1-mutant ALS patient iPSCs recapitulates hallmarks of disease progression.

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.

Myelination and Node of Ranvier Formation in a Human Motoneuron-Schwann Cell Serum-Free Coculture.

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.

Differential Monocyte Actuation in a Three-Organ Functional Innate Immune System-on-a-Chip.

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.

Inverse liquid-solid chromatography to evaluate drug interactions with organosilane-modified polydimethylsiloxane for use in body-on-a-chip systems.

Body-on-a-chip and organ-on-a-chip systems utilize polydimethylsiloxane (PDMS) because of the relative suitability of the material for fabrication of microfluidic channels and chambers used in these devices. However, hydrophobic molecules, especially therapeutic compounds, tend to adsorb to PDMS, which may distort the dose-response curves that feed into the pharmacokinetic/pharmacodynamic models used to translate preclinical data into predictions of clinical outcomes. Surface modification by organosilanes is one method being explored to modify PDMS, but the effect of organosilanes on drug adsorption isotherms is not well characterized. We utilized Inverse Liquid-Solid Chromatography to characterize the adsorption parameters of the drugs acetaminophen, diclofenac, and verapamil with native PDMS and organosilane-modified (fluoropolymer (13F) and polyethylene glycol) PDMS surfaces, to correlate the modifications with changes in drug adsorption. It was determined that the organosilane modifications significantly changed the energy of adsorption of the test drug utilizing our methodology.