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.

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.

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 Human-Based Functional NMJ System for Personalized ALS Modeling and Drug Testing.

Loss of the neuromuscular junction (NMJ) is an early and critical hallmark in all forms of ALS. The study design was to develop a functional NMJ disease model by integrating motoneurons (MNs) differentiated from multiple ALS-patients' induced pluripotent stem cells (iPSCs) and primary human muscle into a chambered system. NMJ functionality was tested by recording myotube contractions while stimulating MNs by field electrodes and a set of clinically relevant parameters were defined to characterize the NMJ function. Three ALS lines were analyzed, 2 with SOD1 mutations and 1 with a FUS mutation. The ALS-MNs reproduced pathological phenotypes, including increased axonal varicosities, reduced axonal branching and elongation and increased excitability. These MNs formed functional NMJs with wild type muscle, but with significant deficits in NMJ quantity, fidelity and fatigue index. Furthermore, treatment with the Deana protocol was found to correct the NMJ deficits in all the ALS mutant lines tested. Quantitative analysis also revealed the variations inherent in each mutant lines. This functional NMJ system provides a platform for the study of both fALS and sALS and has the capability of being adapted into subtype-specific or patient-specific models for ALS etiological investigation and patient stratification for drug testing.

Mechanical Properties, Cytocompatibility and Manufacturability of Chitosan:PEGDA Hybrid-Gel Scaffolds by Stereolithography.

Extracellular matrix mimetic hydrogels which hybridize synthetic and natural polymers offer molecularly-tailored, bioactive properties and tunable mechanical strength. In addition, 3D bioprinting by stereolithography allows fabrication of internal pores and defined macroscopic shapes. In this study, we formulated a hybrid biocompatible resin using natural and synthetic polymers (chitosan and polyethylene glycol diacrylate (PEGDA), respectively) by controlling molecular weight of chitosan, feed-ratios, and photo-initiator concentration. Ear-shaped, hybrid scaffolds were fabricated by a stereolithographic method using a 405 nm laser. Hybrid hydrogel scaffolds of chitosan (50-190 kDa) and PEGDA (575 Da) were mixed at varying feed-ratios. Some of the cationic, amino groups of chitosan were neutralized by dialysis in acidic solution containing chitosan in excess of sodium acetate solution to inhibit quenching of newly formed photoradicals. A feed-ratio of 1:7.5 was found to be the most appropriate of the formulations considered in this study in terms of mechanical properties, cell adhesion, and printability. The biofabricated hybrid scaffold showed interconnected, homogeneous pores with a nominal pore size of 50 µm and an elastic modulus of ~400 kPa. Moreover, long-term cell viability and cell spreading was observed via actin filament staining. Printability of the biocompatible resin was confirmed by printing thresholded MR images of an ear and the feed ratio of 1:7.5 provided the most faithful reproduction of the shape. To the best of our knowledge, this is the first report of stereolithographic printing hybridizing cell-adhesive properties of chitosan with mechanical robustness of PEG in scaffolds suitable for repair of complex tissue geometries, such as those of the human ear.