Development of their connections was evaluated with microscopic observations, immunochemical analysis, and calcium imaging
February 12, 2022
Development of their connections was evaluated with microscopic observations, immunochemical analysis, and calcium imaging. mm regions were captured. Speed of the movie is 3 times faster than the real time.(WMV) pone.0148559.s004.wmv (3.6M) GUID:?D9F8187E-120E-4287-9247-6821548632DA Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is usually warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that this bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions. Introduction The nervous system consists of the central and peripheral systems that are connected with each other, and thus form an electrical signaling network throughout the body. Although each neuron type is usually differentiated from different stem/progenitor cell pools, interactions between various cell types are well-coordinated both morphologically and functionally. The peripheral nervous system (PNS) is usually connected to the central nervous system (CNS), and this functional system is responsible for the homeostasis of various tissues and organs. Indeed, peripheral neuropathies caused by genetic disorders , autoimmune diseases , or diabetes [3,4] induce functional abnormalities in the entire body. Owing to the complexity of causes and symptoms, peripheral neuropathy is usually treated with symptomatic approaches such as surgical intervention or pain management. Therefore, understanding the molecular mechanism of peripheral neuropathy progression and the interaction of the PNS with target organs might contribute to the development of novel therapeutic methods aiming for a complete remedy. Co-culture systems can be used to model inter-organ communications model system for studying peripheral neuron-related diseases. In this study, we created co-culture networks using human PNS and CNS neurons. First, we fabricated a PDMS-based co-culture chamber, which consisted of two culture compartments connected with 20 microtunnels, and we cultured induced PNS and CNS neurons differentiated from human iPS cells. Development of their connections was evaluated Ruscogenin with microscopic observations, immunochemical analysis, and calcium imaging. Furthermore, we prepared a co-culture system using PNS neurons and cardiomyocytes, both derived from the same human iPS cells, to confirm that our microfabricated device can be used with various cell types. Materials and Methods Ethnic statement The use of human iPS cells was approved by the Ethics Committee of National Institute of Advanced Industrial Science and Technology (AIST). Device fabrication The co-culture device was fabricated from PDMS using Gfap soft lithography and replica molding technique. For producing the master mold, SU-8 3005 (Microchem) was spin-coated on a 76 silicon wafer (Matsuzaki Seisakusyo., Ltd.) at 4000 rpm for 60 s to reach a height of 5 m. The coated wafer was pre-baked at 95C for 3 min. Then, the wafer was exposed to ultraviolet (UV) light with a UV crosslinker (CL-1000L; UVP) through a custom-made photomask. The photomask was designed to fabricate 20 microtunnels Ruscogenin with a width of 50 m and a length of 3 mm. After UV exposure, the wafer was developed Ruscogenin with the SU-8 programmer (Microchem), and then it was rinsed with 2-propanol (Wako Pure Chemical Industries). After its development, the wafer was placed in a conventional culture dish (100 mm; Corning). Mixture of the PDMS-prepolymer and curing catalyst (10:1 weight ratio; Silpot 184, Dow Corning) was poured over the fabricated wafer to achieve a thickness of.