In vitro microfluidic platform for co-culture of myocytes with mouse embryonic stem cell-derived interneurons and motor neurons

Lee, Jaewon
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Central pattern generators (CPGs) in the spinal cord are motor circuits that oversee controlling rhythmic motor activities. CPGs are composed of different types of interneurons (INs) that interact with the motor neurons (MNs) to produce the patterns of MN activity that can coordinate limb and body movements. Elucidating how different populations of INs contribute to the spinal CPG circuitry in different ways is crucial to understanding neural circuit formation and function. In addition, to study how neural circuits, such as CPGs recruit muscle and dictate locomotion, a functional neuromuscular junction (NMJ) model is essential. As CPG and NMJ are both important components in understanding spinal locomotion, a cell-culture based model where it is possible to precisely modulate cellular, molecular, or transcriptional cues in well-defined time and space is highly desirable. In this study, an in vitro NMJ and a V2a IN-MN-myotube (MT) circuit were developed as model platforms to study CPGs using the unique cell lines developed previously in the lab: transgenic mouse embryonic stem cell (mESC) lines that can produce enriched cultures of Hb9⁺ MNs and excitatory Chx10⁺ V2a INs. We established and confirmed a co-culture condition for the formation of NMJ between the unique mESC-derived Hb9⁺ MNs and the C2C12-derived myotubes in vitro and qualitatively and quantitatively assessed the functional connection between the two cell types. Our platform was further advanced to include a model IN population, Chx10⁺ V2a INs to develop a platform that allows for the characterization of IN-MN connections in situ. The in vitro circuit was made by co-culturing V2a INs, MNs, and MTs in separate chambers of the microdevice that are connected with the microchannels. A co-culture protocol for all three cell types was established using different seeding strategies and medium combinations. To evaluate the possible network formation in the dissociated cultures of different cell types, microelectrode arrays (MEAs) were used. Utilizing the techniques developed in this project, a more systematic study of rhythm and pattern generation according to the IN composition can be carried out to further elucidate the role of each IN population in circuit formation and function