Functional innervation of human induced pluripotent stem cell-derived cardiomyocytes by co-culture with sympathetic neurons developed using a microtunnel technique.

Microelectrode array (MEA) based-drug screening with human induced pluripotent stem cell-derived cardiomyocytes (hiPSCM) is a potent pre-clinical assay for efficiently assessing proarrhythmic risks in new candidates. Furthermore, predicting sympathetic modulation of the proarrhythmic side-effects is an important issue. Although we have previously developed an MEA-based co-culture system of rat primary cardiomyocyte and sympathetic neurons (rSNs), it is unclear if this co-culture approach is applicable to develop and investigate sympathetic innervation of hiPSCMs. In this study, we developed a co-culture of rSNs and hiPSCMs on MEA substrate, and assessed functional connections. The inter-beat interval of hiPSCM was significantly shortened by stimulation in SNs depending on frequency and pulse number, indicating functional connections between rSNs and hiPSCM and the dependency of chronotropic effects on rSN activity pattern. These results suggest that our co-culture approach can evaluate sympathetic effects on hiPSCMs and would be a useful tool for assessing sympathetic modulated-cardiotoxicity in human cardiac tissue.

A device for co-culturing autonomic neurons and cardiomyocytes using micro-fabrication techniques.

It is still unclear how the activity of sympathetic and parasympathetic neurons influences the activity of cardiomyocytes in culture. We developed a device for co-culturing sympathetic neurons, parasympathetic neurons, and cardiomyocytes using micro-fabrication techniques. Morphological connections between each type of autonomic neuron and the cardiomyocytes were observed by immunostaining. The inter-beat-interval (IBI) of the cardiomyocytes was modulated after electrically stimulating each type of autonomic neuron. Modulation of the IBI was blocked by the addition of pharmacological blockers to the culture medium. These results suggest that the co-culture device can be utilized to understand how the activity of sympathetic neurons and parasympathetic neurons influences the activity of cardiomyocytes in the culture environment.

Signal transfer within a cultured asymmetric cortical neuron circuit.

OBJECTIVE: Simplified neuronal circuits are required for investigating information representation in nervous systems and for validating theoretical neural network models. Here, we developed patterned neuronal circuits using micro fabricated devices, comprising a micro-well array bonded to a microelectrode-array substrate. APPROACH: The micro-well array consisted of micrometre-scale wells connected by tunnels, all contained within a silicone slab called a micro-chamber. The design of the micro-chamber confined somata to the wells and allowed axons to grow through the tunnels bidirectionally but with a designed, unidirectional bias. We guided axons into the point of the arrow structure where one of the two tunnel entrances is located, making that the preferred direction. MAIN RESULTS: When rat cortical neurons were cultured in the wells, their axons grew through the tunnels and connected to neurons in adjoining wells. Unidirectional burst transfers and other asymmetric signal-propagation phenomena were observed via the substrate-embedded electrodes. Seventy-nine percent of burst transfers were in the forward direction. We also observed rapid propagation of activity from sites of local electrical stimulation, and significant effects of inhibitory synapse blockade on bursting activity. SIGNIFICANCE: These results suggest that this simple, substrate-controlled neuronal circuit can be applied to develop in vitro models of the function of cortical microcircuits or deep neural networks, better to elucidate the laws governing the dynamics of neuronal networks.

Microfabricated device for co-culture of sympathetic neuron and iPS-derived cardiomyocytes.

Induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CMs) has been expected as a cell source for therapy of serious heart failure. However, it is unclear whether the function of iPS-CMs is modulated by the host sympathetic nervous system. Here we developed a device for co-culture of sympathetic neurons and iPS-CMs using microfabrication technique. The device consisted of a culture chamber and a microelectrode-array (MEA) substrate. The superior cervical ganglion (SCG) neurons were co-cultured with iPS-CMs in a microfabricated device, which had multiple compartments. Several days after seeding, synapses were formed between SCG neurons and iPS-CMs, as confirmed by immunostaining. Spontaneous electrical activities of the SCG neurons and the iPS-CMs were observed from the electrode of the MEA substrate. The beat rate of iPS-CMs increased after electrical stimulation of the co-cultured SCG neurons. Such changes in the beat rate were prevented in the presence of propranolol, a ß-adrenoreceptor antagonist. These results suggest that the microfabricated device will be utilized for studying the functional modulation of iPS-CMs by connected sympathetic neurons.

Bidirectional synaptic connection between primary and stem cell-derived neurons in co-culture device.

Regenerative medicine is expected to be a potent therapeutic option for the disorders or injuries of the central nervous system. However, little is known about how the newly formed neurons derived from grafted stem cells integrate into the host established tissue. The aims of this study are to make functional connection between primary neurons and stem cell-derived neurons via chemical synapses and maintain the connection for a long time in in vitro. We employed an in vitro co-culture device to cultivate two different neuronal populations and evaluate the interaction between them. Mouse cortical neurons and P19 cell-derived neurons were co-cultured in the co-culture device. The synchronous activities were maintained for at least 4 weeks. Evoked responses to electrical stimulation suggested that bi-directional connections were formed between cortical and P19-derived neurons. The responses were changed after pharmacological treatment. These results showed that cortical neurons and P19 cell-derived neurons formed bidirectional synaptic connections via glutamate receptors and the connection was maintained for at least 4 weeks.

Sympathetic neurons modulate the beat rate of pluripotent cell-derived cardiomyocytes in vitro.

Although stem cell-derived cardiomyocytes have great potential for the therapy of heart failure, it is unclear whether their function after grafting can be controlled by the host sympathetic nervous system, a component of the autonomic nervous system (ANS). Here we demonstrate the formation of functional connections between rat sympathetic superior cervical ganglion (SCG) neurons and pluripotent (P19.CL6) cell-derived cardiomyocytes (P19CMs) in compartmentalized co-culture, achieved using photolithographic microfabrication techniques. Formation of synapses between sympathetic neurons and P19CMs was confirmed by immunostaining with antibodies against ß-3 tubulin, synapsin I and cardiac troponin-I. Changes in the beat rate of P19CMs were triggered after electrical stimulation of the co-cultured SCG neurons, and were affected by the pulse frequency of the electrical stimulation. Such changes in the beat rate were prevented when propranolol, a ß-adrenoreceptor antagonist, was added to the culture medium. These results suggest that the beat rate of differentiated cardiomyocytes can be modulated by electrical stimulation of connected sympathetic neurons.

Device for co-culture of sympathetic neurons and cardiomyocytes using microfabrication.

Rat superior cervical ganglion (SCG) neurons and ventricular myocytes (VMs) were co-cultured separately in a minichamber placed on a microelectrode-array (MEA) substrate. The minichamber, fabricated photolithographically using polydimethylsiloxane (PDMS), had 2 compartments, 16 microcompartments and 8 microconduits. The SCG neurons were seeded into one of the compartments and all of the microcompartments using a glass pipette controlled by a micromanipulator and a microinjector. The VMs were seeded into the other compartment. Three days after seeding of the VMs, the SCG neurons were still confined to one compartment and all of the microcompartments, and the neurites of the SCG neurons had connected with the VMs via the microconduits. Constant-voltage stimulation, using a train of biphasic square pulses (1 ms at +1 V, followed by -1 ms at 1 V), was applied to the SCG neurons in the microcompartments using 16 electrodes. Evoked responses were observed in several electrodes while electrical stimulation was applied to the SCG neurons. Two-way analysis of variance (ANOVA) revealed that the frequency of the stimulation pulses had significant effects in increasing the beat rate of the VMs, and that the interaction between the frequency and the number of the pulses also had a significant effect on the ratio. No significant increases in the beat rate were observed when propranolol, a ß-adrenergic receptor antagonist, was added to the culture medium. These results suggest that synaptic pathways were formed between the SCG neurons and the VMs, and that this co-culture device can be utilized for studies of network-level interactions between sympathetic neurons and cardiomyocytes.

Autonomic nervous system driven cardiomyocytes in vitro.

Rat superior cervical ganglia (SCG), which are sympathetic ganglia, neurons and ventricular myocytes (VMs) were co-cultured separately in a minichamber placed on a microelectrode-array (MEA) substrate. The minichamber was fabricated photolithographically and had 2 compartments, 16 microcompartments and 8 microconduits. The SCG neurons were seeded into one of the compartments and all of the microcompartments using a glass pipette controlled by a micromanipulator and a microinjector. The VMs were seeded into the other compartment. Three days after seeding of the VMs, the neurites of the SCG neurons had connected with the VMs via the microconduits. Electrical stimulations, trains of biphasic square pulses, were applied to the SCG neurons in the microcompartments using 16 electrodes. Evoked responses were observed in several electrodes while electrical stimulation was applied to the SCG neurons. According to the two-way analysis of variance (ANOVA), the beat rate after electrical stimulation was affected by the frequency and the number of the stimulation pulses. These results suggest that pulse number and the frequency of the electrical stimulation contribute to modulation of the beat rate of the cardiomyocytes.

Development of semi-separated co-culture system of sympathetic neuron and cardiomyocyte.

Rat superior cervical ganglion (SCG) neurons and ventricular myocytes (VMs) were co-cultured in chambers made of polydimethylsyloxane. The chambers were placed on a microelectrode-array (MEA) substrate and connected with a pathway. 24 hours after dissemination of the VMs, neuntes of the SCG neurons outgrew through the pathway and reached the VMs. Spontaneous electrical activities of the SCG neurons and the VMs were observed several days after the dissemination. Constant-voltage stimualtion (1 V, 1 ms, biphasic square pulses) was applied to the SCG neurons at the frequency of 10 Hz using 32 electrodes. Contraction rate of the VMs increased by 153 +/-110 % immediately after the stimulation to the SCG neurons was stopped. Then contraction rate gradually decreased and returned to almost the same rate as before the stimulation 5 minutes after the 1-min stimulation. Propranolol (beta-adrenergic receptor antagonist) prevented contraction rate of the VMs from increasing after electrical stimulation to the SCG neurons. These results suggest that neuromuscular junctions were formed between the SCG neurons and the VMs. Overall the semi-separated co-culture system in this study is available in research on changes in contraction rate of the VMs after applying electrical stimulation to the SCG neurons.