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 β-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.

Cancer-associated fibroblasts (CAFs) are the key components of the densely proliferated stroma in pancreatic ductal adenocarcinoma (PDAC) and contribute to tumor progression and drug resistance. CAFs comprise heterogeneous subpopulations playing unique and vital roles. However, the commonly used mouse models have not been able to fully reproduce the histological and functional characteristics of clinical human CAF. Here, we generated a human cell-derived stroma-rich CDX (Sr-CDX) model, to reproduce the clinical tumor microenvironment. By co-transplanting human adipose-derived mesenchymal stem cells (AD-MSCs) and a human PDAC cell line (Capan-1) into mice, the Sr-CDX model recapitulated the characteristics of clinical pancreatic cancer, such as accelerated tumor growth, abundant stromal proliferation, chemoresistance, and dense stroma formed from the heterogeneous CAFs. Global RNA sequencing, single-cell based RNA sequencing, and histological analysis of CAFs in the Sr-CDX model revealed that the CAFs of the Sr-CDX mice were derived from the transplanted AD-MSCs and composed of heterogeneous subpopulations of CAF, including known and unknown subtypes. These lines of evidences suggest that our new tumor-bearing mouse model has the potential to address an open question in CAF research, that is the mechanism of CAF differentiation.

The autonomic nervous system (ANS) regulates tissue homeostasis and remodelling through antagonistic effects of noradrenergic sympathetic and cholinergic parasympathetic signalling. Despite numerous reports on the induction of sympathetic neurons from human pluripotent stem cells (hPSCs), no induction methods have effectively derived cholinergic parasympathetic neurons from hPSCs. Considering the antagonistic effects of noradrenergic and cholinergic inputs on target organs, both sympathetic and parasympathetic neurons are expected to be induced. This study aimed to develop a stepwise chemical induction method to induce sympathetic-like and parasympathetic-like ANS neurons. Autonomic specification was achieved through restricting signals inducing sensory or enteric neurogenesis and activating bone morphogenetic protein (BMP) signals. Global mRNA expression analyses after stepwise induction, including single-cell RNA-seq analysis of induced neurons and functional assays revealed that each induced sympathetic-like or parasympathetic-like neuron acquired pharmacological and electrophysiological functional properties with distinct marker expression. Further, we identified selective induction methods using appropriate seeding cell densities and neurotrophic factor concentrations. Neurons were individually induced, facilitating the regulation of the beating rates of hiPSC-derived cardiomyocytes in an antagonistic manner. The induction methods yield specific neuron types, and their influence on various tissues can be studied by co-cultured assays.

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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 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 in vitro 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 the 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.