ABSTRACT
Food-derived biological signals are transmitted to the brain via peripheral nerves through the paracrine activity of gastrointestinal (GI) hormones. The signal transduction circuit of the brain-gut axis has been analyzed in animals; however, species-related differences and animal welfare concerns necessitate investigation using in vitro human experimental models. Here, we focused on the receptors of five GI hormones (CCK, GLP1, GLP2, PYY, and serotonin (5-HT)), and established human induced pluripotent stem cell (iPSC) lines that functionally expressed each receptor. Compared to the original iPSCs, iPSCs expressing one of the receptors did not show any differences in global mRNA expression, genomic stability, or differentiation capacities of the three germ layers. We induced parasympathetic neurons from these established iPSC lines to assess vagus nerve activity. We generated GI hormone receptor-expressing neurons (CCKAR, GLP1R, and NPY2R-neuron) and tested their responsiveness to each ligand using Ca2+ imaging and microelectrode array recording. GI hormone receptor-expressing neurons (GLP2R and HTR3A) were generated directly by gene induction into iPSC-derived peripheral nerve progenitors. These receptor-expressing neurons promise to contribute to a better understanding of how the body responds to GI hormones via the brain-gut axis, aid in drug development, and offer an alternative to animal studies.
Subject(s)
Gastrointestinal Hormones , Induced Pluripotent Stem Cells , Animals , Humans , Induced Pluripotent Stem Cells/metabolism , Gastrointestinal Hormones/metabolism , Neurons , Cell Differentiation , Signal TransductionABSTRACT
In vitro derivation of human neurons in the autonomic nervous system (ANS) is an important technology, given its regulatory roles in maintaining homeostasis in the human body. Although several induction protocols for autonomic lineages have been reported, the regulatory machinery remains largely undefined, primarily due to the absence of a comprehensive understanding of the molecular mechanism regulating human autonomic induction in vitro. In this study, our objective was to pinpoint key regulatory components using integrated bioinformatics analysis. A protein-protein interaction network construction for the proteins encoded by the differentially expressed genes from our RNA sequencing data, and conducting subsequent module analysis, we identified distinct gene clusters and hub genes involved in the induction of autonomic lineages. Moreover, we analyzed the impact of transcription factor (TF) activity on target gene expression, revealing enhanced autonomic TF activity that could lead to the induction of autonomic lineages. The accuracy of this bioinformatics analysis was corroborated by employing calcium imaging to observe specific responses to certain ANS agonists. This investigation offers novel insights into the regulatory machinery in the generation of neurons in the ANS, which would be valuable for further understanding and precise regulation of autonomic induction and differentiation.
Subject(s)
Autonomic Nervous System , Neurons , Humans , Neurons/metabolism , Homeostasis , Gene Regulatory NetworksABSTRACT
Raman scattering represents the distribution and abundance of intracellular molecules, including proteins and lipids, facilitating distinction between cellular states non-invasively and without staining. However, the scattered light obtained from cells is faint and cells have complex structures, making it difficult to obtain a Raman spectrum covering the entire cell in a short time using conventional methods. This also prevents efficient label-free cell classification. In the present study, we developed the Paint Raman Express Spectroscopy System, which uses two fast-rotating galvano mirrors to obtain spectra from a wide area of a cell. By using this system and applying machine learning, we were able to acquire broad spectra of a variety of human and mouse cell types, including pluripotent stem cells and confirmed that each cell type can be classified with high accuracy. Moreover, we classified different activation states of human T cells, despite their similar morphology. This system could be used for rapid and low-cost drug evaluation and quality management for drug screening in cell-based assays.
Subject(s)
Cells/classification , Spectrum Analysis, Raman/methods , Animals , Humans , Machine Learning , Mice , Single-Cell Analysis/methodsABSTRACT
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.
Subject(s)
Adipose Tissue/cytology , Cell Differentiation , Fibroblasts/cytology , Heterografts , Mesenchymal Stem Cells/cytology , Stromal Cells/cytology , Animals , Carcinoma, Pancreatic Ductal/pathology , Humans , Mice , Pancreatic Neoplasms/pathology , Pancreatic NeoplasmsABSTRACT
Neural crest cells (NCCs) are a promising source for cell therapy and regenerative medicine owing to their multipotency, self-renewability, and capability to secrete various trophic factors. However, isolating NCCs from adult organs is challenging, because NCCs are broadly distributed throughout the body. Hence, we attempted to directly induce NCCs from human adipose-derived mesenchymal stem cells (ADSCs), which can be isolated easily, using small molecule cocktails. We established a controlled induction protocol with two-step application of small molecule cocktails for 6 days. The induction efficiency was evaluated based on mRNA and protein expression of neural crest markers, such as nerve growth factor receptor (NGFR) and sex-determining region Y-box 10 (SOX10). We also found that various trophic factors were significantly upregulated following treatment with the small molecule cocktails. Therefore, we performed global profiling of cell surface makers and identified distinctly upregulated markers, including the neural crest-specific cell surface markers CD271 and CD57. These results indicate that our chemical treatment can direct human ADSCs to developing into the neural crest lineage. This offers a promising experimental platform to study human NCCs for applications in cell therapy and regenerative medicine.
Subject(s)
Cell Culture Techniques , Culture Media , Mesenchymal Stem Cells , Neural Crest , Regenerative Medicine/methods , CD57 Antigens/metabolism , Cell Differentiation , Cell Proliferation , Cells, Cultured , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Nerve Tissue Proteins/metabolism , Neural Crest/cytology , Neural Crest/metabolism , Receptors, Nerve Growth Factor/metabolism , SOXE Transcription Factors/metabolismABSTRACT
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.
Subject(s)
Heart Rate/physiology , Myocytes, Cardiac/physiology , Neurons/physiology , Parasympathetic Nervous System/physiology , Sympathetic Nervous System/physiology , Autonomic Pathways/metabolism , Autonomic Pathways/physiology , Bone Morphogenetic Proteins/metabolism , Cells, Cultured , Humans , Interneurons/metabolism , Interneurons/physiology , Male , Myocytes, Cardiac/metabolism , Neurons/metabolism , Parasympathetic Nervous System/metabolism , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/physiology , RNA, Messenger/metabolism , Signal Transduction/physiology , Sympathetic Nervous System/metabolismABSTRACT
Although various production methods for 3D vascularised tissues have been developed, constructing capillary-like structures branching from perfusable large channels remains difficult. This study describes a method to fabricate tube-shaped 3D liver-like tissue (tubular liver tissue) with large channels and capillary-like structures using a perfusion device. The perfusion device functions as an interface between the tissue and an external pump, as it has connectors equipped with anchors that hold the tissue in response to its shrinkage, which is accompanied by the self-organisation of capillary-like structures. Histological analysis revealed that perfusion via the large channel induced capillary formation around the channel and maintained proper tissue functions. Accompanied by structural examinations, global gene expression analysis supported this finding; specifically, genes involved in angiogenesis were enriched in the perfused condition. Furthermore, we confirmed the penetrability of the capillary-like structures by infusing India ink, as well as substance exchange by measuring the amounts of secreted albumin. These lines of evidence indicate that our method can be used to construct 3D tissues, which is useful for fields of in vitro tissue regeneration for drug development and regenerative medicine.
Subject(s)
Artificial Organs , Liver/blood supply , Tissue Engineering/methods , Blood Vessels/anatomy & histology , Capillaries/anatomy & histology , Hep G2 Cells , Human Umbilical Vein Endothelial Cells , Humans , Mesenchymal Stem Cells , PerfusionABSTRACT
We have shown that pharmacological inhibition of HSP90 ATPase activity induces apoptosis of myoblasts during their differentiation. However, the signaling pathways remain not fully characterized. We report that pharmacological targeting of HSP90 with 17-AAG activates the intrinsic pathway including caspase-dependent and caspase-independent pathways. 17-AAG induces the typical apoptotic phenotypes including PARP cleavage, chromatin condensation, and nuclear fragmentation with mitochondrial release of cytochrome c, Smac/DIABLO, procaspase-9 processing, and caspase-3 activation. AIF and EndoG redistribute from the mitochondria into the cytosol and are partially translocated to the nucleus in 17-AAG-treated cells. These results suggest that caspase-dependent and caspase-independent pathways should be considered in apoptosis of myogenic cells induced by inhibition of HSP90 ATPase activity.
Subject(s)
Adenosine Triphosphatases/antagonists & inhibitors , Apoptosis/drug effects , Benzoquinones/pharmacology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Lactams, Macrocyclic/pharmacology , Myoblasts/drug effects , Animals , Apoptosis Inducing Factor/metabolism , Apoptosis Regulatory Proteins , Blotting, Western , Caspase 3/metabolism , Caspase 9/metabolism , Cell Nucleus/drug effects , Chromatin/metabolism , Cytochromes c/metabolism , Endodeoxyribonucleases/metabolism , Enzyme Activation , HSP70 Heat-Shock Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mitochondria/drug effects , Mitochondria/enzymology , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Myoblasts/cytology , Poly(ADP-ribose) Polymerases/metabolism , Proteolysis , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
Synapse elimination and neurite pruning are essential processes for the formation of neuronal circuits. These regressive events depend on neural activity and occur in the early postnatal days known as the critical period, but what makes this temporal specificity is not well understood. One possibility is that the neural activities during the developmentally regulated shift of action of GABA inhibitory transmission lead to the critical period. Moreover, it has been reported that the shifting action of the inhibitory transmission on immature neurons overlaps with synapse elimination and neurite pruning and that increased inhibitory transmission by drug treatment could induce temporal shift of the critical period. However, the relationship among these phenomena remains unclear because it is difficult to experimentally show how the developmental shift of inhibitory transmission influences neural activities and whether the activities promote synapse elimination and neurite pruning. In this study, we modeled synapse elimination in neuronal circuits using the modified Izhikevich's model with functional shifting of GABAergic transmission. The simulation results show that synaptic pruning within a specified period like the critical period is spontaneously generated as a function of the developmentally shifting inhibitory transmission and that the specific firing rate and increasing synchronization of neural circuits are seen at the initial stage of the critical period. This temporal relationship was experimentally supported by an in vitro primary culture of rat cortical neurons in a microchannel on a multi-electrode array (MEA). The firing rate decreased remarkably between the 18-25 days in vitro (DIV), and following these changes in the firing rate, the neurite density was slightly reduced. Our simulation and experimental results suggest that decreasing neural activity due to developing inhibitory synaptic transmission could induce synapse elimination and neurite pruning at particular time such as the critical period. Additionally, these findings indicate that we can estimate the maturity level of inhibitory transmission and the critical period by measuring the firing rate and the degree of synchronization in engineered neural networks.
Subject(s)
Action Potentials/physiology , Models, Neurological , Nerve Net/physiology , Neuronal Plasticity/physiology , Synaptic Transmission/physiology , Animals , Animals, Newborn , Axons/physiology , Cerebral Cortex/cytology , Cerebral Cortex/physiology , Cerebrum/cytology , Cerebrum/physiology , Computer Simulation , Microelectrodes , Neurites/physiology , Primary Cell Culture , Rats , Receptors, GABA-A/physiology , Receptors, GABA-B/physiology , Synapses/physiology , Time FactorsABSTRACT
In this study, we propose a novel method for inducing neuronal cells by briefly exposing them to small-molecule cocktails in a step-by-step manner. Global gene expression analysis with immunohistochemical staining and calcium flux assays reveal the generation of neurons from mouse embryonic fibroblasts. In addition, time-lapse imaging of neural precursor-specific enhancer expression and global gene expression analyses show that the neurons are generated by passing through a neural crest-like precursor stage. Consistent with these results, the neural crest-like cells are able to differentiate into neural crest lineage cells, such as sympathetic neurons, adipocytes, osteocytes, and smooth muscle cells. Therefore, these results indicate that brief exposure to chemical compounds could expand and induce a substantial multipotent cell population without viral transduction.
Subject(s)
Cell Differentiation/drug effects , Fibroblasts/drug effects , Neural Crest/drug effects , Neural Stem Cells/drug effects , Small Molecule Libraries/pharmacology , Adipocytes/drug effects , Adipocytes/metabolism , Animals , Cell Differentiation/genetics , Cells, Cultured , Colforsin/pharmacology , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Profiling/methods , Gene Ontology , Immunohistochemistry , Mice, Inbred C57BL , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Neural Crest/cytology , Neural Crest/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurons/drug effects , Neurons/metabolism , Osteocytes/drug effects , Osteocytes/metabolism , Pyridines/pharmacology , Pyrimidines/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Time-Lapse Imaging/methods , Tranylcypromine/pharmacology , Valproic Acid/pharmacologyABSTRACT
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.
Subject(s)
Nerve Net , Calcium/metabolism , Calcium Signaling , Cell Differentiation , Central Nervous System/cytology , Coculture Techniques/instrumentation , Electric Stimulation , Humans , Immunochemistry , Induced Pluripotent Stem Cells/cytology , Microtechnology , Myocytes, Cardiac/cytology , Peripheral Nervous System/cytologyABSTRACT
The body's motion and function are all in part effected by a vital tissue, the tendon. Tendon injury often results in limited functioning after postoperative procedures and even for a long time after rehabilitation. Although numerous studies have reported surgical procedures using animal models which have contributed to both basic and clinical research, modeling of tendon sutures or postoperative immobilizations has not been performed on small experimental animals, such as mice. In this study we have developed an easy Achilles tendon suture and postoperative ankle fixation model in a mouse. Right Achilles tendons were incised and 10-0 nylons were passed through the proximal and distal ends using a modified Kessler method. Subsequently, the right ankle was immobilized in a plantarflexed position with novel splints, which were made from readily available extension tubes. Restriction of the tendon using handmade splints reduced swelling, as opposed to fixating with the usual plaster of Paris. Using this method, the usage of the right Achilles tendons began on postoperative days 13.5 ± 4.6, which indicated healing within two weeks. Therefore our simple short-term murine Achilles tendon suture procedure is useful for studying immediate tendon repair mechanisms in various models, including genetically-modified mice.
Subject(s)
Achilles Tendon/surgery , Immobilization/methods , Suture Techniques , Tendon Injuries/surgery , Achilles Tendon/injuries , Animals , Mice , Models, Animal , Postoperative Period , Wound HealingABSTRACT
Controversy exists as to whether the muscle-specific E3 ubiquitin ligases MAFbx and MuRF1 are transcriptionally upregulated in the process of sarcopenia. In the present study, we investigated the effects of ageing on mRNA/protein expression of muscle-specific E3 ubiquitin ligases and Akt/Foxo signalling in gastrocnemius muscles of female mice. Old mice exhibited a typical sarcopenic phenotype, characterized by loss of muscle mass and strength, decreased amount of myofibrillar proteins, incidence of aberrant muscle fibres, and genetic signature to sarcopenia. Activation levels of Akt were lower in adult and old mice than in young mice. Consequently, Akt-mediated phosphorylation levels of Foxo1 and Foxo3 proteins were decreased. Nuclear levels of Foxo1 and Foxo3 proteins showed an overall increasing trend in old mice. MAFbx mRNA expression was decreased in old mice relative to adult mice, whereas MuRF1 mRNA expression was less affected by ageing. At the protein level, MAFbx was less affected by ageing, whereas MuRF1 was increased in old mice relative to adult mice, with ubiquitin-protein conjugates being increased with ageing. In conclusion, we provided evidence for no mRNA upregulation of muscle-specific E3 ubiquitin ligases and disconnection between their expression and Akt/Foxo signalling in sarcopenic mice. Their different responsiveness to ageing may reflect different roles in sarcopenia.
Subject(s)
Aging/metabolism , Muscle, Skeletal/physiology , Proto-Oncogene Proteins c-akt/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Aging/genetics , Animals , Female , Gene Expression , Mice , Mice, Inbred C57BL , Muscle, Skeletal/enzymology , Muscle, Skeletal/metabolism , PhosphorylationABSTRACT
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.
Subject(s)
Action Potentials/physiology , Cerebral Cortex/physiology , Nerve Net/physiology , Neurons/physiology , Animals , Axons/physiology , Cell Culture Techniques/methods , Cells, Cultured , Electric Stimulation/methods , RatsABSTRACT
Stem cell transplantation is a promising therapy to treat neurodegenerative disorders, and a number of in vitro models have been developed for studying interactions between grafted neurons and the host neuronal network to promote drug discovery. However, methods capable of evaluating the process by which stem cells integrate into the host neuronal network are lacking. In this study, we applied an axonal conduction-based analysis to a co-culture study of primary and differentiated neurons. Mouse cortical neurons and neuronal cells differentiated from P19 embryonal carcinoma cells, a model for early neural differentiation of pluripotent stem cells, were co-cultured in a microfabricated device. The somata of these cells were separated by the co-culture device, but their axons were able to elongate through microtunnels and then form synaptic contacts. Propagating action potentials were recorded from these axons by microelectrodes embedded at the bottom of the microtunnels and sorted into clusters representing individual axons. While the number of axons of cortical neurons increased until 14 days in vitro and then decreased, those of P19 neurons increased throughout the culture period. Network burst analysis showed that P19 neurons participated in approximately 80% of the bursting activity after 14 days in vitro. Interestingly, the axonal conduction delay of P19 neurons was significantly greater than that of cortical neurons, suggesting that there are some physiological differences in their axons. These results suggest that our method is feasible to evaluate the process by which stem cell-derived neurons integrate into a host neuronal network.
Subject(s)
Action Potentials/physiology , Nerve Net/physiology , Neural Conduction/physiology , Neural Stem Cells/physiology , Neurons/physiology , Pluripotent Stem Cells/physiology , Animals , Axons/physiology , Cell Differentiation/physiology , Cells, Cultured , Mice , Nerve Net/cytology , Neural Stem Cells/cytology , Neurons/cytology , Pluripotent Stem Cells/cytologyABSTRACT
Investigating the mechanisms of the neuron-glia interaction is important in the basic research of neuroscience and neural transplantation. Synaptic transmission is modulated by astrocyte activation in the pre- and post-synaptic terminals, and this phenomenon is spread to the surrounding astrocytes through gap junctions. However, the modulation of network-wide neuronal activity dependent on extensive astrocyte activation is not well understood. In this study, we show network-wide neuronal modulation associated with a newly developed three-dimensional neuronal and astrocytic network co-culture method. To establish widespread neuronal and astrocytic network interactions in vitro, we performed integration of magnetic nanoparticle-injected astrocytes (Mag-AS) onto the matured monolayer of neuronal networks using an external magnetic force. The neuronal electrical activity was dynamically synchronized at 24 h after integration of the Mag-AS network. In addition, Mag-AS network activation using a caged calcium compound rapidly induced suppression and subsequent synchronization of neuronal electrical activity. These results indicate that the high-density astrocytic network integration onto the neuronal network can induce widespread neuronal modulation, and our in vitro co-culture method contributes to the advancement of neuronal and astrocytic transplantation research.
Subject(s)
Astrocytes/chemistry , Calcium Compounds/chemistry , Gap Junctions/chemistry , Gap Junctions/genetics , Magnetite Nanoparticles/chemistry , Neurons/chemistry , Neurons/metabolism , Astrocytes/cytology , Gap Junctions/metabolism , Neurons/cytology , Synaptic TransmissionABSTRACT
Cultured myotubes induced in vitro from myoblast cell lines have been widely used to investigate muscle functional properties and disease-related biological phenotypes. Until now, several cell patterning techniques have been applied to regulate in vitro myotube structures. However, these previous studies required specific geometry patterns or soft materials for inducing efficient myotube formation. Thus, more simple and easy handling method will be promising. In this study, we aimed to provide a method to form C2C12 myotubes with regulated sizes and orientations in simple line patterns. We used a poly(dimethylsiloxane) (PDMS) stamp and a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer solution to fabricate line patterns for myotube formation onto a culture dish. We confirmed that C2C12 myotubes of well-defined size and orientation were reproducibly formed. In particular, myotubes formed in the micropatterned lines showed the increased fusion efficiency. Then, functional dynamics in the micropatterned myotubes were detected and analyzed using a calcium imaging method. We confirmed micropatterning in line patterns enhanced the responsiveness of myotubes to external electrical stimulations. These results indicate that micropatterning myoblasts with the MPC polymer is a simple and effective method to form functional myotube networks.
Subject(s)
Cell Engineering/methods , Electric Stimulation , Muscle Fibers, Skeletal/cytology , Animals , Calcium/metabolism , Cell Line , Mice , Microscopy, Fluorescence , Microtechnology , Muscle Fibers, Skeletal/radiation effects , Myoblasts, Skeletal/cytology , Myoblasts, Skeletal/radiation effectsABSTRACT
Magnetic stimulation noninvasively modulates neuronal activity through a magnetically induced current. However, despite the usefulness and popularity of this method, the effects of neuronal activity in the nonstimulated regions on the stimulus responses are unknown. Here, we report that the induced current-evoked responses were affected by neuronal activities in the nonstimulated regions. Our experiment used a Mu-metal-based localized induced current stimulation (LICS) system combined with the microfabricated cell culture chamber system and a microelectrode array (MEA). The cell culture chamber system has radiating microtunnels connecting one central and eight outer chambers, which were fabricated using soft lithography and a replica modeling technique with SU-8 photoresist and polydimethylsiloxane (PDMS). Rat cortical neurons were separately cultured in the chambers and formed functional synaptic connections through the microtunnels. By applying a biphasic alternating pulsed magnetic field to the Mu-metal located in the central chamber, induced currents were mainly generated near the cultured neurons and modified the neuronal activities, which were recorded through MEA. Furthermore, we confirmed that the evoked responses were modified by localized pharmacological stimulation (LPS) in the outer chambers. These results suggest that our system would be promising tool for analyzing the effect of magnetic stimulation on interacting neuronal activity.
Subject(s)
Brain/physiology , Electric Stimulation , Electromagnetic Radiation , Models, Neurological , Animals , Brain/cytology , Brain/drug effects , Brain/radiation effects , Cells, Cultured , Evoked Potentials , Immunohistochemistry , Microelectrodes , Nerve Net , Neurons , Pharmacology , Rats , Rats, WistarABSTRACT
Reconstruction of skeletal muscle myotubes in vitro using myogenic cell lines have been widely carried out to study functional properties and disease-related biological changes of myotubes, such as intracellular calcium dynamics. However, the analysis of biological signals in isolated single myotubes or interactions among several myotubes is quite difficult problem because of the randomness in size, morphology and orientation of differentiated myotubes cultured on a conventional tissue culture dish. In the present study, we attempted to form uniform-size myotubes and detect intracellular calcium dynamics from the fabricated myotubes. We modified surfaces of culture dishes using a poly(-dimethylsiloxane) (PDMS) stamp and a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer solution to form line patterns for myotube formation. We could form uniform-size and -orientation C2C12 myotubes and detect intracellular calcium dynamics from it. This simple method would be a useful for studying properties in myotubes with specific sizes and morphologies.
Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/metabolism , Animals , Cell Differentiation/physiology , Cell Line , Dimethylpolysiloxanes/chemistry , Methacrylates/chemistry , Mice , Nylons/chemistry , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/chemistryABSTRACT
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.