Neural Differentiation Of PC12 Cells Research Articles

Effect of Electrical Stimulation on PC12 Cells Cultured in Different Hydrogels: Basis for the Development of Biomaterials in Peripheral Nerve Tissue Engineering.

Extensive damage to peripheral nerves is a health problem with few therapeutic alternatives. In this context, the development of tissue engineering seeks to obtain materials that can help recreate environments conducive to cellular development and functional repair of peripheral nerves. Different hydrogels have been studied and presented as alternatives for future treatments to emulate the morphological characteristics of nerves. Along with this, other research proposes the need to incorporate electrical stimuli into treatments as agents that promote cell growth and differentiation; however, no precedent correlates the simultaneous effects of the types of hydrogel and electrical stimuli. This research evaluates the neural differentiation of PC12 cells, relating the effect of collagen, alginate, GelMA, and PEGDA hydrogels with electrical stimulation modulated in four different ways. Our results show significant correlations for different cultivation conditions. Electrical stimuli significantly increase neural differentiation for specific experimental conditions dependent on electrical frequency, not voltage. These backgrounds allow new material treatment schemes to be formulated through electrical stimulation in peripheral nerve tissue engineering.

Exosomes from magnetic particles-primed mesenchymal stem cells enhance neural differentiation of PC12 cells

This study aimed to investigate the effects of mesenchymal stem cell exosomes loaded with Fe3O4 magnetic particles (Fe3O4@ MSC-exo) on the survival and neural differentiation of PC12 cells. Exosomes were separated from Fe3O4 magnetic nanoparticles-primed umbilical cord mesenchymal stem cells condition medium by ultracentrifugation and characterized by transmission electron microscopy, flow nano analysis, and western blotting. PC12 cells were treated with culture medium containing exosomes. The effects of Fe3O4@ MSC-exo on PC12 cell proliferation, migration, and neural differentiation were analyzed using CCK-8 assay, transwell migration assay, RT-qPCR, and immunofluorescence, respectively. Additionally, miRNA sequencing was performed on Fe3O4@ MSC-exo, followed by bioinformatic analysis of the results. We found that Fe3O4@ MSC-exo can promote PC12 cell proliferation, migration, and neural differentiation. According to the sequencing results, there were a total of 43 differentially expressed miRNAs. The present study indicated that Fe3O4@ MSC-exo might enhance nerve cell function, laying the foundation for targeted therapy of nerve injury.

Human adipose derived stem cell exosomes enhance the neural differentiation of PC12 cells.

Human adipose stem cells (hADSCs) are proper cell sources for tissue regeneration. They mainly mediate their therapeutic effects through paracrine factors as exosomes. The exosomes contents are protein, lipid and RNA. Exosomes are effective in restoring the function of neurons and astrocytes in neurodegenerative diseases, and improve the therapeutic outcomes. We investigated the effect of hADSCs derived exosomes on survival and neural differentiation of PC12 cells in vitro. The isolated hADSCs, were characterized by flow cytometry. Exosomes were separated from hADSC-condition medium using Exo-spinTM kit and characterized by DLS and TEM. Then acridine orange staining was performed to confirm entrance of exosomes into PC12 cells. PC12 cells were treated with culture medium containing NGF and exosome. Cell viability was assessed by MTT assay, and neural differentiation by ICC technique and qRT-PCR. TEM and DLS data confirmed the isolation of exosomes according to their size (30-100nm) and acridine orange staining indicated entrance of exosomes to target cells. MTT assay showed that cell viability was significantly increased in exosome treated group. ICC technique revealed that the expression of Map2 was superior in the exosome treated group. Based on qRT-PCR data, Map2 and β-tub III gene expression was increased in the exosome treated group. Significant expression of Gfap was seen in the NGF and NGF/EXO treated groups. Present study indicated that hADSCs derived exosomes might enhance cell viability and promote neuronal differentiation and expression of mature neural marker in PC12 cells.

3D-Printed PCL/rGO Conductive Scaffolds for Peripheral Nerve Injury Repair.

The incidence of peripheral nerve injuries is on the rise and the current gold standard for treatment of such injuries is nerve autografting. Given the severe limitations of nerve autografts which include donor site morbidity and limited supply, neural guide conduits (NGCs) are considered as an effective alternative treatment. Conductivity is a desired property of an ideal NGC. Reduced graphene oxide (rGO) possesses several advantages in addition to its conductive nature such as high surface area to volume ratio due to its nanostructure and has been explored for its use in tissue engineering. However, most of the works reported are on traditional 2D culture with a layer of rGO coating, while the native tissue microenvironment is three-dimensional. In this study, PCL/rGO scaffolds are fabricated using electrohydrodynamic jet (EHD-jet) 3D printing method as a proof of concept study. Mechanical and material characterization of the printed PCL/rGO scaffolds and PCL scaffolds was done. The addition of rGO results in softer scaffolds which is favorable for neural differentiation. In vitro neural differentiation studies using PC12 cells were also performed. Cell proliferation was higher in the PCL/rGO scaffolds than the PCL scaffolds. Reverse transcription polymerase chain reaction and immunocytochemistry results reveal that PCL/rGO scaffolds support neural differentiation of PC12 cells.

Fabricating Muscle-Neuron Constructs with Improved Contractile Force Generation.

In this study, we fabricated innervated skeletal muscle tissue constructs comprising C2C12 myoblasts and PC12 neural cells using a magnetic force-based tissue engineering technique. We found that the C2C12/PC12 co-culture enhanced neural differentiation of PC12 cells and sarcomere formation of C2C12 myotubes, accompanying with neuromuscular junction formation. The innervated skeletal muscle tissue constructs generated significantly higher contractile forces compared with aneural (C2C12 monoculture) skeletal muscle tissue constructs. These innervated skeletal muscle tissue constructs can be a useful tool for drug testing and biological research for neuromuscular diseases.

Biocompatible Electroactive Tetra(aniline)-Conjugated Peptide Nanofibers for Neural Differentiation.

Peripheral nerve injuries cause devastating problems for the quality of patients' lives, and regeneration following damage to the peripheral nervous system is limited depending on the degree of the damage. Use of nanobiomaterials can provide therapeutic approaches for the treatment of peripheral nerve injuries. Electroactive biomaterials, in particular, can provide a promising cure for the regeneration of nerve defects. Here, a supramolecular electroactive nanosystem with tetra(aniline) (TA)-containing peptide nanofibers was developed and utilized for nerve regeneration. Self-assembled TA-conjugated peptide nanofibers demonstrated electroactive behavior. The electroactive self-assembled peptide nanofibers formed a well-defined three-dimensional nanofiber network mimicking the extracellular matrix of the neuronal cells. Neurite outgrowth was improved on the electroactive TA nanofiber gels. The neural differentiation of PC-12 cells was more advanced on electroactive peptide nanofiber gels, and these biomaterials are promising for further use in therapeutic neural regeneration applications.

A novel method of neural differentiation of PC12 cells by using Opti-MEM as a basic induction medium.

The PC12 cell line is a classical neuronal cell model due to its ability to acquire the sympathetic neurons features when deal with nerve growth factor (NGF). In the present study, the authors used a variety of different methods to induce PC12 cells, such as Opti-MEM medium containing different concentrations of fetal bovine serum (FBS) and horse serum compared with RPMI-1640 medium, and then observed the neurite length, differentiation, adhesion, cell proliferation and action potential, as well as the protein levels of axonal growth-associated protein 43 (GAP-43) and synaptic protein synapsin-1, among other differences. Compared with the conventional RPMI-1640 medium induction method, the new approach significantly improved the neurite length of induced cells (2.7 times longer), differentiation rate (30% increase), adhesion rate (21% increase) and expression of GAP-43 and synapsin-1 (three times), as well as reduced cell proliferation. The morphology of induced cells in Opti-MEM medium containing 0.5% FBS was more like that of neurons. Additionally, induced cells were also able to motivate the action potential after treatment for 6 days. Therefore, the research provided a novel, improved induction method of neural differentiation of PC12 cells using Opti-MEM medium containing 0.5% FBS, resulting in a better neuronal model cell line that can be widely used in neurobiology and neuropharmacology research.

Three-Dimensional Laminin Mimetic Peptide Nanofiber Gels for In Vitro Neural Differentiation.

The extracellular matrix (ECM) provides biochemical signals and structural support for cells, and its functional imitation is a fundamental aspect of biomaterial design for regenerative medicine applications. The stimulation of neural differentiation by a laminin protein-derived epitope in two-dimensional (2D) and three-dimensional (3D) environments is investigated. The 3D gel system is found to be superior to its 2D counterpart for the induction of neural differentiation, even in the absence of a crucial biological inducer in nerve growth factor (NGF). In addition, cells cultured in 3D gels exhibits a spherical morphology that is consistent with their form under in vivo conditions. Overall, the present study underlines the impact of bioactivity, dimension, and NGF addition, as well as the cooperative effects thereof, on the neural differentiation of PC-12 cells. These results underline the significance of 3D culture systems in the development of scaffolds that closely replicate in vivo environments for the formation of cellular organoid models in vitro.

Vimentin is important in the neural differentiation of PC12 cells promoted by sialylation.

Sialic acid modification is a kind of post-translational modification. To investigate the regulation effect of sialic acid on neural differentiation, we used CycloManN propanyl perac (CycloManN pro), a metabolic precursor of sialic acid, to treat PC12 cells. We noted that CycloManN pro indeed robustly promoted global sialylation detected by MAL II lectin blot in PC12 cells. Simultaneously, we interestingly found that the neurite outgrowth of PC12 cells was significantly promoted by the CycloManN pro treatment. The profile analysis of sialylated proteins showed that a protein band at 55KD was greatly enhanced especially in PC12L cells after CycloManN pro treatment. After enrichment with lectin MAL II, the proteins in this band were analyzed by mass spectrometry. The results showed that 23 proteins were in the band, but the score of vimentin was the highest among them. To investigate further the role of vimentin in the process of neurite differentiation, vimentin construct was transfected into PC12 cells. We interestingly observed that ectopic expression of vimentin significantly enhanced the neurite outgrowth induced by CycloManN pro. However, after three potential glycosylation sites (Ser-7, Thr-33, Ser-34:) of vimentin were mutated to alanine, overexpression of the mutated vimentin completely lost the enhancement activity for the neural differentiation even in the presence of CycloManN pro. Taken together, our study demonstrated that vimentin was important in the induction of neural differentiation by CycloManN pro.

Effects and mechanisms of melatonin on the proliferation and neural differentiation of PC12 cells

Melatonin, a lipophilic molecule that is mainly synthesized in the pineal gland, performs various neuroprotective functions. However, the detailed role and mechanisms of promoting neuronal differentiation remains limited. This study demonstrated that 10 μM melatonin led to significant increases in the proliferation and neurite outgrowth of PC12 cells. Increased expression of microtubule-associated protein 2 (MAP2, a neuron-specific protein) was also observed. However, luzindole (melatonin receptor antagonist) and PD98059 (MEK inhibitor) attenuated these increases. LY294002 (AKT inhibitor) inhibited melatonin-mediated proliferation in PC12 cells and did not affect melatonin-induced neural differentiation. The expression of p-ERK1/2/ERK1/2 was increased by melatonin treatment for 14 days in PC12 cells, whereas luzindole or PD98059 reduced the melatonin-induced increase. These results suggest that the activation of both the MEK/ERK and PI3K/AKT signaling pathways could potentially contribute to melatonin-mediated proliferation, but that only the MEK/ERK pathway participates in the melatonin-induced neural differentiation of PC12 cells. Altogether, our study demonstrates for the first time that melatonin may exert a positive effect on neural differentiation via melatonin receptor signalling and that the MEK/ERK1/2 signalling may act down stream from the melatonin pathway.

Nanofiber containing carbon nanotubes enhanced PC12 cell proliferation and neuritogenesis by electrical stimulation.

The nervous system is an important regulator of the human body because it adapts our responses to the external environment and provides people the ability of thought, memory, and emotion. PC12 is a cell line that is commonly used to study the behavior of neural differentiation. PC12 cells further differentiate into nerve cells when stimulated by nerve growth factor (NGF), which have neurite, dendrite, and axon, and form synapses with neighboring cells to build neural networks. Micropatterns and electric stimulation can significantly influence cellular attachment, proliferation, orientation, extracellular matrix (ECM) expression, neural differentiation, and cellular motion. We fabricated polycaprolactone (PCL) nanofiber with or without carbon nanotubes (CNTs) by electrospinning and promoted the neural differentiation of PC12 cells by electric stimulation. We used scanning electron microscope (SEM) and fluorescence microscope to observe the NGF-induced growth of PC12 cells on PCL nanofiber. Axon formation and cellular activity expression, that confirm that PC12 cells can grow well on PCL nanofiber, and the gene expressions of MAP1b and GAP43 significantly increased after electric stimulation. Based on the results, the structure of nanofibers containing CNTs can effectively induce neural differentiation of PC12 cells in an electric field. This experimental model can be used for future clinical applications.

Effective Spatial Separation of PC12 and NIH3T3 Cells by the Microgrooved Surface of Biocompatible Polymer Substrates

Most organs and tissues are composed of more than one type of cell that is spatially separated and located in different regions. This study used a microgrooved poly(lactic-co-glycolic acid) (PLGA) substrate to guide two types of cocultured cells to two spatially separated regions. Specifically, PC12 pheochromocytoma cells are guided to the inside of microgrooves, whereas NIH3T3 fibroblasts are guided to the ridge area in between neighboring parallel microgrooves. In addition, the microgrooved structures can significantly promote the proliferation and neural differentiation of PC12 cells as well as the osteogenic differentiation of NIH3T3 cells. Therefore, the microgrooved PLGA surface with separated PC12 and NIH3T3 cells can serve as a potential model system for studying nerve reconstruction in bone-repairing scaffolds.

Up-regulation of Idh3α causes reduction of neuronal differentiation in PC12 cells

The PC12 is the widely used cell line to study neuronal differentiation. We had extensively investigated the details of protein expression in differentiated PC12 cells by proteomic analysis. The cells were incubated at the presence of nerve growth factor. We had analyzed the expression changes in the differentiating PC12 cells by 2-dimensional electrophoresis and the identification of the proteins using MALDI-TOF MS. By comparing expression pattern in the time course, we identified the candidate genes which are associated with neuronal differentiation. Among these genes, we performed real-time PCR analysis to validate Idh3alpha expression by the time course. To identify the function of Idh3alpha in neuronal differentiation stage, the transfection of Idh3alpha to PC12 cells was performed. As a result, we proved that up-regulation of Idh3alpha causes reduction in neural differentiation of PC12 cells. Based on these data, we suggest that Idh3alpha plays a role to the neuronal differentiation.

Evaluation of anti-Fas ligand-induced apoptosis and neural differentiation of PC12 cells treated with nerve growth factor using small interfering RNA method and sampling by microdialysis

The small interfering RNA (siRNA) method is an effective technique for silencing gene expression and is a useful tool for screening the gene functions in drug discovery. Our study found that nerve growth factor (NGF) can increase the cell viability of PC12 cells and that NGF induction up-regulates the expression of Bcl-2 detected by real-time reverse transcription–polymerase chain reaction (RT–PCR). To further investigate the role of Bcl-2 expression in NGF-treated PC12 cells, the plasmid of Bcl-2 siRNA was then transfected into PC12 cells. Moreover, to investigate and continuously monitor the real-time dynamic neurotransmitter release, and to compare with the time course of Bcl-2 expression, a liquid chromatography coupled with electrochemical detection (LC–ED) and with a microdialysis device was used. After 6 h of NGF being added to the PC12 cell culture medium, the dopamine (DA) concentrations were significantly increased ( P < 0.05). This result is simultaneously compatible with the up-regulated messenger RNA (mRNA) expressions of tyrosine hydroxylase (TH), aromatic acid decarboxylase (AADC), and Bcl-2 by RT–PCR. Using the Bcl-2 siRNA method, our data revealed that NGF can inhibit Fas ligand (FasL)-induced apoptosis in PC12 cells through the activation of Bcl-2. The in vitro observation further demonstrated that NGF can stimulate the neurite development in PC12 cells through the activation of Bcl-2. Moreover, the DA concentrations of NGF induction were decreased specifically by Bcl-2 siRNA ( P < 0.05). In sum, our data support that NGF prevents Fas-induced apoptosis, facilitates neural differentiation, promotes dendritic formation, and increases DA release in PC12 cells through activation of Bcl-2.

Collaboration of JNKs and ERKs in nerve growth factor regulation of the neurofilament light chain promoter in PC12 cells.

Nerve growth factor (NGF) induces transcription-dependent neural differentiation of PC12 cells, and the ERK family of MAPKs has been implicated as the dominant signal pathway that mediates this response. We employed a neurofilament light chain (NFLC) promoter-luciferase (NFLC-Luc) reporter to define the role of the ERKs as well as additional MAPK pathways in NGF induction of this neural specific gene. Constitutive active forms of c-Raf-1, MEKK1 and MKK6, proximal regulators of the ERKs, JNKs, and p38 MAPKs, respectively, all stimulated NFLC-Luc activity. NFLC-Luc activity stimulated by NGF, however, was partially (approximately 50%) inhibited by the MEK inhibitor, PD098059, or by co-transfection of kinase-inactive MEK1 but not by the p38 MAPK inhibitor, SB203580, indicating a role for the ERKs, but not the p38 MAPKs, in NGF regulation of the NFLC promoter. Importantly, a gain-of-function MKK7-JNK3 fusion protein stimulated NFLC-Luc and synergized with gain-of-function c-Raf-1 to activate the NFLC promoter. In addition, transfection of kinase-inactive forms of MEK1 and MKK7 produced an additive inhibition of NGF-stimulated NFLC-Luc relative to either inhibitor alone. These findings indicate that the ERK and JNK pathways collaborate downstream of the NGF receptor for regulation of the NFLC promoter. Truncation analysis and electromobility shift assays established the requirement for a cAMP-response element/activating transcription factor-like site in the NFLC promoter that minimally interacts with constitutively expressed cAMP-response element-binding protein and JunD as well as c-Jun which is induced by NGF in an ERK-dependent manner. Cumulatively, these findings indicate that the ERK pathway requires collaboration with the JNK pathway for maximal activation of the NFLC gene in PC12 cells through the integrated control of c-Jun function.

Lysophosphatidylserine potentiates nerve growth factor-induced differentiation of PC12 cells

Since lysophosphatidylserine (LPS) is required for nerve growth factor (NGF)-induced secretion of histamine from rat mast cells, we investigated whether LPS might potentiate the effects of NGF in inducing neural differentiation of PC12 cells. Cell morphology was evaluated 48 h after addition of NGF, LPS or NGF+LPS. LPS alone was ineffective, but strongly promoted NGF-induced differentiation to give rise to cells that more closely resembled neurons in primary culture. LPS increased the number of PC12 cells that developed neurites in response to NGF (0.01–40 ng/ml), with the response to 1.0 ng/ml increasing from 17.8±2.2 to 50.8±4.1% when LPS was also present. Neurite length was also greater in PC12 cells receiving NGF+LPS: 17.8±2.2% of cells had neurites longer than three cell body diameters with 1.0 ng/ml NGF+1 μg/ml LPS, compared to 1.6±1.6% with NGF alone. Further, cells responding to NGF+LPS typically developed only 1–2 neurites per cell (90.9%, 1 μg/ml LPS), compared with the multipolar appearance with NGF alone (71.1% with 3–6 neurites, 10 ng/ml NGF). LPS occurs at sites of tissue damage where NGF can also be present, and therefore may be a naturally-occurring modifier of neuronal structure and/or function.

Cloning of cDNA and expression of the gene encoding rat presenilin-2

We have cloned the rat homologue of the presenilin-2 ( PS-2) cDNA. PS-2 is responsible for chromosome 1-linked familial Alzheimer's disease. Sequence analysis predicted that the rat PS-2 encodes a 448 amino acid (aa) protein, and there was a very high degree of amino acid identity between rat and human PS-2 (95%). All the mutated codons in PS-2 and PS-1 in chromosome 1- or 14-linked familial Alzheimer's disease patients were conserved in rat PS-2. The expression of PS-2 was weaker than that of PS-1. The alternatively spliced short form of PS-2 mRNA, which was detected in human tissues was not detected in various rat tissues. During brain development, the expression level of both PS-2 and PS-1 increased but decreased in the adult. No remarkable change was observed in neural differentiation of PC12 cells.

Nerve growth factor induces changes in (2′–5′)oligo(A) synthetase and 2′-phosphodiesterase activities during differentiation of PC12 pheochromocytoma cells

Treatment of rat pheochromocytoma cell line PC12 with Vipera lebetina (snake) nerve growth factor (NGF) induces a rapid increase (from 5 to 25-fold) in the level of (2′–5′)oligo(A) synthetase activity and a simultaneous decrease (from 2 to 5-fold) in the activity of 2′–5′A degrading enzymes—2′-phosphodiesterases (2′-PDE). These changes in the enzyme activities led to the significant increase in the intracellular concentration of 2′–5′A. We have found that the serum starvation of PC12 cells causes a 1.5 to 2.0-fold increase in the level of 2′–5′A-synthetase activity, but the ativities of 2′-PDE and the intracellular concentration of 2′–5′A remain unaltered. These results show that NGF modulates the activity of (2′–5′)oligo(A) enzymes and intracellular concentration of 2′–5′A during the neural differentiation of PC12 cells.