Quantitative Assessment of Neuronal Differentiation in Three-dimensional Collagen Gels Using Enhanced Green Fluorescence Protein Expressing PC12 Pheochromocytoma Cells
- 220 Downloads
There is a paucity of quantitative methods for evaluating the morphological differentiation of neuronal cells in a three-dimensional (3-D) system to assist in quality control of neural tissue engineering constructs for use in reparative medicine. Neuronal cells tend to aggregate in the 3-D scaffolds, hindering the application of two-dimensional (2-D) morphological methods to quantitate neuronal differentiation. To address this problem, we developed a stable transfectant green fluorescence protein (GFP)-PC12 neuronal cell model, in which the differentiation process in 3-D can be monitored with high sensitivity by fluorescence microscopy. Under 2-D conditions, the green cells showed collagen adherence, round morphology, proliferation properties, expression of the nerve growth factor (NGF) receptors TrkA and p75NTR, stimulation of extracellular signal-regulated kinase phosphorylation by NGF and were able to differentiate in a dose-dependent manner upon NGF treatment, like wild-type (wt)-PC12 cells. When grown within 3-D collagen gels, upon NGF treatment, the GFP-PC12 cells differentiated, expressing long neurite outgrowths. We describe here a new validated method to measure NGF-induced differentiation in 3-D. Having properties similar to those of wt-PC12 and an ability to grow and differentiate in 3-D structures, these highly visualized GFP-expressing PC12 cells may serve as an ideal model for investigating various aspects of differentiation to serve in neural engineering.
KeywordsGreen fluorescent PC12 cells NGF Neuronal differentiation Three-dimensional collagen gel
This study was supported by grants from the Stein Family Foundation, Philadelphia, PA (PIL and PL), the Nanotechnology Institute of Southeastern Pennsylvania (PIL), and the United States–Israel Binational Science Foundation (PL). PL is affiliated with and supported in part by the David R. Bloom Center for Pharmacy and the Dr. Adolf and Klara Brettler Center for Research in Molecular Pharmacology and Therapeutics at The Hebrew University of Jerusalem, Israel. SL is supported by an “Eshkol” fellowship from The Israel Ministry of Science, Culture and Sport.
- Arien-Zakay, H., Nagler, A., Galski, H., & Lazarovici, P. (2007). Neuronal conditioning medium and nerve growth factor induce neuronal differentiation of collagen-adherent progenitors derived from human umbilical cord blood. Journal of Molecular Neuroscience, 32, 179–191. doi: 10.1007/s12031-007-0027-2.PubMedCrossRefGoogle Scholar
- Bieberich, E., & Anthony, G. E. (2004). Neuronal differentiation and synapse formation of PC12 and embryonic stem cells on interdigitated microelectrode arrays: Contact structures for neuron-to-electrode signal transmission (NEST). Biosensors & Bioelectronics, 19, 923–931. doi: 10.1016/j.bios.2003.08.016.CrossRefGoogle Scholar
- Guterman, E., Cheng, S., Palouian, K., Bidez, P. R., Lelkes, P. I., & Wei, Y. (2002). Peptide-modified electroactive polymers for tissue engineering applications. Polymer Preprints, 43, 766–767.Google Scholar
- Holmes, T. C., de Lacalle, S., Su, X., Liu, G., Rich, A., & Zhang, S. (2000). Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. Proceedings of the National Academy of Sciences of the United States of America, 97, 6728–6733. doi: 10.1073/pnas.97.12.6728.PubMedCrossRefGoogle Scholar
- Leach, J. B., Brown, X. Q., Jacot, J. G., Dimilla, P. A., & Wong, J. Y. (2007). Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity. Journal of Neural Engineering, 4, 26–34. doi: 10.1088/1741-2560/4/2/003.PubMedCrossRefGoogle Scholar
- Moxon, K. A., Hallman, S., Aslani, A., Kalkhoran, N. M., & Lelkes, P. I. (2007). Bioactive properties of nanostructured porous silicon for enhancing electrode to neuron interfaces. Journal of Biomaterials Science. Polymer Edition, 18, 1263–1281. doi: 10.1163/156856207782177882.PubMedCrossRefGoogle Scholar
- Sales, V. L., Mettler, B. A., Lopez-Ilasaca, M., Johnson Jr, J. A., & Mayer Jr., J. E. (2007). Endothelial progenitor and mesenchymal stem cell-derived cells persist in tissue-engineered patch in vivo: Application of green and red fluorescent protein-expressing retroviral vector. Tissue Engineering, 13, 525–535. doi: 10.1089/ten.2006.0128.PubMedCrossRefGoogle Scholar
- Schenke-Layland, K., Riemann, I., Damour, O., Stock, U. A., & Konig, K. (2006). Two-photon microscopes and in vivo multiphoton tomographs—Powerful diagnostic tools for tissue engineering and drug delivery. Advanced Drug Delivery Reviews, 58, 878–896. doi: 10.1016/j.addr.2006.07.004.PubMedCrossRefGoogle Scholar
- Simons, D. M., Gardner, E. M., & Lelkes, P. I. (2006). Dynamic culture in a rotating-wall vessel bioreactor differentially inhibits murine T-lymphocyte activation by mitogenic stimuli upon return to static conditions in a time-dependent manner. Journal of Applied Polymer Science, 100, 1287–1292. doi: 10.1152/japplphysiol.00887.2005.Google Scholar
- Takman, R., Jiang, H., Schaefer, E., Levine, R. A., & Lazarovici, P. (2004). Nerve growth factor pretreatment attenuates oxygen and glucose deprivation-induced c-Jun amino-terminal kinase 1 and stress-activated kinases p38alpha and p38beta activation and confers neuroprotection in the pheochromocytoma PC12 Model. Journal of Molecular Neuroscience, 22, 237–250. doi: 10.1385/JMN:22:3:237.PubMedCrossRefGoogle Scholar
- Tatard, V. M., Venier-Julienne, M. C., Benoit, J. P., Menei, P., & Montero-Menei, C. N. (2004). In vivo evaluation of pharmacologically active microcarriers releasing nerve growth factor and conveying PC12 cells. Cell Transplantation, 13, 573–583. doi: 10.3727/000000004783983675.PubMedCrossRefGoogle Scholar