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Journal of Molecular Neuroscience

, Volume 37, Issue 3, pp 225–237 | Cite as

Quantitative Assessment of Neuronal Differentiation in Three-dimensional Collagen Gels Using Enhanced Green Fluorescence Protein Expressing PC12 Pheochromocytoma Cells

  • Hadar Arien-Zakay
  • Shimon Lecht
  • Anat Perets
  • Blair Roszell
  • Peter I. Lelkes
  • Philip LazaroviciEmail author
Article

Abstract

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.

Keywords

Green fluorescent PC12 cells NGF Neuronal differentiation Three-dimensional collagen gel 

Notes

Acknowledgments

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.

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Copyright information

© Humana Press 2008

Authors and Affiliations

  • Hadar Arien-Zakay
    • 1
  • Shimon Lecht
    • 1
  • Anat Perets
    • 2
  • Blair Roszell
    • 2
  • Peter I. Lelkes
    • 2
  • Philip Lazarovici
    • 1
    • 2
    Email author
  1. 1.Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of MedicineThe Hebrew University of JerusalemJerusalemIsrael
  2. 2.School of Biomedical Engineering, Science and Health SystemsDrexel UniversityPhiladelphiaUSA

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