Quantitative assessment of neuronal differentiation in three-dimensional collagen gels using enhanced green fluorescence protein expressing PC12 pheochromocytoma cells.
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 p75(NTR), 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.
Arien-Zakay
Hadar
H
Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, 91120, Israel.
Lecht
Shimon
S
Perets
Anat
A
Roszell
Blair
B
Lelkes
Peter I
PI
Lazarovici
Philip
P
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation; D003094:Collagen / Q000378:metabolism*; D005782:Gels / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D016716:PC12 Cells / Q000166:cytology / Q000502:physiology; D051381:Rats; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism
D005782:Gels; D017475:Receptors, Nerve Growth Factor; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003094:Collagen