J Mol Neurosci (2009) 37:225–237
DOI 10.1007/s12031-008-9123-1
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 Lazarovici
Received: 27 May 2008 / Accepted: 5 June 2008 / Published online: 16 July 2008
# Humana Press 2008
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 differ-
entiation. 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, ex-
pression 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
H. Arien-Zakay : S. Lecht : P. Lazarovici (*)
Department of Pharmacology and Experimental Therapeutics,
School of Pharmacy, Faculty of Medicine,
The Hebrew University of Jerusalem,
P.O. Box 12065, Jerusalem 91120, Israel
e-mail: philipl@ekmd.huji.ac.il
A. Perets : B. Roszell : P. I. Lelkes : P. Lazarovici
School of Biomedical Engineering, Science and Health Systems,
Drexel University,
Philadelphia, PA 19122, USA
an ideal model for investigating various aspects of differen-
tiation to serve in neural engineering.
Keywords Green fluorescent PC12 cells . NGF .
Neuronal differentiation . Three-dimensional collagen gel
Introduction
The PC12 cell line has been extensively used in neuroscience
research as a neuronal model for studying neuronal signaling
(Vaudry et al. 2002) and has become the model of choice for
the study of neuronal differentiation (Fujita et al. 1989;
Ravni et al. 2006). When treated in conventional two
dimensional (2-D) tissue culture conditions with nanomolar
concentrations of nerve growth factor (NGF), PC12 cells
stop dividing, elaborate neurite processes (outgrowths), and
become electrically excitable (Fujita et al. 1989). NGF
biological effects are mediated by two types of receptors:
the NGF high-affinity tyrosine kinase (TrkA) receptor
(Kaplan and Miller 2000) and the low-affinity neurotrophin
receptor p75 (p75NTR), a member of the tumor necrosis
factor receptor superfamily (Chao et al. 1998). Activation
of these receptors by NGF initiates differentiation of the
cells with a time course of several weeks, characterized by
processes such as proliferation arrest and initiation of the
neurite outgrowths in the first 2 days of treatment;
continuous, progressive elongation of the neurites for up
to 3 weeks of treatment, including extensive sprouting
(branching) and synapse connections between the neurites;
and generation of neurite networks and ganglia-like cell
organization due to aggregation of neuronal cell bodies
(Fujita et al. 1989).
Lately, PC12 cells have become an important cellular
tool in neural engineering. Their morphological features,
226
such as growth, branching, differentiation, and synapse
formation were found to be influenced by different matrix-
resident biophysical cues such as micro- and nanotopog-
raphy (Foley et al. 2005; Moxon et al. 2007), including
substrate stiffness (Leach et al. 2007). Furthermore, PC12
cells were used to explore the microenvironmental adequacy
for neuronal cells of new bioengineering materials such as
agarose scaffolds (Yu et al. 1999), peptide scaffolds (Holmes
et al. 2000), neuron–microelectrode interfaces (Bieberich and
Anthony 2004; Moxon et al. 2007), microchannel-containing
substrates (Mahoney et al. 2005), and microfabricated
silicon-based nanostructures (Lopez et al. 2006; Moxon
et al. 2007).
A major issue in neuroengineering is the investigation
of the biocompatibility of scaffolds with neuronal cells,
aimed at generating a three-dimensional (3-D) neuronal
constructs for implantation in patients in order to repair
defective neural pathways. To achieve this goal, it is
important to use in vitro models in which it is possible to
visualize cell growth in the 3-D construct. In general, the
methods used to date include fixation of the neuronal-
containing scaffolds and histological evaluation of the
morphology of the fixed cells, allowing temporal “snap-
shots” at defined time points, but not a continual real time
monitoring of neuronal development. As an alternative,
nondestructive fluorescent labeling of the living cells
populating the 3-D construct may be advantageous. Such
labeling methods must also be compatible with the
histochemical methods commonly used for routine mor-
phological analysis (Tohill et al. 2004) and facilitate
noninvasive visualization of neuronal development.
The goal of the present study was to generate a
fluorescent PC12 cell model to be used in 3-D neuronal
engineering in order to address fundamental issues of
optimization of neuronal differentiation. In this study, we
generated a stable PC12 cell line expressing the green
fluorescent protein (GFP) from the jellyfish Aequorea
victoria. GFP does not naturally exist in mammalian
species and can be easily detected by UV light and
fluorescence microscopy. It also has the advantage of
being a “vital dye” whose presence can be monitored in
living organisms and unfixed tissue (Okabe et al. 1997). It
has the potential to elucidate nervous system development
(Niell and Smith 2004) and to clarify the complex
biological process of neurite outgrowth regulation (Laketa
et al. 2007). In this study, using the GFP-PC12 cells, we
defined a new morphological analysis for measurement of
neuronal cell differentiation in a 3-D environment and
validated this method according to a commonly used
bioassay for NGF-induced differentiation under 2-D
conditions.
J Mol Neurosci (2009) 37:225–237
Materials and Methods
Cell Cultures
PC12 cells (NIH clone, originally provided by Dr. G.
Guroff, NICHD, NIH) were grown in 25 cm2 flasks in
Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 7% fetal bovine serum (FBS), 7% horse
serum, 20 U/ml penicillin and 20 μg/ml streptomycin, all
purchased from Biological Industries (Beit Haemek, Israel).
Cells were incubated at 37°C in 6% CO2 (Katzir et al.
2002). The cells were detached from the flask mechanically
and washed twice with phosphate-buffered saline (PBS)
before reseeding.
Transduced 293T (human kidney epithelial, ATCC,
Manassas, VA, USA) cells were cultured in DMEM
containing 400 μg/ml of a neomycin analogue, G418
(Gibco BRL, Rockville, MD, USA), 10% FBS, 20 U/ml
penicillin, and 20 μg/ml streptomycin (Kosaka et al. 2004).
All experiments were carried out in a clean room, according
to ISO7 requirements (10,000 particles/m3) using a P2+
facility and lentiviral vector generation and transduction
according to NIH biosafety guidelines.
Lentivirus Production and Transduction
HIV-1-derived lentiviral vectors were generated by transient
transfection of a human kidney 293T cell line by a three-
plasmid expression system. The packaging plasmid encodes
HIV-1 gag, pol, tat, and rev proteins; the envelope plasmid
encodes the VSV/G glycoprotein gene; and the transfer
vector (TK113) encodes a CMV promoter driving the
expression of GFP (Kosaka et al. 2004). YTK 113 (vector),
VSV-G (packaging), and deltaNRF (envelope) plasmids
were transiently transfected into 293T cells using a calcium
phosphate transfection method (Kosaka et al. 2004). All
transfections were carried out using 60-mm dishes coated
with poly-L-lysine where 1.5–2.5×106 293T cells were
plated in 5 ml of complete medium 24 h before transfection.
One hour before transfection, the medium was replaced
with serum-free DMEM containing antibiotics. The DNA/
CaCl2 solution was prepared and left at room temperature
for 30 min before being added to the cells. The cells were
then returned to the 37°C incubator (5% CO2) for 24 h
when the medium was replaced with 7 ml of fresh
complete medium. After 48 h, the medium was removed
and filtered through a 0.45-μm filter to collect the viral
particles secreted into the medium. The viral titer was
adjusted to 4.0×107 IU/ml by dilution.
PC12 transduction with the virus was performed as
previously described for CD34+ human cells (Miyoshi et al.
J Mol Neurosci (2009) 37:225–237
1999). Briefly, cells were seeded at a density of 4×105
cells/well in six-well tissue culture plates coated with 10%
poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA). The
cells were transduced with 1 ml of 4.0×107 IU/ml TK113
viral particles (HIV-based vector from which the GFP
reporter gene is constitutively expressed under the control
of the cytomegalovirus promoter) and supplemented with
2 ml of PC12 medium. The cells were incubated for 3 days
post transduction in a 37°C incubator, 6% CO2, at high
humidity.
FACS Sorting
Flow cytometric analysis of the PC12-GFP/lentivirus-
transduced PC12 cell population was performed using a
FACS with fluorescence excitation at 488 nm wavelength
(Simons et al. 2006). In brief, the cell culture was
dissociated from the dish by agitation and resuspended in
PBS without magnesium and calcium, supplemented with
3% FBS and filtered through a 20-μm filter to generate
individually dispersed cells. Cell density was adjusted to
20×106 cells/ml. For analysis, 50,000 events were acquired
on a FACSCanto (Becton-Dickinson, San Jose, CA, USA)
flow cytometer using FACS Diva software (Becton-
Dickinson). Evaluation of flow data was performed using
FlowJo (Tree Star, Ashland, OR, USA). GFP-positive cells
were collected under aseptic conditions in 5 ml FACS
polystyrene tubes (BD Biosciences, Bedford, MA, USA)
containing 400 μl of FBS. Wild-type PC12 (wt-PC12) cells,
at the same density, served as the negative control.
Clonal Expansion of GFP-positive PC12 Cells
GFP-positive cells (3.5×105), aseptically sorted by the
FACS, were returned to culture. After 7 days of culture,
cloning of individual GFP-PC12 expressing cells was
performed by limiting dilution. The cells were then diluted
to a concentration of 1 cell/100 μl and aliquoted into 480
wells of five 96-well plates. One day later, a well
containing a single fluorescent cell was identified by
fluorescence microscopy to ensure that each colony would
be derived from a single cell. To promote proliferation, the
GFP-PC12 cells were grown in PC12 growth medium
supplemented with 10% conditioning medium collected
from a confluent wt-PC12 cell culture. The clones were
monitored every other day and split when the colony
reached 50% confluence. After 5 weeks of expansion, the
chosen clones were collected and frozen in PC12 medium
supplemented with 5% dimethylsulfoxide (Sigma-Aldrich).
For biological characterization, different parameters were
evaluated over a period of several weeks in samples of each
227
clone and wild-type cells. GFP expression was estimated by
fluorescence microscopy. Clone adherence was assessed by
monolayer generation; nonadherent cells grew in suspen-
sion or partially attached to the dish. The wt-PC12 cultures
contained six different morphotypes (round, monopolar,
bipolar, tripolar, multipolar, and fibroblast like). The
selected GFP-PC12 clones showed a similar heterogeneous
morphology.
2-D Cell Growth Conditions
wt-PC12 or GFP-PC12 cells were seeded on 24-well plates
coated with 200 μg/ml type I rat tail collagen (BD
Biosciences) and cultured as described above.
3-D Cell Growth Conditions
Sandwiched 3-D type I collagen gels at a final concentra-
tion of 1.5 mg/ml solution were constructed as previously
described (Dietrich and Lelkes 2006; Mondrinos et al.
2007). Briefly, depending on the final volume required,
calculated amounts of 10× Dulbecco’s PBS (Mediatech,
Herndon, VA, USA), 1 N NaOH, cell culture grade H2O,
and type I rat collagen (BD Biosciences) were mixed, and
the pH was adjusted under sterile conditions to 7.4±0.05.
To generate the bottom layer of the gel, 400 μl of the
collagen solution was added to each well of a 24-well plate
and allowed to polymerize for 15 min at 37°C. For the
second layer, wt-PC12 or GFP-PC12 cells were transferred
into one volume of serum-free medium, which was then
mixed with the collagen solution. A 500-μl volume of this
suspension was added on top of the first layer. The cells
were evenly distributed by brief agitation of the culture
plates. These were then incubated for 15 min in a
hybridization oven (37°C). After complete polymerization
of the various gel assemblies, 1 ml of growth medium,
supplemented as described for the 2-D cell culture
conditions, was added to the sandwich-like gel assemblies.
The cells were then cultured for various periods of time in a
conventional CO2 incubator (6% CO2, 37°C), and the
medium was replaced every 2 days.
Cell Proliferation
The proliferation of wt-PC12 and GFP-PC12 cells was
estimated daily over a period of up to 14 days using the
Alamar blue (BD Biosciences) staining method essentially
as previously described (Nikolaychik et al. 1996). Briefly,
in both 2-D and 3-D cell culture conditions, the wells were
incubated for 4 h with the Alamar blue reagent (10% v/v in
complete medium). At the end of the incubation period,
228
media samples were collected and their fluorescence
intensity was evaluated in an ELISA reader at a excitation
of 560 nm and emission of 595 nm at constant gain. Cell
proliferation was calculated as the relative increase in
fluorescence compared with day 0 (control).
NGF-induced Neuronal Differentiation
To assess neuronal differentiation, wt-PC12 or GFP-PC12
cells were seeded in glass chamber slides (Nunc, Roskilde,
Denmark) at a density of 4×105/cm2 on either collagen-
coated surfaces (2-D) or into 3-D collagen gels (1.5 mg/ml).
One day after seeding, the culture medium was replaced
with either the same PC12 medium (control) or with
differentiation medium (PC12 medium supplemented with
50 ng/ml mouse β-subunit, nerve growth factor 2S-mNGF;
Alomone Labs, Jerusalem, Israel). In the 3-D gel, NGF
diffused through the gel allowing interaction with the cells
(Takezawa et al. 2007). The cells were then cultured for up
to 45 days in a conventional CO2 incubator (6% CO2, 37°C).
Neuronal Differentiation Analysis
To assess neuronal differentiation, neurite outgrowth length
was quantitated using the SigmaScanPro 5.0 program as
previously described by us (Katzir et al. 2002). Briefly, the
total length of outgrowths from each cell in the region of
interest (ROI) was divided by its diameter, thus generating
an elongation factor, denoted elongation (E) parameter. The
experiments were performed at least three times and in
duplicate. In each well, three ROIs were photographed in
randomly chosen regions. In each ROI, 15–25 cells were
measured, i.e., at least 270 cells were measured for each
treatment. Since neuronal cells in 3-D cultures generate
aggregates rendering it difficult to measure the perykarion
diameter of individual cells, we characterized a fractal
dimension (Df) as a suitable parameter for quantitative
measurements of neuronal differentiation. This approach,
introduced here for the first time for analyzing neuronal
outgrowth, is based on a method utilized for blood capillary
network sprouting measurements (Kirchner et al. 1996;
Lazarovici et al. 2006). ROIs were acquired in 2,560×
1,920 pixel dimensions and with resolution of 300 pixels/
inch. Each image was transformed to 0–255 gray scale, and
a new transparent layer was generated using Photoshop™.
Using a 3-pixel wide digital pencil tool, all outgrowths
were overlaid. The layer containing outgrowth overlays
(outgrowths network) was saved separately as an 8-bit
JPEG file. The length and complexity of each outgrowth
networks image were measured using ImageJ software,
utilizing fractal box count analysis to facilitate visual
inspection of the neuronal network morphology, and
“skeletonized” to calculate the fractal dimension (Df). The
J Mol Neurosci (2009) 37:225–237
Df is a statistical descriptor of fractal space (area and
length) filled by neurite outgrowth (Kirchner et al. 1996). In
our experiments Df ranged from 0.6 to 1.25. According to this
method, the neuronal network of neurite outgrowths is
overlaid with a series of square boxes of decreasing size
(denoted in pixels, p) and the number of boxes (Np) containing
at least 1 pixel is counted. The negative value of the least-
squares regression slope of the plot of log Np vs log p yields
Df (Kirchner et al. 1996). The structure was considered fractal
if the r2 value of the regression line was >0.95.
Reverse Transcriptase Polymerase Chain Reaction
(RT-PCR)
The total RNA of NGF-treated and -untreated GFP-PC12
cells was isolated and genomic DNA was degraded from
the RNA preparations, using the SV total RNA isolation
system (Promega, Madison, WI, USA). A quantity of 1 μg
of total RNA was reverse transcribed using the Reverse
Transcription System (Promega), according to the manu-
facturer’s instructions. Then, PCR was performed in a final
volume of 50 μl containing 5 μg complementary DNA
(cDNA), 50 pmol of upstream sense and downstream sense
primers, and 25 μl of GoTaq® Green Master Mix
(Promega). The cDNA was amplified by 35 cycles. To
generate various cDNA fragments, a Mastercycler gradient
(Eppendorf, Germany) was programmed as follows: dena-
turation of cyclic parameters at 94°C for 1 min, annealing at
65°C for 1 min, and elongation at 72°C for 2 min (Arien-
Zakay et al. 2007). To identify the various messenger RNA
(mRNA) transcripts, the following primers were used:
& β-actin (285 bp, internal control)
sense: TCATGAAGTGTGACGTTGACATCCGT-3′
antisense: CTTAGAAGCATTTGCGGTGCACGATG-3′
& TrkA (571 bp)
sense: CACTAACAGCACATCAAGAGAC-3′
antisense: GAAGACCATGAGCAATGGG-3′
& p75NTR (663 bp)
sense: AGCCAACCAGACCGTGTGTG-3′
antisense: TTGCAGCTGTTCCACCTCTT-3′
All PCR products were analyzed by electrophoresis on
agarose gel (2%) containing ethidium bromide for UV
visualization. Wt-PC12-derived mRNA was used as a
positive control for validation.
Western Blot
Total cellular protein was isolated using lysis buffer
(Clontech, Mountain View, CA, USA). The amount of total
J Mol Neurosci (2009) 37:225–237
protein extracted was determined by Bio-Rad protein assay
(Bio-Rad, Hercules, CA, USA). Protein samples of 40 μg
were separated by SDS-PAGE (8–10%). The proteins were
electrotransferred (100 V, 1.5 h, 4°C) to nitrocellulose
membranes and incubated for 2 h at room temperature (RT)
with 5% nonfat powdered milk in Tris-buffered saline
containing 0.1% Tween 20. Immunodetection of phosphory-
lated ERK was performed overnight at 4°C using rabbit
monoclonal anti-phospho-p44/42 MAPK (extracellular signal-
regulated kinase—ERK; 1:1,000; Cell Signaling Technology
Inc, Danvers, MA, USA) for ERK phosphorylation
activity. The membranes were then washed and incubated
for 1 h at RT with the secondary antibody/horseradish-
peroxidase-conjugated goat anti-rabbit antibody (1:10,000,
Jackson ImmunoResearch, West Grove, PA, USA) and
developed using the ECL reagent (Pierce, Rockford, IL,
USA), allowing visualization of the proteins. Then, the
membranes were washed and incubated for 30 min at RT
in Restore Western Blot Stripping Buffer (Pierce) and
reincubated with rabbit monoclonal anti-pan-ERK
(1:2,000; Invitrogen) antibody to quantify the nonphos-
phorylated protein, using a two-step procedure similar to
that previously described (Takman et al. 2004). Quantita-
tive analysis of ERK phosphorylation activity was per-
formed as previously described (Arien-Zakay et al. 2007).
Light and Fluorescence Microscopy
Cell cultures were examined under an inverted Nikon
contrast microscope (Eclipse TE-2000-U and Eclipse
TS100; Nikon, Melville, NY, USA). All images were
acquired digitally with either a Hamamatsu black-and-white
high-resolution camera or a Nikon Coolpix5000 camera
and analyzed using Northern Eclipse software (Empix
Imaging Inc., Mississauga, ON, Canada) and SigmaScan
software (Sigma). GFP fluorescence was quantified with
the aid of a confocal laser scanning fluorescence micro-
scope (Olympus 300 IX-70, Japan and Axiovert 200, Zeiss,
Göttingen, Germany), using excitation and emission wave-
lengths of 488 and 509 nm, respectively. Pictures were
acquired with a SensiCam digital camera (PCD, CCD
Imaging, Kelhein, Germany) and processed with Image-Pro
Plus 6.0 (Media Cybernetics®, Silver Spring, MD, USA).
Statistics
Each experiment was repeated in at least duplicates three to
four times. Where possible, the data are presented as the
mean ± SEM as evaluated using the GraphPad InStat 3
program (GraphPad Software Inc, San Diego, CA, USA).
Statistically significant differences between the experimen-
tal groups were determined by analysis of variance; the
results were considered significant when p<0.05.
229
Results
FACS Sorting Followed by Cloning of Stable Transfectant
GFP-PC12 Cells
PC12 cells transduced with the lentivirus GFP vector
underwent sterile separation using the FACS to separate
the population of labeled from the unlabeled cells (Fig. 1a).
In a representative flow cytometric analysis of GFP-
transduced cells, 65.4% of the population was fluorescent
(Fig. 1a—left, insert). Using appropriate gating, 55.8% of
this population, characterized by very high fluorescence
intensity (105 fluorescent units) was collected (Fig. 1a—
left). The population, representing 84.2% highly intense
GFP-fluorescent cells (Fig. 1a—right) was re-analyzed.
This cell population was collected aseptically and cultured
under conventional 2-D culture conditions. Stably trans-
fected GFP clones were selected by limited dilution. Out of
∼500 clones, four were selected according to the following
criteria: (1) ability to proliferate with a mean population
doubling time of 50–80 h, (2) high intensity of GFP
expression, (3) “normal” PC12 morphology, (4) monolayer
formation upon adherence to collagen-coated dishes, and
(5) “normal” differentiation: neurite outgrowth in response
to stimulation with NGF. Each of the four clones selected
adhered to collagen and exhibited a morphology similar to
that of the wt-PC12 cells as exemplified by one of the
clones (Fig. 1b). Some differences between the four clones
were found in terms of proliferation rate, intensity of GFP
expression, and the ability to undergo NGF-induced
differentiation (Table 1). The GFP-PC12-4f clone chosen
for the present study was the one that was most similar in
all the relevant criteria to the wt-PC12, expressed high and
stable GFP intensity (for about 2 years upon freezing and
reculturing; Fig. 1b), and was characterized by a prolifer-
ation rate similar to that of the wt-PC12 (Fig. 1c).
GFP-PC12 NGF Receptors Expression and Signaling
NGF-induced differentiation in the PC12 cells is mediated
by activation of the NGF receptors: TrkA and p75NTR and
activation of downstream signaling pathways (Kaplan and
Miller 2000). Since GFP-PC12 clones were derived from
individual cells of the parental wt-PC12 cell population and
considering the large heterogeneity of NGF receptors level
in different neurons, it was extremely important to validate
NGF receptor expression and signaling in the GFP-PC12
cells. This characterization was performed by evaluating the
level of NGF receptor mRNA expression, NGF-induced
activation of ERK, and NGF-induced neurite outgrowth
under 2-D culture conditions.
mRNA expression levels of both TrkA and p75NTR
receptors in GFP-PC12 cells were assessed using semi-
230 J Mol Neurosci (2009) 37:225–237

Figure 1 Development and

characterization of stable GFP-
PC12 clonal cells. a FACS
analysis (number of cells with
different fluorescence intensity)
and sorting of GFP-transduced
PC12 cells: left immediately
after transfection; right after 7
days of cell culture and before
cloning by limited dilution. b
Phase contrast and fluorescent
micrographs of wild-type
(wt-PC12) and green fluorescent
protein (GFP)-transduced PC12
cells after several weeks in
culture. c Time course of cell
proliferation (fold increase vs
control—day 0) of GFP-PC12
(gray) in comparison with wt-
PC12 (white) under 2-D condi-
tions, estimated by the Alamar
blue assay. The results represent
the mean ± SEM of triplicate
experiments. *p<0.01 vs day 0

quantitative RT-PCR. The results, normalized to the
expression of β-actin, suggest similar levels of expression
for both neurotrophin receptors as found in wt-PC12 cells
(Fig. 2a). Activation of the TrkA receptors by NGF triggers
intracellular signaling cascades, such as the ras-activated,
mitogen-activated protein kinase (MAPK) pathway, result-
ing in neuronal differentiation as evidenced by neurite
outgrowth (Kaplan and Miller 2000). To quantitatively
evaluate the NGF-induced TrkA activation in the GFP-
PC12 compared with that in wt-PC12 cells, we measured
the phosphorylated form of the downstream activated
MAPK member: the extracellular signal-regulated kinase
(Fig. 2b). The basal phosphorylation level of ERK1/2 was
very low in both the untreated wt-PC12 and GFP-PC12
cells, while under NGF treatment their phosphorylation was
highly elevated in both cell lines. In the GFP-PC12 cells,
treatment with NGF significantly increased the relative
phosphorylation of ERK1 and ERK2 by 10.5- and 10-fold
vs the control, respectively (p<0.01; Fig. 2b). As expected,
in the wt-PC12 cells, treatment with NGF significantly
increased the relative phosphorylation of ERK1 and ERK2
by 11.5- and 12-fold vs the control, respectively (p<0.01;
Fig. 2b).
These results clearly indicate a significant similarity in
NGF receptor mRNA expression and NGF-induced ERK
phosphorylation in GFP-PC12 clonal cells compared with
that of the parental wt-PC12 cells.
Characterization of NGF-induced Differentiation
in GFP-PC12 in Comparison with that in wt-PC12 Cells
in 2-D
To quantitatively assess NGF-induced differentiation of the
GFP-PC12 cells under 2-D culture conditions, we used the
same bioassay developed in our laboratory for wt-PC12
cells (Katzir et al. 2002). GFP-PC12 cells at low density
J Mol Neurosci (2009) 37:225–237 231

Table 1 Biological characterization of four different GFP-PC12 clones

PC12 Proliferation GFP expression Morphologyd Adherencee NGF response

clonea (doubling time, h)b (fluorescence intensity)c (% of cells with outgrowths)f

wt-PC12 48 0 Neuronal Monolayer 100

PC12-4c 50–60 103 Neuronal Monolayer 80
PC12-1d 50–60 105–106 Neuronal Monolayer 60
PC12-4d 70–80 104 Heterogeneousg Monolayer 100
PC12-4f 50–60 105 Neuronal Monolayer 100
a
PC12 cells transduced with a GFP-expressing lentiviral vector were isolated by FACS, cloned by limiting dilution into 500 wells, and grown for
1 month. Four clones were isolated and expanded. For biological characterization, samples of each clone and wild-type cells were evaluated for
different parameters over a period of several weeks.
b
Proliferation was estimated, followed daily for 5 days, using the Alamar blue method.
c
GFP expression was estimated by FACS measurement of fluorescence intensity.
d
wt-PC12 cultures contain about six different morphotypes (round cells, monopolar, bipolar, tripolar, multipolar, and a fibroblast-like
morphotype); their presence in the clones indicates plasticity similar to that of the parental cells.
e
Adherence was estimated by flattening of the cells and generation of a monolayer. Nonadherent cells grow in clusters in suspension or are
partially attached to the monolayer.
f
NGF-induced differentiation was evaluated for 3 weeks. The cells were treated with 50 ng/ml NGF, and the cultures were photographed every 3
days by phase contrast and fluorescence microscopy. Elongation of the neurites and formation of the neural networks were estimated as described
in “Materials and Methods”, using the E parameter.
g
This clone spontaneously generates neurite outgrowths.

were treated with 50 ng/ml mouse β-NGF for 7 days or left GFP-PC12 cells extended neurite outgrowths of different
untreated as control. In parallel experiments, wt-PC12 cells lengths (Fig. 3a, lower panel) similar to wt-PC12 cells. A
at the same density were also treated under identical typical dose response of the NGF-induced differentiation is
conditions. Upon NGF treatment, the majority of the presented in Fig. 3b. The similarity of the response in GFP-

Figure 2 Characterization of NGF receptor mRNA transcript expres-
sion and activity in GFP-PC12 cells compared with that in wild-type
parental cells under 2-D conditions. a mRNA expression of TrkA and
p75 receptors in relation to mRNA expression of β-actin (internal
control). Total RNA was extracted, RT-PCR was applied to equal
amounts of cDNA from each cell type using specific primers (carried
out as described in “Materials and Methods”). b NGF-induced ERK
activity in the two cell types. The cells were treated with 50 ng/ml
mNGF for 30 min or left untreated. Phosphorylation activity was
evaluated by Western blot, using antibodies directed to the phosphor-
ylated (upper) or unphosphorylated (lower) ERK proteins. In the
histograms, the relative phosphorylation (%), calculated as described
in “Materials and Methods”, is presented as: white bars ERK1, gray
bars ERK2. The results represent the mean ± SEM of triplicate
experiments.*p<0.01 vs untreated
232
Figure 3 NGF-induced differentiation in GFP-PC12 cells under 2-D
conditions is dose dependent and blocked by K252a. a Phase contrast
and fluorescent micrographs of GFP-PC12 cells compared with those
of wt-PC12 under 2-D cell growth conditions, upon treatment with 50
ng/ml mNGF (+NGF) or untreated (−NGF). b NGF-induced dose-
dependent differentiation response, measured by elongation of neurite
outgrowths (E), as described in “Materials and Methods”; gray bars
PC12 cells vs that in wt-PC12 cells is evident, indicating
EC50 of 2.9 and 2.6 ng/ml, respectively. A hallmark of
NGF-induced differentiation of the PC12 cells is the
inhibitory effect induced by K252a, the NGF-TrkA receptor
antagonist (Koizumi et al. 1988). Therefore, we evaluated
the ability of K252a to block NGF-induced differentiation
in GFP-PC12 cells (Fig. 3c). As expected, the data clearly
indicate that 100 nM K252a inhibited by about 85% the
NGF-induced differentiation.
These results indicate both qualitatively and quantita-
tively that NGF-induced differentiation of GFP-PC12 is
very similar to that of the wt-PC12 cells. Therefore, GFP-
PC12 cells provide a new valid model for the visualization
and imaging of neurite extension as a surrogate marker of
neuronal differentiation.
J Mol Neurosci (2009) 37:225–237
GFP-PC12 cells; white bars wt-PC12 cells. The results represent the
mean ± SEM of triplicate experiments. *p<0.001 vs untreated
cultures. c The inhibitory effect of 100 nM K252a, the NGF-TrkA
receptor antagonist, on NGF-induced differentiation of GFP-PC12
cells. The results represent the mean ± SEM of triplicate experiments.
*p<0.001 vs control; **p<0.001 vs NGF
GFP-PC12 Proliferation in 3-D Collagen Gels
To evaluate the proliferation of the GFP-PC12 cells in a 3-D
culture venue, the cells were seeded in 3-D type I collagen
gels, and their morphology was evaluated by light and
fluorescence microscopy (Fig. 4). Due to their intensive,
intrinsic (GFP) fluorescence, the GFP-PC12 cells were
easily detectable in a fluorescence microscope within the
3-D collagen gels (Fig. 4, upper panel). As previously
reported (Baldwin et al. 1996), in 3-D hydrogels wt-PC12
cells attached to the matrix and in a few days form
aggregates (Fig. 4, upper panel). This process of aggrega-
tion, which is not observed in 2-D conditions, is probably
facilitated by the mechanical forces generated by the cells
in 3-D culture. GFP-PC12 cells grown at low density in the
J Mol Neurosci (2009) 37:225–237
Figure 4 Phase contrast and fluorescent micrographs of GFP-PC12 cells compared with those of wt-PC12 under 3-D cell growth conditions,
treated with 50 ng/ml mNGF (+NGF) or untreated (−NGF)
3-D collagen gels showed increased proliferation for up to
21 days (Fig. 5). Although their proliferation in 3-D culture
conditions was somewhat slower than that of wt-PC12 as
also observed in 2-D, the increase in cell number with time
was similar to that of wt-PC12.
NGF-induced Differentiation of GFP-PC12 Cells in 3-D
Collagen Gels
When grown in collagen gels in the presence of 50 ng/ml
NGF for up to 14 days, both wt-PC12 and GFP-PC12 cells
Figure 5 Time course of cell proliferation (fold increase vs control—
day 0) of GFP-PC12 (gray) in comparison with that of wt-PC12
(white) under 3-D conditions, estimated by the Alamar blue assay. The
results represent the mean ± SEM of triplicate experiments. *p<0.01
vs day 0; **p<0.05 vs wt-PC12 at the same time point
233
extended very long and dense neurite outgrowths that
emanated from the 3-D cell aggregates (Fig. 4, lower
panel). Since both the small and the large neurite out-
growths of GFP-PC12 cells were highly visible in the 3-D
gels, we were also able to measure their length and compare
it with NGF-induced differentiation of GFP-PC12 cells in a
2-D environment (Fig. 3). However, due to cell aggregation
in the 3-D collagen gels (Baldwin et al. 1996), the
estimation of neurite outgrowth, as the ratio between
neurite length and cell diameter, using the commonly
available 2-D method is not accurate. Furthermore, out-
growths of many neurites in three dimensions made it
difficult to trace them from individual cells. This problem
required the adaptation of a novel measurement assay of
NGF-induced differentiation under 3-D conditions. For this
purpose, we applied NGF-induced differentiation using a
fractal dimension (Df) method used for assessing blood
capillary sprouting and network formation in vitro and in
vivo (Kirchner et al. 1996; Lazarovici et al. 2006). In the
first step, we validated the method by comparing the E and
Df parameters from measurements of the same images upon
2 days of NGF treatment, at the beginning of the
differentiation process (Fig. 6a). A correlation coefficient
of 0.94 of the differentiation effects measured by these two
methods indicates a high similarity in neurite outgrowth
elongation estimated by the fractal dimension method and
the elongation method. Furthermore, the Df parameter was
checked for its ability to reflect a dose-dependent elonga-
tion of neurite outgrowth (Fig. 6b). By increasing the NGF
concentration, the differentiation process was enhanced, as
evident from the parallel change in Df compared with the E
parameter. We then compared NGF-induced differentiation
234
Figure 6 Validation of NGF-induced differentiation of GFP-PC12
cells under 2-D conditions by comparing the fractal dimension
parameter (Df) with the elongation parameter (E). E and Df were
calculated as described in “Materials and Methods”. a The relationship
between Df and E with a correlation coefficient of 0.94 was calculated
from the regression line. b NGF-induced dose-dependent differentia-
of GFP-PC12 cells cultured in 2-D and 3-D conditions, using
the Df method. Our data suggest that at 8 days the cells
maintained in 2-D underwent a 3.5-fold more pronounced
differentiation that those cultured in 3-D (Fig. 7). After
15 days of treatment, NGF induced similar degrees of
differentiation in both 2-D and 3-D cultures. Extension of
the culture period to 45 days did not further enhance NGF-
induced differentiation of GFP-PC12 cells in 3-D beyond
that observed at day 15.
Figure 7 NGF-induced differentiation of GFP-PC12 cells under 3-D
(gray) conditions is delayed compared with those under 2-D (white)
conditions as measured by Df. Df was calculated as described in
“Materials and Methods”. The values represent the mean ± SEM of
triplicate experiments. *p<0.01 vs untreated cells; **p<0.05 vs 2-D
conditions at the same time point
J Mol Neurosci (2009) 37:225–237
tion responses, analyzed by Df and E. Upper panel Representative
pictures of NGF-induced neural networks and their representative
fractal dimension images. Lower panel Comparison between the dose-
dependent differentiation responses measured using Df and E. The
values represent the mean ± SEM of quadruplicate experiments. *p<
0.01 vs untreated cells
Discussion
Neurons, with their long axons and elaborate dendritic
arborization, establish the complex circuitry that is essential
to the proper functioning of the nervous system. A list of
structural, molecular, and functional differences between
axons and dendrites is emerging. However, the mechanisms
involved in early events of neuronal differentiation, such as
neurite initiation and elongation, are less well understood,
mainly because of a lack of quantitative methods to
accurately measure neuronal differentiation. In tissue
engineering research, fluorescent cells derived from GFP-
transgenic rodents or engineered in vitro to express the GFP
transgene have been recently used to elucidate the cell–
microenvironment interaction (Tan et al. 2007), for both
regeneration and repair purposes (Boldrin et al. 2007; Sales
et al. 2007), and to study bone differentiation (Zhou et al.
2006). We, therefore, continued this research approach and
developed a GFP-PC12 cell model suitable for studying
neuronal differentiation in a 3-D microenvironment.
In contrast to some molecular approaches in which the
GFP transgene is targeted to the mitochondria (Sirk et al.
2003) or coupled to a specific intracellular protein (Niell
and Smith 2004), in the present study, the GFP transgene
protein accumulated in the cytosol of the green PC12 cells,
as previously shown for hematopoietic cells (Miyoshi et al.
1999). This enabled both intensive visualization of the
perikarya and the neurite outgrowth cytoplasm of very
small neurite branches. These GFP-PC12 cells similar to
J Mol Neurosci (2009) 37:225–237
wt-PC12 cells under 2-D conditions proliferated, expressed
NGF receptors, and activated the ERK pathway in response
to NGF. They also showed NGF-induced differentiation, a
process inhibited by K252a, the NGF-TrkA receptor
antagonist.
Under 3-D conditions, GFP-PC12 cells also proliferate,
form aggregates (Baldwin et al. 1996), and respond to NGF
by neurite outgrowths of different lengths and complexity,
again very similar to wt-PC12 cells. Since the techniques
for quantifying the NGF-induced differentiation in 2-D
were not applicable for 3-D conditions, we employed and
validated a novel approach for measuring neurite outgrowth
based on fractal dimension (Df). This method was validated
by the 2-D method in which the elongation parameter (E) is
measured and used to quantify NGF-induced differentiation
of GFP-PC12 cells in 2-D by dose response and in 3-D by
time course. At day 8, NGF-induced differentiation of GFP-
PC12 cells in 3-D collagen gels was significantly retarded
compared to cells cultured in 2-D on collagen-coated
surfaces. By contrast after 15 days, neurite extension in
both 2-D and 3-D was statistically indistinguishable (Fig. 7).
Two plausible explanations for the delayed NGF-induced
elongation of the neurites in 3-D vs 2-D conditions are
proposed: (a) the relative high stiffness rendered by
collagen gels (1.5 mg/ml), known to generate about 15–17
Pa (Willits and Skornia 2004), inhibited neurite elongation;
and (b) the process of metaloprotease secretion by the cells,
known to generate the necessary space in the gel by
collagen hydrolysis, required for the elongation of the
neurite outgrowths, was limited. Indeed, PC12 cells over-
expressing the tissue plasminogen activator, which pro-
motes proteolytic degradation of the matrix, grow neurites
to a greater extent than do control cells (Pittman and
DiBenedetto 1995).
Since collagen gels and other hydrogel scaffolds are
relatively translucent, it is feasible to visualize by confocal
fluorescent microscopy the morphological differentiation of
GFP-expressing neurons inside the gels and to quantify
neuritogenesis using the Df method, thus avoiding conven-
tional time-consuming histological methods. Over the last
decade, the genes for GFP and other color proteins have
been cloned and used for biological purposes (Suzuki et al.
2007), providing an elegant approach which facilitates
concomitant measurement of multiple proteins involved in
the NGF-induced differentiation under 3-D conditions.
Rationally designed matrices for neural engineering and
cell therapy rely on a comprehensive understanding of 3-D
cellular differentiation induced by neurotrophins, such as
NGF. Under these conditions, both biochemical and matrix
mechanical cues (such as stiffness) will influence neuronal
differentiation. In extending previous work with wt-PC12
cells (Saltzman et al. 1992), the present GFP-PC12 cell
model may be suitable to address this issue. Indeed, several
235
studies provided preliminary, qualitative evidence that
neurite outgrowth and branching of PC12 cells was
inhibited with decreasing substrate rigidity (Leach et al.
2007). On the other hand, attachment, morphology, and
functions of PC12 cells were superior in 3-D compared to
2-D culture conditions (Chia et al. 2005). These observa-
tions deserve further careful quantitative studies, for which
the present GFP-P12 cells might be well suited.
In the past, NGF-induced neuronal differentiation, in
particular neurite outgrowth, has been assessed qualitatively
in 3-D cultures using PC12 cells cultured on a variety of
chemically tailored biomaterials (Pittier et al. 2005; Yu
et al. 1999; Holmes et al. 2000) and polymers modified
with integrin-binding motifs (Park and Yun 2004). These
studies emphasized the enhanced biocompatibility of these
biomaterials and their prospects for neural regeneration.
However, to be suited for clinical neuroregenerative
therapy, such designed 3-D matrices will require quantita-
tive evaluation of their differentiative properties using novel
analytical tools such as the Df method in conjunction with
GFP-PC12 cells introduced by this study.
We expect that this GFP-PC12 cell model will continue
to serve in the development of novel bioactive matrices,
such as electrically conductive polymers for enhanced
neuronal differentiation (Guterman et al. 2002; Guo et al.
2007; Gomez and Schmidt 2007), for studying neuro-
trophin-induced differentiation in/on 3-D polymeric scaf-
folds (Levenberg et al. 2005), and for in vitro evaluation of
pharmacologically active microcarriers releasing NGF or
other drugs conveying PC12 cells (Tatard et al. 2004). The
NGF-induced differentiation of GFP neurons, in conjunc-
tion with near-infrared multiphoton microscopy and in vivo
laser tomography, should provide novel models for drug
screening and high-resolution structural imaging (Schenke-
Layland et al. 2006) for measurements of neuronal
differentiation in situ.
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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